characterization of the consensus dna binding site for ... · function that requires a dna binding...
TRANSCRIPT
Characterization of the consensus DNA binding site for human
KRAB-zinc finger protein KS1
Jennifer M. Grants
Thesis submitted to the
Department of Biochemistry & Microbiology
in partial fulfillment of the requirements for the degree of
Bachelor of Science (Hon.)
University of Victoria
Victoria, British Columbia, Canada
April 2010
© Jennifer M. Grants, April 2010
ii
Abstract
The zinc finger domain is a common eukaryotic DNA binding motif, and is often
found in transcription factors and transcriptional repressor proteins. KS1 (KRAB Suppressor
of Transformation 1) is a member of the Krüppel-Associated Box (KRAB) family of zinc
finger proteins. Its consensus DNA binding site (KSE) contains two regions of high sequence
conservation: a 5 base pair subsite termed the A box, and an 8 base pair subsite termed the B
box. The current study characterized this DNA binding site quantitatively, using
nitrocellulose filter binding assays (FBAs) to determine equilibrium constants for KS1
binding to the consensus site and scanning mutants. Numerous diagnostic and
troubleshooting procedures were utilized in the cloning and protein purification of the KS1
zinc finger region. A modified FBA protocol substituting polyA instead of poly(d[IC]) as the
carrier was developed in order to measure specific protein-DNA binding. The apparent
dissociation constant (Kd) for KS1 binding to KSE was 7.10 ± 1.28 nM. Scanning mutations
in the A or B boxes of KSE reduced the affinity of KS1 by 10- or 50-fold, respectively, while
mutations in flanking regions caused an approximate 2-fold reduction. Binding of KS1 to a
truncated binding site could not be detected. A model for KS1-DNA binding was proposed,
in which all zinc fingers may potentially bind to the entire 30 base pair consensus site, but the
strongest contacts are made by a subset of zinc fingers that bind to the A and B box sites.
iii
Acknowledgements
I would like to thank Dr. Paul Romaniuk for providing me with so many opportunities
over the past three years, and for his patience and guidance during my time in his lab. I also
thank Tristen Weiss for passing on her expertise in laboratory protocols, and I thank Tristen
Weiss and Erin Flanagan for many helpful discussions. Finally, I thank my parents, Jan and
Arvid Grants, for their continuing support throughout my time at university.
iv
Table of Contents
Abstract……………………………………………………………………….. ii
Acknowledgements…………………………………………………………… iii
List of Tables…………………………………………………………………. vi
List of Figures………………………………………………………………… vii
Introduction…………………………………………………………………… 1
Materials and Methods………………………………………………………...
• PCR amplification of KS1 gene constructs from a pTOPO vector.......
• Diagnostic restriction digests………………………………................
• Diagnostic PCR using universal primers……………………………..
• Plasmid sequencing……………………………………………………
• Cloning of KS1 gene constructs from human genomic DNA…………
• Plasmid isolation……………………………………………………....
• Protein purification…………………………………………………....
• Radiolabelling of DNA…………………………………………….......
• Standard nitrocellulose filter binding assay………………………......
• DNA excess nitrocellulose filter binding assay……………………......
• Cloning of KSE and mutant oligonucleotides……………………........
11
11
13
13
13
13
14
16
16
17
18
18
Results…………………………………………………………………………
• The pTOPO vector did not contain the KS1 gene……………………..
• KS1 zinc fingers can be cloned and purified from human genomic
DNA…………………………………………………………………....
• DNA binding by KS1 zinc fingers is not sequence-specific under
assay conditions……………………………………………………....
• Effect of monovalent salt concentration……………………………....
• Effect of competitor DNA or RNA………………………………….....
• Kd values for specific binding determined by FBAs with polyA
carrier. ………………………………………………………………
• Poly(d[IC]) interferes with binding reported in the literature………
19
19
24
29
33
33
35
37
v
Discussion……………………………………………………………………..
• KS1 protein constructs………………………………………………...
• Filter binding assays…………………………………………………..
40
40
43
Conclusion………………………………………………………….................. 54
References…………………………………………………………………….. 55
vi
List of Tables
Table 1: PCR programs and sequence of KS1-specific primers employed in
the amplification of two KS1 gene constructs………………………………...
12
Table 2: Standard Taq PCR program and sequence of pTOPO universal PCR
primers…………………………………………………………………………
15
Table 3: Sequence of pET universal PCR primers…………………………… 15
Table 4: Expected and actual sizes of restriction fragments in the diagnostic
digest…………………………………………………………………………...
23
Table 5: Sequences of oligonucleotides employed in standard and DNA
excess FBAs…………………………………………………………………...
31
Table 6: Kd values and relative affinities as determined by standard FBAs….. 31
Table 7: Relative affinities as determined by DNA excess FBAs………......... 32
Table 8: Relative affinities of KSE scanning mutants as determined by
standard FBAs with polyA carrier……………………………………………..
36
Table 9: Sequences of oligonucleotides used by Gebelein and Urrutia
(2001)…………………………………………………………………………..
38
Table 10: Dissociation constants of literature oligonucleotides as determined
by standard FBA with polyA carrier.………………………………………….
39
vii
List of Figures
Figure 1: Crystal structures of Zif268 bound to DNA……………………... 3
Figure 2: Schematic of hydrogen bonds formed in C-capping and
stabilization of the TGEKP linker……………………………………………..
4
Figure 3: Comparison of consensus sequences for the KS1 DNA binding
site…………………………………………………………………………......
7
Figure 4: Hydrogen bond donors and acceptors presented at the major
groove by DNA base pairs…………………………………………………….
Figure 5: Diagnostic PCR reactions with the putative pTOPO-KS1
plasmid...............................................................................................................
8
21
Figure 6: Diagnostic restriction digests of the putative pTOPO-KS1
plasmid………………………………………………………………………...
22
Figure 7: PCR of KS1-zf1 and KS1-zf2 constructs from human genomic
DNA…………………………………………………………………………...
26
Figure 8: Colony PCR identification of clones positive for KS1 gene
constructs……………………………………………………………………...
26
Figure 9: Confirmation of colony PCR results using specific and universal
primers. …………………………………………………………………….....
27
Figure 10: BLASTp (NCBI) analysis of KS1-zf1 #18(4) ………………..... 27
Figure 11: Induction and purification of KS1-zf1 and KS1-zf2………….... 28
Figure 12: Effect of competitor DNA or RNA on apparent Ka, as determined
by standard FBA………………………………………………………………
34
Figure 13: Standard FBAs with 1 µg/ml polyA for KS1-zf1 binding to
scanning mutants M1-M4……………………………………………………..
36
Figure 14: Comparison of standard FBAs with 1 µg/ml poly(d[IC]) or polyA
for KS1-zf1 binding to literature mutants……………………………………..
38
Figure 15: Comparison of IC and GC base pairs…………………………….. 49
1
Introduction
Eukaryotic gene expression is a tightly controlled process, in which each step can be
up- or downregulated, at a gene-specific level or on a global scale within the cell. The first
step in this process, transcription, is modulated by a variety of transcription factors ranging
from the general factors needed to guide RNA polymerase to promoters, to highly specific
factors that act only at a unique address within the genome. In general, transcription factors
serve as adaptors for other proteins which lack the ability to bind to DNA on their own, a
function that requires a DNA binding domain and one or more protein binding domains.
The zinc finger is a common DNA binding motif which consists of approximately 30
amino acids folded into two N-terminal β-strands and a C-terminal α-helix, surrounding a
central zinc ion. This zinc ion is generally coordinated by four cysteine and/or histidine
residues; the so-called “classical” arrangement of ligands is C2H2 (Iuchi, 2001). From the
perspective of DNA binding, the key residues in a zinc finger motif are found in the α-helix,
as these form sequence-specific hydrogen bonds with DNA bases. When a zinc finger binds
to DNA, the α-helix is brought into close contact with the major groove so that these bonds
may form. Crystal structures have shown that each zinc finger typically contacts three to four
DNA bases, and while the majority of contacts are formed with one strand of the DNA
duplex, some cross-strand contacts are also formed (Isalan et al., 1997).
Within transcription factors, zinc fingers are not found as discrete units, but instead
are organized into domains of tandem zinc fingers. Perhaps one of the best characterized zinc
finger transcription factors is Zif268 (also known as EGR1), which contains three tandem
zinc fingers. When bound to DNA, the zinc finger domain is said to be oriented in an
“antiparallel” fashion to the DNA, meaning that its N-terminus is directed toward the 3’ end
of the DNA strand to which the majority of bonds are formed, while its C-terminus is found
at the 5’ end (Isalan et al., 1997). In order for each zinc finger to make specific contacts with
DNA, the entire domain must wrap around the major groove, as is shown in Figure 1(a).
Contacts are made with nine consecutive bases: each zinc finger forms hydrogen bonds with
a three base pair subsite on one strand of the double helix, as well as reaching over and back
to a fourth base on the opposite strand, upstream of the core subsite (Isalan et al., 1997).
Five amino acid linkers, with the canonical sequence TGEKP, separate individual zinc
2
fingers and serve to control their orientation and spacing for more facile interaction with
DNA (Peisach and Pabo, 2003).
Transcription factors may have many more than just three zinc fingers. In these
polyzinc finger proteins, individual zinc finger motifs still interact specifically with bases at
the major groove; however, natural polyzinc finger proteins generally do not wrap
indefinitely around the major groove. Instead, some zinc fingers adopt the role of a
“structured linker” that crosses the minor groove, bringing the next zinc finger back to the
major groove on the same side of the DNA duplex. Notably, zinc finger proteins which use
this mode of binding recognize discontinuous DNA binding sites. Xenopus transcription
factor IIIA (TFIIIA), a nine-zinc finger protein, provides a well characterized example of this
type of DNA binding: zinc fingers 1 to 3 bind to the so-called “A-block” of the internal
control region of the 5S rRNA gene, finger 5 binds to the “intermediate element”, and fingers
7 to 9 bind to the “C-block”, while fingers 4 and 6 cross the minor groove (Wuttke et al.,
1997).
Artificial polyzinc finger proteins with six or nine zinc fingers have been designed to
recognize continuous binding sites of up to 18 or 30 base pairs, respectively, by wrapping
around the major groove of the DNA duplex with all zinc fingers (Kamiuchi et al., 1998).
However, this mode of binding requires an extended period of time to reach equilibrium, and
gives dissociation constants that are considerably higher than expected for the number of zinc
fingers present. Artificial zinc finger proteins with much lower dissociation constants can be
achieved by using longer linkers (Kim and Pabo, 1998), or “structured linkers” consisting of
an non-DNA binding zinc finger (Moore et al., 2001).
The problem of recognizing an extended DNA binding site stems from a difference in
the periodicities of DNA and the zinc finger domain. When a three-zinc finger protein such
as Zif268 binds to DNA, the double helix is slightly underwound (Peisach and Pabo, 2003).
The greater the number of zinc fingers that bind to the major groove, the longer the segment
of DNA that must unwind, and so the entire complex is strained. This begs the question, why
did the canonical linker between zinc fingers evolve to be only five amino acids long? If the
linker were longer, it would allow the zinc finger domain to conform to the natural
periodicity of B-DNA, which would be particularly beneficial in polyzinc finger proteins.
However, a crystal structure of two individual Zif268 zinc finger domains bound to adjacent
3
sites on DNA (Figure 1(b)) demonstrated that the distance spanned by a TGEKP linker is
naturally the optimal distance between zinc fingers in a polyzinc finger complex. It is
assumed that longer linkers increase the flexibility of the protein in solution, to the point that
DNA binding carries a high entropic penalty (Peisach and Pabo, 2003).
(a)
(b)
Figure 1: Crystal structures of Zif268 bound to DNA. (a) A single Zif268 zinc finger domain
bound to DNA. Zinc fingers are separated by canonical TGEKP linkers, and the double helix
is slightly unwound to 11.3 bp/turn (Elrod-Erickson et al., 1996). (b) Two Zif268 zinc finger
domains bound to DNA. Individual domains bind such that they are separated by the distance
spanned by a canonical TGEKP linker (Peisach and Pabo, 2003). N- and C-termini of the
zinc finger proteins are indicated by letters N and C, and 5’ and 3’ ends of the DNA strand
of interest are indicated by the appropriate numbers.
Primary contacts with brown DNA strand
N
C
5’
3’
N
N
C C
5’
3’
Primary contacts with pink DNA strand
4
TGEKP linkers make significant enthalpic contributions to the free energy of DNA
binding, by the stabilizing the zinc finger α-helices and locking the zinc finger domain in its
DNA binding conformation. Upon DNA binding, the linker stabilizes the C-terminus of the
α-helix in the preceding zinc finger domain, through an interaction referred to as “C-
capping”. Figure 2 is a schematic of this interaction, in which the glycine α-amino proton
forms a key hydrogen bond with the α-carbonyl oxygen of the third residue from the C-
terminal end of the α-helix, which is not stabilized by internal hydrogen bonds within the α-
helix itself. Also shown in Figure 2 is an internal hydrogen bond within the linker, between
the glutamate α-amino proton and the threonine Oγ, which converts the linker from a flexible,
unstructured region to a rigid structure upon DNA binding (Laity et al., 2000). Due to this
dramatic increase in protein rigidity upon DNA binding, low entropy in solution is
favourable. Thus, these two hydrogen bonds likely provided selective pressure for the
maintenance of both the sequence and length of the TGEKP linker throughout evolution. It
should be noted that similar N-capping interactions often form as well, to stabilize the N-
terminal end of a zinc finger α-helix; however, these interactions involve other residues
within the zinc finger, and not linker residues (Laity et al., 2000).
Figure 2: Schematic of hydrogen bonds formed in C-capping and stabilization of the TGEKP
linker. The C-capping hydrogen bond (dashed arrow) forms between the glycine α-amino
proton and the α-carbonyl group of the third amino acid from the C-terminal end of the α-
helix. An additional hydrogen bond (dotted arrow) between the glutamate glycine α-amino
proton and the threonine Oγ stabilizes the linker in a rigid conformation (Laity et al., 2000).
Thr
Gly
Hydrogen bonding interactions
α-helix residues linker residues
Glu
5
The protein which is investigated in this study, KS1 (KRAB suppressor of
transformation 1, also referred to in the literature as ZNF382), is an interesting zinc finger
protein in many regards. It contains a HCH2 zinc finger which is separated by a 56-amino
acid spacer from nine tandem C2H2 zinc fingers at its C-terminus (Luo et al., 2002); refer to
the bottom of this page for a schematic of the KS1 protein. Zinc finger 1 has been referred to
as a vestigial zinc finger (Luo et al., 2002), implying that its DNA binding activity has been
abolished by a cysteine to histidine mutation which rendered it a HCH2 zinc finger. However,
histidine is also capable of coordinating zinc, and experimental evidence to date has not
demonstrated definitively whether or not this zinc finger is capable of binding DNA.
Interestingly, a KS1 homolog in rats (83% amino acid identity in the zinc finger domain) has
ten C2H2 zinc fingers and a true vestigial zinc finger within the 56-amino acid spacer, in
which a cysteine to aspartate mutation and a histidine to leucine mutation prevent the
coordination of a zinc ion (Gebelein et al., 1998). In human KS1, this vestigial zinc finger
within the spacer retains only one histidine, as it has the same C/D and H/L mutations as the
rat homolog, as well as an additional cysteine to tryptophan mutation (Luo et al., 2002; refer
to Figure 10 for clarification). The remaining nine zinc fingers of human KS1 follow the
consensus sequence CX2CX3FX5LX2HX3H (Luo et al., 2002), which conforms to the
classical C2H2 zinc finger consensus, CX2-4CX12HX2-6H (Iuchi, 2001). A rather unusual
feature of the KS1 zinc fingers is that most are separated by canonical TGEKP linkers (Luo
et al., 2002); this feature suggests that C-capping may contribute significantly to the strength
of KS1-DNA binding.
Schematic of the domain organization of the KS1 protein.
Zinc finger domain KRAB N C
56 amino acid spacer
2 3 1 4 6 7 8 9 10 5
6
The N-terminus of KS1 contains a Krüppel-associated box (KRAB) domain, which
recruits proteins involved in transcriptional repression, such as KAP-1 (Luo et al., 2002).
KS1 was the first KRAB-zinc finger protein proven capable of bringing about site-specific
transcriptional repression in vivo by binding to a high affinity site determined in an in vitro
random oligonucleotide selection process. Interestingly, the consensus binding site for KS1
was determined to be 27 base pairs long, and repression of promoters containing this
sequence required the presence of zinc fingers 2 through 10 (Gebelein and Urrutia, 2001).
Thus, prior research indicates that all nine zinc fingers may bind to DNA by wrapping around
the double helix at the major groove. If this postulation can be proved, then KS1 would be
the first example of a natural polyzinc finger protein to use such a mechanism of DNA
binding.
To date, the DNA binding site of KS1 has largely been studied in an indirect and/or
qualitative manner. Electrophoretic mobility shift assays (EMSAs) and reporter gene assays
were used to demonstrate that KS1 binds to the consensus site determined by random
oligonucleotide selection (Gebelein and Urrutia, 2001); however, these assays could not be
used to determine the equilibrium constants for the binding of KS1 to its consensus site or
mutants. An additional pitfall of these studies was that the consensus KS1 binding element
(KBE) was derived from 31 high affinity oligonucleotide sequences identified an in vitro
selection process, but it was not taken into account that some of these sequences were
identical; therefore, certain bases within the KBE were unduly biased. In the current study, a
slightly modified consensus KS1 sequence element (KSE) was used instead, which was
derived from one representative of each unique sequence identified in the original random
oligonucleotide selection process. The differences between KBE and KSE are outlined in
Figure 3; notably, KBE is 27 base pairs long, while KSE is a 30 base pair consensus
sequence. If each zinc finger is presumed to contact consecutive groups of three base pairs, as
the model for Zif268 binding would indicate (Isalan et al., 1997), then the length of KSE
would suggest that all ten KS1 zinc fingers are involved in DNA binding.
7
KBE TCCTACAGTACCAACCCTACAGAGTAA AGGATGTCATGGTTGGGATGTCTCATT
KSE CTATCATACTACCCACCCTACAGATGCACA GATAGTATGATGGGTGGGATGTCTACGTGT Figure 3: Comparison of consensus sequences for the KS1 DNA binding site. KBE was
determined by Gebelein and Urrutia (2001) from 31 sequences identified in a random
oligonucleotide selection process, all of which were not unique. KSE was used in this study,
and was derived from only the unique sequences identified by the Gebelein group;
differences from KBE are indicated in red. The A and B box regions are underlined, and
other conserved residues of KBE are highlighted in gray.
Despite these differences, KBE and KSE share two common motifs, termed the A and
B box regions. These were highly conserved among the 31 sequences identified by random
oligonucleotide selection, and this is reflected by the fact that KBE and KSE differ by only
one base pair in the A box and are identical in the B box (Figure 3). Despite a high degree of
conservation, preliminary mutagenesis studies of KBE indicated that while the A and B
boxes participate in protein-DNA binding, KS1 likely makes contact with bases flanking
these sites as well. That is, mutating one or both of the A and B boxes appeared to diminish
the affinity of protein-DNA binding, whereas deleting five or six base pairs at the 5’ or 3’
end of KBE abrogated binding entirely (Gebelein and Urrutia, 2001). This seemed to indicate
that KS1 strictly requires a 27 base pair minimum binding site; however, the study lacked
both quantitative data and a complete set of mutants. The strength of the binding interaction
observed between KS1 and mutants of the KBE A and/or B boxes could have been hundreds
or thousands of times weaker than binding to the consensus KBE, which would render the
binding interactions irrelevant in vivo. Indeed, KBE scanning mutants were not tested in
reporter assays; deletions of KBE were the only mutants investigated. In addition, a complete
set of mutants should have included scanning mutations in the 5’ and 3’ ends, as these would
A B
8
have ruled out the possibility that the deletion mutations simply eliminated residues which
were crucial to high-affinity binding.
The current study provides a first step toward a more complete set of DNA mutant
data. These included scanning mutations of the A or B boxes, as well as scanning mutations
in the 5’ and 3’ flanking regions of KSE. In this way, it was possible to assess the
contribution of each of these key regions to the free energy of protein-DNA binding. By
using scanning mutations of the 5’ and 3’ flanking sites, instead of simple deletions as in the
previous study, it was possible to distinguish between the sequence requirements and the
binding site length requirements of the KS1 protein. In addition, the scanning mutations of
the A and B boxes employed in this study were more informative than those used in previous
research: the mutations were designed to alter the pattern of hydrogen bonds donors and
acceptors presented at the major groove by each base pair (Figure 4), whereas previous
studies had simply mutated the sequences of interest to polyG.
Figure 4: Hydrogen bond donors and acceptors presented at the major groove by DNA base
pairs. D indicates hydrogen bond donors, A indicates hydrogen bond acceptors.
Major groove side
Minor groove side
D D A A A A
9
This study also provides the first quantitative data for the binding of KS1 to its DNA
element. Nitrocellulose filter binding assays (FBA) were used to determine the equilibrium
constants for the binding of KS1 to the KSE consensus sequence and scanning mutants. This
technique (Romaniuk, 1985) relies upon the ability of a nitrocellulose filter to trap protein
and any complexed labelled oligonucleotides, while a nylon filter is used to trap unbound
labelled oligonucleotides. The fraction of DNA bound by varying protein concentrations can
thus be determined by autoradiography, and then normalized to account for minimal retention
of free oligonucleotide by the nitrocellulose; this fraction is subsequently plotted as a
function of protein concentration to generate an equilibrium binding curve (with curve fitting
software).
From this curve, it is possible to determine the dissociation constant (Kd) from the
following bimolecular equilibrium:
DNA-protein DNAfree + Proteinfree (1)
In an FBA, it is not possible to measure the concentration of free protein directly. However,
assuming that the concentration of bound protein is significantly lower than the Kd, the
concentration of free protein can be approximated by the total protein concentration:
[protein]free = [protein]total – [DNA-protein] Where [DNA-protein] << Kd
[protein]free ≈ [protein]total (2)
Bearing this assumption in mind, the Kd can be determined as the total protein concentration
at 50% saturation:
[DNA-protein] = [protein]total (3)
[DNA]total [protein]total + Kd
A drawback to this method is that the protein is assumed to be 100% active. In order
to circumvent this assumption, an alternate FBA can be used in which the Kd is determined
by titrating constant a concentration of protein and labelled oligonucleotide with an
increasing concentration of identical, unlabelled competitor oligonucleotide. Plotting the
amount of bound DNA as a function of the amount of unbound DNA gives a curve in which
the Kd is the free DNA concentration that gives 50% protein saturation:
10
[DNA-protein] = [DNA]free (4)
[Protein]total [DNA]free + Kd
Thus, this alternate assay (termed the DNA excess FBA) does not assume 100% protein
activity, since the concentration of protein is held constant. However, standard FBAs are still
valid when comparing the relative binding affinity of a single protein preparation to different
DNA mutants.
The results of these FBAs demonstrated that the KS1 zinc finger region bound to
KSE with an apparent Kd of 7.10 ± 1.28 nM. The A and B boxes made the greatest
contribution to the free energy of binding, as mutations in these regions caused an
approximate 10- or 50 fold reduction in apparent Kd, respectively, while mutations in the 5’
and 3’ flanking regions caused only an approximate 2-fold reduction. Conversely, binding of
KS1 to a 3’ deletion of KBE could not be detected. From these data, a modified model for
KS1-DNA binding emerged, in which all zinc fingers may potentially bind to the entire 30
base pair consensus site, but the strongest contacts are formed by the binding of certain zinc
fingers binding to the A and B box sites.
11
Materials and Methods
PCR amplification of KS1 gene constructs from a pTOPO vector1
A pTOPO vector containing the KS1 gene (Thermo Scientific) was isolated using a
QIAGEN miniprep kit. PCR primers (IDT) unique to KS1 were used to amplify two
constructs of the zinc finger region: zinc fingers 1 through 10 (henceforth referred to as KS1-
zf1), and zinc fingers 2 through 10 (referred to as KS1-zf2). Reactions contained 2x Pfx
DNA polymerase buffer and 0.5 units Pfx DNA polymerase (Invitrogen), 1 mM MgSO4, 0.3
mM dNTPs, 1 ng plasmid, 0.3 µM upstream NcoI primer, and 0.3 µM downstream XhoI
primer (Table 1). A standard 35-cycle Pfx PCR program outlined in Table 1 was employed,
using a Biometra T-personal thermocycler. Alternately, the annealing temperature of the
standard PCR program was raised to 62°C, or a touchdown PCR program was employed
(Table 1). Samples from each reaction were analyzed on a 1.5% agarose gel.
1 The pTOPO vector did not contain the KS1 gene, but instead the gene for a Rab kinase GTPase activating
protein (TBC1D13). However, this was unknown to me at the time.
12
Table 1: PCR programs and sequence of KS1-specific primers employed in the amplification
of two KS1 gene constructs.
Standard Pfx PCR program Touchdown PCR program
i) 94°C
ii) 94°C
iii) 60°C
iv) 72°C
5 min
30 sec
30 sec
5 min
v) repeat ii-iv 30x, decreasing
annealing temp. 0.3°C per cycle
i) 94°C
ii) 94°C
iii) 55°C or 62°C
iv) 68°C
v) repeat ii-iv 35x
vi) 68°C
vii) 4°C
2 min
10 sec
10 sec
90 sec
2 min
hold vi) 94°C
vii) 55°C
viii) 72°C
ix) repeat vi-viii 10x
x) 72°C
xi) 4°C
30 sec
30 sec
5 min
5 min
hold
Upstream NcoI primer Downstream XhoI primer
KS1-zf1 CGGCCGCCATGGAACCATTTG
-ACCATAATGAATGTGAAAAATCC
KS1-zf2 CGGTCACCATGGAACCCT
-TTCACTGTCCTTACTGTGG
CGCCAGCTCGAGTCATTACTGAA
-TTCCCATAGTTTCTACCTTGTG
13
Diagnostic restriction digests
The pTOPO-KS1 plasmid was digested with NotI (Invitrogen), ScaI (Pharmacia),
NdeI, AflIII, PstI, AvaII, NcoI, or EcoRI (NEB). Reactions contained appropriate buffers (as
specified by the manufacturer), 200 ng plasmid, and 1 to 4 units of restriction enzyme.
Additional digests were conducted with an excess of restriction enzyme (15 units NotI, 10
units NcoI, or 20 units EcoRI). Digests were incubated for 90 minutes at 37°C. Samples were
analyzed on a 0.8% agarose gel, along with undigested plasmid.
Diagnostic PCR using universal primers
Universal M13 primers (IDT; Table 2), complementary to the pTOPO vector, were
used to detect the presence of a gene within the pTOPO vector. Reactions contained 1x Taq
DNA polymerase buffer and 0.25 units Taq DNA polymerase (ABM), 1.5 mM MgCl2, 0.1
mM dNTPs, 1 ng plasmid, 0.3 µM forward primer, and 0.3 µM reverse primer. Additional
reactions combined universal primers with the KS1-specific primers, to detect the
functionality of the KS1 primers. A 35-cycle standard Taq PCR program was employed
(Table 2). Samples were analyzed on a 0.8% agarose gel stained with ethidium bromide.
Plasmid sequencing
Plasmids were sequenced by the University of Victoria Centre for Biomedical
Research. The identity of gene sequences were determined by BLAST analysis (NCBI).
Cloning of KS1 gene constructs from human genomic DNA
The two KS1 constructs were amplified from 1 µg of human genomic DNA, using the
same KS1-specific primers and the standard Pfx PCR reaction conditions that were employed
with the pTOPO vector. PCR products were purified by gel extraction (QIAGEN kit),
digested with NcoI and XhoI, purified with the QIAquick PCR purification kit (QIAGEN),
and ligated into pET30a digested with the same restriction enzymes. Restriction digests
contained the appropriate buffers (as specified by NEB) and 1 unit of each enzyme, and were
incubated for 90 minutes at 37°C. Ligation reactions contained 1x T4 DNA ligase buffer and
400 units T4 DNA ligase (NEB), 60 ng NcoI/XhoI-digested pET30a, and 5.0 µl of the
14
digested and purified PCR product; the reaction was incubated for 24 hours at room
temperature.
Plasmid isolation
Competent Escherichia coli DH5α cells were transformed with one half of each
ligation reaction, and plated on selective agar media containing 50 µg/ml kanamycin.
Colonies positive for the desired gene were identified by colony PCR, using 0.25 units Taq
DNA polymerase (ABM), 1.5 mM MgCl2, 0.1 mM dNTPs, 1 ng plasmid, 0.3 µM forward
primer, and 0.3 µM reverse primer. The pET-specific universal primers (IDT) detailed in
Table 3 were employed. Plasmids were purified using the QIAprep Spin Miniprep Kit
(QIAGEN).
15
Table 2: Standard Taq PCR program and sequence of pTOPO universal PCR primers.
Standard Taq PCR program
i) 94°C
ii) 94°C
iii) 55°C or 62°C
iv) 68°C
v) repeat ii-iv 35x
vi) 68°C
vii) 4°C
2 min
10 sec
10 sec
90 sec
2 min
hold
M13 forward primer M13 reverse primer GGCGATTAAGTTGGGTAACGCCAG CTCGTATGTTGTGTGGAATTGTGAGCG
Table 3: Sequence of pET universal PCR primers. A standard Taq PCR program was
employed (Table 2).
pET16 forward primer pET16 reverse primer CGGCGGTGTGAGCGGATAACAATTCCC GGCGCGATGCTAGTTATTGCTCAGCGG
16
Protein purification
Competent E. coli BL21(DE3) cells were transformed with 50 ng of each plasmid,
and plated on selective agar media containing 50 µg/ml kanamycin. Overnight cultures were
prepared by inoculating 5 ml 1x LB containing 50 µg/ml kanamycin with a single
transformed colony and incubating at 37°C for approximately 20 hours. Half of each
overnight culture was subcultured (1/10 dilution) and then grown at 37°C to an optical
density between 0.5 and 0.6 before induction with 1 mM IPTG. Induced cells were grown at
37°C to an optical density between 1.1 and 1.6; for incubation times longer than 5.5 hours,
the temperature was reduced to room temperature for the remainder of the period. Cells were
harvested by centrifugation (5000 rpm, Beckman JLA 16.250). Cells were lysed by French
press in a resuspension buffer consisting of 1x TAB (20 mM MgCl2, 5 mM ZnCl2, 250 mM
NaCl, pH 7.5), 1 mM PMSF, 10.2 % glycerol, 5 mM DTT, and 5 mM imidazole. The lysate
was centrifuged (11,500 rpm, Beckman JA-20), and inclusion bodies in the insoluble fraction
were resuspended overnight in a denaturation buffer, consisting of resuspension buffer plus
5M urea. The supernatant was isolated by centrifugation (11,500 rpm, Beckman JA-20), and
loaded on a Nickel-Agarose 6 Resin (ABM) column equilibrated with denaturation buffer.
Protein was eluted with 150 mM and 250 mM imidazole in denaturation buffer, and the
protein concentration was determined by the method of Bradford (1976). Samples from
inductions and column purification were analyzed on SDS-PAGE gels (3.5% stacking, 15%
separating) stained with Coomassie blue.
Radiolabelling of DNA
DNA oligonucleotides (IDT) to be used as the substrate in filter binding assays were
annealed by heating a solution of top and bottom strand oligonucleotides (40 µM each) in a
5x concentrated annealing buffer2 to 95°C for 5 minutes and then cooling to room
temperature for 1 hour. The mixture was subsequently diluted 1/10 in 1x annealing buffer.
These were designed such that each had a 4 base pair 5’ overhang, to allow end-labelling
with α-32P dATP. Labelling reactions contained 1x React2 buffer and 2.5 units DNA
polymerase I large fragment (Klenow), 4 µM annealed oligonucleotide, 2.0 µl α-32P dATP
(Perkin-Elmer), and 0.2 mM each of dGTP, dTTP and dCTP. The reactions were incubated
2 5x concentrated annealing buffer: 250 mM NaCl, 5 mM EDTA, 15 mM Tris, 35 mM Tris-HCl
17
for 30 minutes at room temperature, and labelled oligonucleotides were subsequently
separated from unincorporated nucleotides on a non-denaturing 20% acrylamide gel. Bands
were visualized on Kodak X-ray film (2 minute exposure), and then excised and eluted
overnight in buffer containing 1 mM EDTA, 0.6 M NH4OAc, and 0.1% SDS. The buffer now
containing labelled DNA was decanted, and precipitated for 1 hour at -80°C with 15 µg
glycogen and 70% ethanol. DNA was harvested by centrifugation (13,300 rpm, Fischer
Scientific tabletop centrifuge), the supernatant was removed, and the pellets were dried
(Savant Speed Vac Concentrator) and then resuspended in distilled DNase-free water
(Gibco). The concentration of each DNA preparation (in cpm/µl) was determined by
scintillation (LKB Wallac).
Standard nitrocellulose filter binding assay
The method developed by Romaniuk (1985) was followed. Briefly, a 12-point protein
dilution series (720 nM, 360 nM, 180 nM, 72 nM, 36 nM, 18 nM, 7.2 nM, 3.6 nM, 1.8 nM,
0.72 nM, 0.36 nM, and 0 protein) was prepared using TMK buffer as the diluent (pH 7.5, 100
mM KCl 0.5 mM TCEP, 0.1 mM BSA, 5 mM MgCl2, 0.01 mM ZnCl2 final concentrations).
Radiolabelled DNA and poly(d[IC]) were added to each well, to a final concentration of 50
cpm/µl and 1 µg/ml, respectively. Mixtures were incubated for 90 minutes at 20°C, and then
80% of the total volume was filtered on nitrocellulose followed by nylon filter paper, using a
Dot Blot apparatus. Dried filters were exposed to a phosphor screen (Molecular Dynamics)
overnight, and the signal was detected using a Storm 820 phosphor imager (Molecular
Dynamics). The fraction of DNA bound at each protein concentration was determined with
Microsoft Excel, using the following formula:
[DNA-protein] = bound DNAx bound DNA0
[DNA]total total DNAx total DNA0
Where total DNA = bound DNA + free DNA and subscript x indicates values for a
given protein concentration, while subscript 0 indicates values for the zero protein wells.
The fraction of DNA bound was plotted as a function of protein concentration, and a curve
was fitted using Kaleidagraph 2.0 software.
18
DNA excess nitrocellulose filter binding assay
Protein was diluted to 200 or 400 nM in TMK buffer, and aliquoted into 11 of 12
wells in each column of a 96-well round-bottom plate; the 12th well was reserved for TMK
buffer alone (no protein). A cold DNA dilution series (81.6 nM, 42.6 nM, 21.7 nM, 10 nM,
7.22 nM, 4.26 nM, 2.17 nM, 1.00 nM, 0.722 nM, 0.426 nM, 0.217 nM, 0.217 nM) was
prepared separately, and then the equivalent radiolabelled DNA oligonucleotide and
poly(d[IC]) were added to each well, to a final concentration of 7,500 cpm/µl and 10 µg/ml,
respectively. Subsequently, DNA from each well of the DNA series was added to the
corresponding well in the diluted protein plate, diluting the DNA 1/10. The mixture was
incubated, filtered, and filters were exposed to a phosphor screen as in the standard filter
binding assay. Data were analyzed using the appropriate formulae, with Microsoft Excel and
Kaleidagraph 2.0 software.
Cloning of KSE and mutant oligonucleotides
Oligonucleotides to be sequenced were ligated into pK19 that had been digested for
90 minutes at 37°C with EcoRI and BamHI (NEB) in appropriate buffers. Ligation reactions
were prepared as described previously, and were incubated overnight at 37°C. Plasmids were
purified as described as above, and were sequenced without identification by colony PCR.
19
Results
The pTOPO vector did not contain the KS1 gene
Attempts to amplify two KS1 gene constructs from a pTOPO vector, using a standard
PCR thermocycler program, failed to yield product. In an effort to increase primer annealing
efficiency, the annealing temperature was raised to 62°C (approximately 5°C below the
primer melting temperature), or a touchdown PCR program was employed; however, these
conditions also failed to yield product. As a positive control, PCR reactions were performed
using plasmids with known inserts and universal plasmid primers; these reactions
successfully yielded product, thus eliminating the possibility that human error was at fault.
This unexpected problem with the initial step in cloning required further investigation of the
pTOPO vector and the gene within it, including diagnostic restriction digests, PCR using
universal primers, and finally, sequencing of the plasmid.
A series of diagnostic PCR reactions were performed with universal primers. M13
primers complimentary to pTOPO were intended to detect whether the plasmid was indeed
the pTOPO vector, and to assess the size of the insert. KS1-specific primers were employed
in combination with universal primers, in order to diagnose potential problems with either the
upstream or the downstream KS1 primers. All reactions were performed in quadruplicate,
using independent preparations of the putative pTOPO-KS1 plasmid as the template; all
replicates behaved identically. PCR using the universal primers produced a strong band
between 2036 and 3054 base pairs. By contrast, the KS1-zf1 upstream primer combined with
the universal reverse primer failed to yield a product. Both the KS1-zf2 upstream primer
combined with the universal reverse primer, and the universal forward primer combined with
the KS1 downstream primer yielded weak bands that were likely artefacts (Figure 5).
Diagnostic restriction digests were designed to target restriction sites within the
pTOPO vector, within the KS1 gene, or within both vector and gene. NotI and NdeI digests
were intended to linearize the plasmid by cutting at a unique restriction site within pTOPO or
within the KS1 gene, respectively. These digests would indicate the size of the plasmid and
insert combined, as well as offering preliminary evidence that the plasmid and insert were
correct, if two unique restriction sites were indeed present. All other digests were intended to
20
cut at multiple sites within both the plasmid and the insert, yielding a specific banding pattern
that could be predicted based on the vector map and the sequence of the KS1 gene (Table 4).
Digests were performed in duplicate or quadruplicate, using independent preparations
of the putative pTOPO-KS1 plasmid. All replicates behaved identically, in that all reactions
failed to yield the expected size and/or number of restriction fragments (Figure 6 and Table
4). Notably, the linearized plasmid appeared to be approximately 6100 base pairs, although
pTOPO-KS1 was supposed to be 6700 base pairs in length. Additionally, EcoRI, NcoI, and
NotI digests were repeated with an excess of restriction enzyme, in order to ensure that the
reaction ran to completion. This, too, yielded bands of unexpected sizes.
The large number of unexpected results obtained in these analyses prompted me to
have all four pTOPO plasmid preparations sequenced (Centre for Biomedical Research,
University of Victoria). The insert within the vector was identified by BLASTn analysis
(NCBI) as the TBC1D13 gene, a human protein with Rab GTPase activator activity (NCBI).
21
Figure 5: Diagnostic PCR reactions with the putative pTOPO-KS1 plasmid. Universal
primers and/or KS1-specific primers were employed, and samples were analyzed on a 0.8%
agarose gel stained with ethidium bromide and imaged under UV light.
22
Figure 6: Diagnostic restriction digests of the putative pTOPO-KS1 plasmid. Digests were
incubated for 90 minutes at 37°C and samples were visualized on 0.8% agarose gels stained
with ethidium bromide and UV light. Reactions were run in quadruplicate or duplicate on
independent plasmid preparations; however, identical results of the quadruplicate trials
(reactions 1-6) are not shown. Table 4 summarizes the products obtained.
23
Table 4: Expected and actual sizes of restriction fragments in the diagnostic digest. Predicted
restriction sites and band sizes are based on the pTOPO vector map and the KS1 gene
sequence (Thermo Scientific).
Restriction
enzyme
Expected site of
restrictions
Expected
fragments
(bp)
Actual fragments
(bp, approx. values)
NotI vector 6700 6100
NdeI insert 6700 6090
ScaI vector and insert 3672, 3026 6100
AflIII vector and insert 5845, 686, 155 6090, 1000
PstI vector and insert 4545, 1739, 285,
113
6090, 1000
AvaII vector and insert 3575, 2294, 608,
219
2000, 1000
Normal Excess
NcoI
insert
3196, 3134, 356 6100, (6000),
(4000), (2100)
6100,
(6000)
EcoRI
insert 3934, 1026, 626,
416, 288, 272,
108
6100, 4000,
2100
4000,
2100
24
KS1 zinc fingers can be cloned and purified from human genomic DNA
The KS1 zinc finger domain is encoded by a single exon on chromosome 19 (Luo et
al., 2002), and so it was possible to amplify the desired gene constructs by PCR using human
genomic DNA as a template (Figure 7). The construct termed “KS1-zf1” was 1161 base pairs
in length, and consisted of zinc fingers 1 through 10, including the spacer region separating
zinc fingers 1 and 2. The construct termed “KS1-zf2” was 909 base pairs long, and consisted
of zinc fingers 2 through 10 and the linker preceding finger 2, but did not include the spacer
region. Following gel extraction, the KS1-zf1 product was not concentrated enough to be
successfully ligated into pET30a. This problem can be visualized, albeit qualitatively, in
Figure 7a: the band for KS1-zf1 is weaker than the band for KS1-zf2, which was successfully
ligated into the pET30a cloning vector. The PCR amplification of KS1-zf1 was repeated,
yielding the product that was subsequently ligated into pET30a (Figure 7b).
Plasmids containing the KS1 gene constructs were identified by colony PCR (Figure
8), and then confirmed by a diagnostic PCR protocol which utilized KS1-specific primers
and universal primers complementary to the pET30a vector (see example in Figure 9).
Positive clones were subsequently sequenced (CBR). However, it was necessary to make two
separate plasmid preparations for the KS1-zf1 construct, as the first (denoted pET30a-KS1-
zf1 #4) was contaminated and could not be sequenced. The second plasmid preparation was
obtained by transforming E. coli DH5α with pET30a-KS1-zf1 #4 and re-purifying the
plasmid. This plasmid preparation was called pET30a-KS1-zf1 #18(4) to reflect that its
sequence was the same as KS1-zf1 plasmid construct #4, barring the occurrence of any
mutations during the re-transformation process. Sequencing and BLASTp (NCBI) analysis
revealed that the KS1-zf1 construct contained three amino acid mismatches: an glutamine to
glutamate mutation near zinc finger 1, a lysine to isoleucine mutation in the linker between
fingers 4 and 5, and an glutamine to arginine mutation between the two cysteine residues of
zinc finger 9 (Figure 10). The KS1-zf2 construct contained a single amino acid mutation: the
first amino acid at the peptide’s N-terminus was mutated from lysine to glutamate (data not
shown).
Before it became apparent that the KS1-zf1 plasmid preparation #4 could not be
sequenced, it was used to express and purify protein. Similarly, KS1-zf1 protein was
expressed and purified from preparation #18(4) before the sequencing results revealed
25
mutations. The protein products of both the KS1-zf1 and KS1-zf2 gene constructs appeared
on a 15% SDS-PAGE gel at an apparent molecular weight between 30 and 46 kDa (Figure
11). These molecular weights are in agreement with predictions based on sequence, including
the 6-His tag: the molecular weight of KS1-zf1 is 44.5 kDa, and that of KS1-zf2 is 34.6 kDa.
Protein expression was unusually slow, requiring up to 22 hours to reach an optical density of
1.6.
26
(a) (b)
Figure 7: PCR of KS1-zf1 and KS1-zf2 constructs from human genomic DNA. Primers
specific to the zinc finger region of the KS1 gene were used to amplify the two gene
constructs by PCR. Samples were visualized with ethidium bromide under UV light, on 0.8%
agarose gels. KS1-zf1 consists of zinc fingers 1 to 10, and KS1-zf2 is zinc fingers 2 to 10.
Note that a second KS1-zf1 reaction, (b), was performed because the first, (a), was not
concentrated enough for subsequent cloning steps.
(a) (b)
Figure 8: Colony PCR identification of clones positive for KS1 gene constructs. (a)
Identification of KS1-zf1 clone. (b) Identification of KS1-zf2 clone. Universal primers
(pET16 forward and reverse) were employed with a standard PCR program, and samples
bands were visualized on 0.8% agarose gels stained with ethidium bromide. Asterisks
indicate positive clones.
* *
27
Figure 9: Confirmation of colony PCR results using specific and
universal primers. This example shows the KS1-zf1 #4 plasmid.
The diagnostic PCR protocol was performed, with both specific
and universal primers. Samples were run on a 0.8% gel, and
visualized with ethidium bromide and imaged under UV light.
Query 46 EPFDHNECEKSFLMKGMLFTHTRAHRGERTFEYNKDGIAFIEKSSLSVHPSNLMEKKPSA 105 +PFDHNECEKSFLMKGMLFTHTRAHRGERTFEYNKDGIAFIEKSSLSVHPSNLMEKKPSA Sbjct 209 QPFDHNECEKSFLMKGMLFTHTRAHRGERTFEYNKDGIAFIEKSSLSVHPSNLMEKKPSA 268 Query 106 YNKYGKFLCRKPVFIMPQRPQTEEKPFHCPYCGNNFRRKSYLIEHQRIHTGEKPYVCNQC 165 YNKYGKFLCRKPVFIMPQRPQTEEKPFHCPYCGNNFRRKSYLIEHQRIHTGEKPYVCNQC Sbjct 269 YNKYGKFLCRKPVFIMPQRPQTEEKPFHCPYCGNNFRRKSYLIEHQRIHTGEKPYVCNQC 328 Query 166 GKAFRQKTALTLHEKTHIEGKPFICIDCGKSFRQKATLTRHHKTHTGEIAYECPQCGSAF 225 GKAFRQKTALTLHEKTHIEGKPFICIDCGKSFRQKATLTRHHKTHTGExAYECPQCGSAF Sbjct 329 GKAFRQKTALTLHEKTHIEGKPFICIDCGKSFRQKATLTRHHKTHTGEKAYECPQCGSAF 388 Query 226 RKKSYLIDHQRTHTGEKPYQCNECGKAFIQKTTLTVHQRTHTGEKPYICNECGKSFCQKT 285 RKKSYLIDHQRTHTGEKPYQCNECGKAFIQKTTLTVHQRTHTGEKPYICNECGKSFCQKT Sbjct 389 RKKSYLIDHQRTHTGEKPYQCNECGKAFIQKTTLTVHQRTHTGEKPYICNECGKSFCQKT 448 Query 286 TLTLHQRIHTGEKPYICNECGKSFRQKAILTVHHRIHTGEKSNGCPRCGKAFSRKSNLIR 345 TLTLHQRIHTGEKPYICNECGKSFRQKAILTVHHRIHTGEKSNGCP+CGKAFSRKSNLIR Sbjct 449 TLTLHQRIHTGEKPYICNECGKSFRQKAILTVHHRIHTGEKSNGCPQCGKAFSRKSNLIR 508 Query 346 HQKTHTGEKPYECKQCGKFFSCKSNLIVHQKTHKVETMGIQ 386 HQKTHTGEKPYECKQCGKFFSCKSNLIVHQKTHKVETMGIQ Sbjct 509 HQKTHTGEKPYECKQCGKFFSCKSNLIVHQKTHKVETMGIQ 549
Figure 10: BLASTp (NCBI) analysis of KS1-zf1 #18(4). The query sequence is compared to
Homo sapiens ZNF382, also known as KS1. Mismatches are in blue, zinc finger amino acids
are in boldface (zinc ligands underlined), and TGEKP linkers are in italics. “Vestigial”
designates an inactivated zinc finger also found in rat KS1 (Luo et al., 2002; Gebelein et al.,
1998).
zinc finger 2
zinc finger 3 zinc finger 4
zinc finger 5 zinc finger 6 zinc finger 7
zinc finger 8 zinc finger 9
zinc finger 10
zinc finger 1 vestigial
28
Figure 11: Induction and purification of KS1-zf1 and KS1-zf2. Caption on next page.
29
Figure 11 caption: Induction gels are shown at left, in which E. coli BL21(DE3) cells were
induced with 1 mM IPTG at T=0 hours, and samples were taken at subsequent time points
until the cells were harvested. Samples from affinity column purification of each protein are
shown at right, with the following abbreviations: SF, soluble fraction of cell lysate; SN,
supernatant (insoluble fraction of cell lysate containing protein of interest); P, pellet; FT,
flow through; W, wash; E1-E4, two elutions with 150 mM imidazole followed by two
elutions with 250 mM imidazole. All samples were visualized on 3.5% stacking, 15%
separating SDS-PAGE gels, stained with Coomassie blue. Arrows indicate the protein of
interest.
DNA binding by KS1 zinc fingers is not sequence-specific under assay conditions
Standard nitrocellulose filter binding assays (FBAs) were performed to determine the
strength of binding of KS1-zf1 and -zf2 to the KS1 consensus DNA binding sequence (KSE),
or to scanning mutants of this sequence (M1-M4; oligonucleotide sequences given in Table
5). These assays involved a protein dilution series from 720 to 0.36 nM and a constant
concentration of radiolabelled oligonucleotide, in a buffer designed to maximize specific
protein-DNA binding and minimize non-specific interactions (Romaniuk, 1985). The
scanning mutants (Table 5) utilized in these assays were designed to test the contribution of
four major features of KSE, identified by Gebelein and Urrutia (2001), to the overall binding
energy: the region upstream of the A box (M1), the A box (M2), the B box (M3), and the
region downstream of the B box (M4). ZFY4R, the consensus DNA binding site of an
unrelated polyzinc finger protein, ZFY (Taylor-Harris et al., 1995), was included as a
negative control. The Kd for the binding of KS1-zf1 or -zf2 to the consensus site were
unexpectedly high: 114 ± 10 nM and 133 ± 13 nM, respectively. In addition, there was very
little difference in the Kd values for binding to mutant sites or to negative control DNA
(Table 6).
At this point, oligonucleotides M1 through M4 were sequenced, in order to rule out
the possibility that the mutants had incorrectly been synthesized by IDT. If all four were
simply replicates of the KSE oligonucleotide sequence, then this would account for the
similarities in binding constants (although it would not explain the binding observed with
ZFY4R and NRSE). However, the sequences of these mutants were found to be correct.
30
This indicated that the protein constructs might instead be at fault. To determine if the
anomalous results of the standard FBAs were caused by low protein activity, the association
constants were also determined by DNA excess FBAs. In these assays, the protein and
radiolabelled DNA oligonucleotide concentrations were held constant, while the equivalent
unlabelled competitor DNA was diluted from 81.6 nM to 0.217 nM. Additional scanning
mutants (MA through MD; Table 5) were tested, which were essentially the inverse of the
original four mutants used in the standard assays. That is, all but one of the four major
regions of the KSE site were mutated in each construct. Another scanning mutant, MX, was
designed to alter every residue within KSE. Finally, a second non-cognate DNA, NRSE
(Mori et al., 1992), was used as a negative control in case ZFY4R shared slight sequence
similarity with KSE. The relative affinities, determined by DNA excess FBA for KS1-zf1
and -zf2 binding to all mutants, are summarized in Table 7. The data were again anomalous,
in that both protein constructs bound with high affinity to negative control DNA, as well as to
the total mutant MX. The apparent Kd values for binding to KSE were approximately 100-
fold lower than the values observed in the standard DNA excess assays: 6.18 ± 3.91 nM for
KS1-zf1 and 3.76 ± 1.30 nM for KS1-zf2.
31
Table 5: Sequences of oligonucleotides employed in standard and DNA excess FBAs. The A
and B box regions are underlined, and scanning mutations are indicated in red. Lower case
letters indicate 5’ overhangs for labelling and/or ligations.
Oligonucleotide sequence
KSE aattCTATCATACTACCCACCCTACAGATGCACAACAG GATAGTATGATGGGTGGGATGTCTACGTGTTGTCctag
M1 aattGGTAGTACTTACCCACCCTACAGATGCACAACAG CCATCATGAATGGGTGGGATGTCTACGTGTTGTCctag
M2 aattCTATCATACCTTTTACCCTACAGATGCACAACAG GATAGTATGGAAAATGGGATGTCTACGTGTTGTCctag
M3 aattCTATCATACTACCCATGTCTGTAGTGCACAACAG GATAGTATGATGGGTACAGACATCACGTGTTGTCctag
M4 aattCTATCATACTACCCACCCTACAGACCGTTTTGGC GATAGTATGATGGGTGGGATGTCTGGCAAAACCGctag
MX aattGGTAGTACTCTTTTGTGTCTGTAGCCGTTTTGGC CCATCATGAGAAAACACAGACATCGGCAAAACCGctag
MA aattCTATCATACCTTTTGTGTCTGTAGCCGTTTTGGC GATAGTATGGAAAACACAGACATCGGCAAAACCGctag
MB aattCCTAGTACTTACCCATGTCTGTAGCCGTTTTGGC CCATCATGAATGGGTACAGACATCGGCAAAACCGctag
MC aattGGTAGTACTCTTTTGCCCTACAGACCGTTTTGGC CCATCATGAGAAAACGGGATGTCTGGCAAAACCGctag
MD aattGGTAGTACTCTTTTGTGTCTGTAGTGCACAACAG CCATCATGAGAAAACACAGACATCACGTGTTGTCctag
ZFY4R gatcCTGTCGATGGAGGCCCGAGTAGGCCTAAAATTGAGGCG GACAGCTACCTCCGGGCTCATCCGGATTTTAACTCCGCttaa
NRSE aattCGGTTCAGCACCCTGGACAGCTCCCGG GCCAAGTCGTGGGACCTGTCGAGGGCCttaa
Table 6: Kd values and relative affinities as determined by standard FBAs. KS1-zf1 or -zf2
binding to the consensus DNA binding sequence (KSE) and four scanning mutants. ZFY4R
was included as a negative control. Oligonucleotide sequences are given in Table 5.
KS1-zf1 KS1-zf2
Kd (nM)a Relative affinityb Kd (nM)a Relative affinityb
KSE 114 ± 10 1.00 133 ± 13 1.00
M1 75 ± 6 1.51 147 ± 14 0.91
M2 55 ± 5 2.05 175 ± 18 0.76
M3 54 ± 5 2.12 215 ± 31 0.62
M4 42 ± 4 2.68 217 ± 26 0.61
ZFY4R 156 ± 18 0.730 not determined not determined a Error values represent the error within a single trial. b Relative affinities were arrived at by dividing the apparent association constant (Ka = 1/Kd) for mutant oligonucleotides by the apparent Ka for the wild type DNA determined in parallel.
32
Table 7: Relative affinities as determined by DNA excess FBAs. KS1-zf1 or -zf2 binding to
the consensus DNA binding sequence (KSE) and scanning mutants. ZFY4R and NRSE were
included as negative controls. Oligonucleotide sequences are given in Table 5.
Relative Affinitya
KS1-zf1 KS1-zf2
KSE 1.00b 1.00b
M1 0.58 ± 0.81 1.34 ± 2.25
M2 0.40 ± 0.64 4.21 ± 1.55
M3 0.95 ± 2.29 4.80 ± 7.20
M4 0.38 ± 2.40 7.62 ± 5.56
MX 2.38 ± 3.23 0.56 ± 0.53
MA 2.83 ± 4.11 5.26 ± 3.15
MB 1.21 ± 2.23 0.16 ± 1.53
MC 1.09 ± 3.35 2.85 ± 1.30
MD 2.26 ± 3.14 2.69 ± 1.00
ZFY4R 3.20 ± 4.11 1.64 ± 1.03
NRSE 1.84 ± 2.85 2.10 ± 0.84 a Relative affinities were arrived at by dividing the mean apparent Ka for mutant oligonucleotides by the mean apparent Ka for the wild type DNA. Error values represent the standard error for at least two independent trials. b The apparent Kd values for the binding of KS1-zf1 and KS1-zf2 to KSE were 6.18 ± 3.91 nM and 3.76 ± 1.30 nM, respectively.
33
Effect of monovalent salt concentration
The contribution of non-specific contacts with the DNA backbone to the observed
binding interactions was tested by increasing the concentration of KCl from 100 mM to 250
mM, and monitoring the effect on the binding of KS1-zf1 and -zf2 to KSE or MX in a DNA
excess FBA. As observed in other trials, both proteins bound to KSE and MX in 100 mM
KCl; however, binding to both DNA oligonucleotides was abolished in the presence of 250
mM KCl.
Effect of competitor DNA or RNA
The sequence specificity, as well as the specificity of KS1 for DNA or RNA, was
probed by monitoring the effect of using carrier RNA, polyA, in place of the usual carrier
DNA, poly(d[IC]). The apparent Kd was measured by standard FBA for the binding of
KS1-zf1 and -zf2 to KSE, MX, or ZFY4R oligonucleotides, in the presence or absence of
each carrier. The Ka values determined in these trials are depicted graphically in Figure 12. In
the absence of carrier, or in the presence of 1 µg/ml poly(d[IC]), the two proteins bound
indiscriminately to all three labelled DNA oligonucleotides. The apparent Ka in the absence
of carrier was approximately 1000-fold higher than in the presence of poly(d[IC]) in all
cases. However, KS1-zf1 appeared to bind specifically to KSE when polyA was used as the
carrier: the apparent Kd for binding to KSE was 7.14 ± 0.70 nM, while the Kd’s for binding to
MX or ZFY4R were in the micromolar range (1.59 ± 1.31 nM and 3.14 ± 4.62 µM,
respectively).
34
(a)
(b)
Figure 12: Effect of competitor DNA or RNA on apparent Ka, as determined by standard
FBA. (a) KS1-zf1 (b) KS1-zf2. No competitor, or 1 µg/ml of either poly(d[IC]) competitor
DNA or polyA competitor RNA were present in each trial. Error bars represent the standard
deviation for two independent trials; where error bars are not shown, useable data could be
obtained in only one trial.
35
Kd values for specific binding determined by FBAs with polyA carrier
Since polyA did not appear to interfere with specific DNA binding by KS1-zf1, I used
it as the carrier in all subsequent FBAs. FBAs were conducted in triplicate, to determine the
apparent Kd values for the binding of KS1-zf1 to mutants M1-M4 (Figure 13). It was
necessary to suppress two outliers in each curve, corresponding to 720 and 360 nM protein,
in order to minimize the margin of error; these points were considerably higher than the fitted
curve. The consensus KSE site was included in each replicate, and the relative affinity of
each mutant was determined as the ratio of the mutant Ka to the consensus Ka (Table 8). In
this way, the fraction of properly refolded, active protein obtained in each assay (a variable)
was removed from the equation. The total mutant MX was also included as a negative control
against non-specific binding. These assays again indicated that KS1-zf1 binds specifically to
KSE, with a Kd of 7.10 ± 1.28 nM. Scanning mutants of the A or B sites affected binding
most profoundly: the A box mutant (M2) displayed an approximate 10-fold reduction in
affinity, while the B box mutant (M3) displayed an approximate 50-fold reduction in affinity
(Table 8). Mutations in the regions upstream and downstream of these sites (M1 and M4)
also displayed decreased affinity for KS1-zf1, but to a lesser extent. The Kd values obtained
for M1 and M4 contained a relatively large margin of error, as the FBA data displayed a
biphasic curve, to which Kaleidagraph software was unable to accurately fit the dissociation
curve.
36
Figure 13: Standard FBAs with 1 µg/ml polyA for KS1-zf1 binding to scanning mutants
M1-M4. Mutant MX was included as a negative control. Error bars represent the standard
deviation among three independent trials. Points corresponding to 720 and 360 nM protein
were suppressed in each curve. See Table 5 for oligonucleotide sequences, and Table 8 for
relative affinities.
Table 8: Relative affinities of KSE scanning mutants as determined by standard FBAs with
polyA carrier. Values were determined from determined from Figure 13. Oligonucleotide
sequences are given in Table 5.
Relative affinitya
KSE 1.00b
M1 0.44 ± 0.21c M2 0.12 ± 0.05 M3 0.04 ± 0.02 M4 0.52 ± 0.20 MX 0.00 ± 0.00
a Relative affinities were arrived at by dividing the mean apparent Ka for mutant oligonucleotides by the mean apparent Ka for the wild type DNA. b The apparent Kd value for the binding of KS1-zf1 to KSE was 7.10 ± 1.28 nM . c Errors were determined by the following formula: ½ error = σmut 2 σwt
2 xmut
xmut xwt xwt
37
Poly(d[IC]) interferes with binding reported in the literature
Standard FBAs were conducted to measure the binding of KS1-zf1 to
oligonucleotides reported in the literature (Gebelein and Urrutia, 2001), with either 1 µg/ml
poly(d[IC]) or 1 µg/ml polyA carrier. Oligonucleotide sequences are outlined in Table 9.
ROB1 was an individual oligonucleotide sequence selected by KS1, during the random
oligonucleotide binding assay in which the high affinity binding site for KS1 was
determined. A GC box (denoted GC) and mutant GC box (GCm) served as negative controls.
The final oligonucleotide that I was able to test was a KS1 consensus binding element in
which the base pairs flanking the B box on the 3’ side were deleted (KBE del 2). Additional
oligonucleotides had been tested by Gebelein and Urrutia ; however, I was unable to obtain
radiolabelled oligonucleotides with these constructs, as the 5’ overhangs were too short.
When poly(d[IC]) was used as the carrier, binding curves appeared to be shifted to the
right, and dissociation constants were accordingly high. Binding appeared to be non-specific,
as KS1-zf1 bound with approximately equal affinity to all oligonucleotides, including those
which were not bound in the literature, as well as the MX negative control (Figure 13). When
polyA was used as the carrier, on the other hand, KS1-zf1 bound specifically to KSE and
ROB1 only, which was in agreement with the literature. The dissociation constant for the
interaction with ROB1 was 5.30 ± 1.72 nM.
38
Table 9: Sequences of oligonucleotides used by Gebelein and Urrutia (2001). Differences
between the GC box and mutant GC box (GCm) are indicated in red.
Oligonucleotide sequence
ROB 1 TCCGTCTTGCTATCATACTACCCACCCTACAGATGCACAACAGTAGGAATT AGGCAGAACGATAGTATGATGGGTGGGATGTCTACGTGTTGTCATCCTTAA
GC ATTCGATCGGGGCGGGGCGAGC TAAGCTAGCCCCGCCCCGCTCG
GCm TTCGATCGGTTCGGGGCGAGC AAGCTAGCCAAGCCCCGCTCG
KBE TCCTACAGTACCAACCCTACAGAGTAA AGGATGTCATGGTTGGGATGTCTCATT
KBE del 1 AGTACCAACCCTACAGAGTAA TCATGGTTGGGATGTCTCATT
KBE del 2 TCCTACAGTACCAACCCTACAG AGGATGTCATGGTTGGGATGTC
Figure 14: Comparison of standard FBAs with 1 µg/ml poly(d[IC]) or polyA for KS1-zf1
binding to literature mutants. Mutant MX was included as a negative control. See Tables 5
and 9 for oligonucleotide sequences, Table 10 for relative affinities.
Poly(d[IC]) PolyA
39
Table 10: Dissociation constants of literature oligonucleotides as determined by standard
FBA with polyA carrier. Refer to Table 9 for oligonucleotide sequences.
Kd (nM) Binding reported in literaturea
ROB 1 5.30 ± 1.72 Yes
GC 2090 ± 1770 No
GCm 1260 ± 811 No
KBE del 2 657 ± 316 No a Gebelein and Urrutia (2001).
40
Discussion
KS1 protein constructs
Initial efforts to clone and express the zinc finger region of KS1 from the pTOPO
vector were fruitless, due to an error on the part of Thermo Scientific, who supplied the
wrong gene. Nevertheless, this was a helpful exercise in plasmid characterization, as well as
a reminder that one should maintain a healthy level of scepticism toward any biological or
chemical reagent that has not been prepared personally. In addition, one of the protocols that
I used to characterize the insert proved to be of use in the verification of later plasmid
preparations, as I will describe in due course.
When PCR reactions involving primers specific to KS1 failed to yield product, I first
investigated a potential problem with primer annealing. The annealing temperature of the
PCR thermocycler program was raised from 55°C to 62°C, which was 4 to 5 degrees below
the melting temperature of all three primers (IDT). It was hoped that this temperature would
be low enough to promote primer annealing to the complimentary region within the KS1
gene, while being high enough to allow rapid breaking of transient bonds between non-
complimentary regions. A touchdown PCR program was also employed; this program began
at a high annealing temperature and gradually decreased (Table 1), thus increasing the
likelihood that the optimal annealing temperature would be achieved during some of the PCR
cycles. Despite the fact that all PCR programs had failed to amplify the desired product,
positive controls (known plasmids and universal primers) were successfully amplified;
therefore, the reaction conditions, enzymes, buffers, and thermocycler were fully functional.
Diagnostic PCR reactions were performed in order to ascertain whether the problem
lay in the primers and/or the insert, or in the vector. The primers specific to KS1 would not
amplify a product if the insert was incorrect, and conversely, the KS1 gene would not be
amplified if the primers were incorrect; thus, the primers and the insert were essentially
indistinguishable in these trials. The universal M13 primers, which are complimentary to the
pTOPO vector, produced a band of between 2036 and 3054 base pairs (Figure 5). This served
to demonstrate that the plasmid did, in fact, contain an insert. If the plasmid had been an
empty pTOPO vector, the band observed using the M13 primers would have been much
smaller. The full length KS1 gene is 2442 base pairs in length, and the M13 primers anneal
41
40 nucleotides upstream and downstream of the vector’s multiple cloning site; therefore, the
band observed in Figure 5 was in the correct size range.
The functionality of each KS1-specific primer was tested individually, using
combinations of specific and universal primers. This attempt was a long shot, at best, as one
would expect to observe large smears if one functional KS1-specific primer were paired with
a non-functional one. If a DNA polymerase is left to terminate replication on its own, without
the “bracketing” function of a second primer, it does so at random, by eventually falling off
the DNA template strand (Berg et al., Biochemistry 6e). This creates amplicons of differing
lengths, which appear as a smear, instead of a band, within the agarose gel. As expected, the
combination of the KS1-zf1 upstream NcoI primer with the universal downstream primer
yielded no product. Surprisingly, the KS1-zf2 upstream NcoI primer combined with the
universal reverse primer produced two faint bands, and the KS1 XhoI primer combined with
the universal forward primer produced a single weak band (Figure 5). By chance, these bands
were within the correct size range to potentially correspond to the KS1 gene. However, this
left crucial questions unanswered: If both the KS1-zf2 upstream primer and the KS1
downstream primer were functional, why did they fail to yield product when combined? Why
did the KS1-zf2 primer produce two bands, and why were bands for both of the primers in
question so faint? It was much more likely that the bands were simply false positives.
Restriction digest characterization of the putative pTOPO-KS1 plasmid confirmed
that something was amiss, and ultimately indicated a problem with the insert. Firstly, the
linearized plasmid did not appear to run at the correct size in a 0.8% agarose gel: pTOPO-
KS1 was expected to be 6700 base pairs in length, while the plasmid at hand ran at
approximately 6100 base pairs (Figure 6). It is important to note that the plasmid was
linearized with two restriction enzymes: NotI was to cut a unique site within the vector, while
NdeI was to cut a unique site within the insert. The NdeI digest was incomplete in all
replicates, as is demonstrated by the faint band, corresponding to the relaxed circular
plasmid, that ran at approximately 12,000 base pairs; however, this was attributed to an
excess of plasmid in the restriction digest reaction.
Secondly, all of the diagnostic restriction digests which were designed to produce
identifiable banding patterns, by cutting at multiple sites within vector and insert, produced
an unexpected number and/or size of bands. This was particularly informative in the case of
42
EcoRI and NcoI, for which all of the expected restriction sites were within or directly
flanking the insert. These enzymes were expected to produce 7 and 3 restriction fragments,
respectively (Table 4). Instead, EcoRI produced only 3 fragments, and the plasmid appeared
to be largely cut only once by NcoI, with 3 other fragments faintly visible (Figure 6). In both
cases, the sum of the sizes of the bands was greater than the size of the linearized plasmid,
indicating that the restriction digests were not running to completion. Increasing the amount
of enzyme remedied this particular problem, but did not produce the banding patterns
expected for pTOPO-KS1 (Figure 6). Specifically, EcoRI produced 2 bands of approximately
4000 and 2100 base pairs, while NcoI appeared largely to linearize the plasmid, creating one
band equal in size to the product of a NotI digest (approximately 6100 base pairs). A very
faint band of approximately 6000 base pairs was also produced by the NcoI digest, perhaps
by partial digestion at a second, imperfect restriction site within the insert. Overall, the faulty
digestion of the insert region by EcoRI and NcoI strongly suggested that the insert within the
pTOPO plasmid was not the KS1 gene. As I have indicated, this hypothesis was ultimately
confirmed by sequencing the plasmid, to find that the insert was the TBC1D13 gene, which
encodes a Rab GTPase activator protein (NCBI).
Despite the fact that the KS1 zinc finger domain could not be cloned from the pTOPO
plasmid, all was not lost in this experience. It was a simple matter to clone the desired
constructs from human genomic DNA (Figure 7), as the zinc finger domain is encoded in a
single exon at the 19q13.13 locus (Luo et al., 2002). Then, when it came time to sequence the
clones, I was able to make use of the KS1-specific and universal PCR primers to confirm that
the plasmids identified by colony PCR (Figure 8) indeed contained inserts (see example in
Figure 9). This was useful, as I had encountered problems with false positives from colony
PCR in the past.
A subsequent difficulty arose in the purification of the pET30a-KS1-zf1 plasmid: the
plasmid preparation was contaminated, and could not be sequenced. While these issues were
being resolved, by re-purification of the plasmid, research went ahead using protein
expressed from the plasmid which could not be sequenced. When the re-purified plasmid was
obtained, new KS1-zf1 protein was expressed and purified from this plasmid; from this point
onward, all FBAs were carried out using the second protein preparation only. What are the
possible implications for the FBA results obtained using these two protein preparations?
43
Ideally, the two preparations should be identical, but the replication of the original plasmid
within E. coli DH5α could have introduced one or more mutations. The samples of protein
expressed from either plasmid appeared at the same molecular weight on SDS-PAGE gels
(Figure 11), indicating that a frameshift mutation leading to the introduction of a premature
stop codon did not occur.
Sequencing of the re-purified KS1-zf1 plasmid was delayed by complications, this
time due to high plasmid concentration causing false peaks. When the sequence was
ultimately obtained, the protein was found to contain three amino acid mutations (Figure 10);
however, at this point it was too late to clone and sequence a new KS1-zf1 construct, and
obtain purified protein with which to conduct FBAs. The point mutations were not expected
to abolish the DNA binding activity of any of the protein’s zinc fingers, as none of the
mutations affected zinc-binding cysteines and histidines. However, all three mutations may
have destabilized the peptide. A glutamine to glutamate mutation in the first residue of the
KS1-zf1 peptide placed a negatively charged side chain at the positively charged N-terminus
of the protein, which may have destabilized protein folding. A lysine to isoleucine mutation
occurred in the linker between zinc fingers 4 and 5, altering the overall charge of the linker
sequence; however, the lysine side chain is not responsible for stabilizing the linker upon
DNA binding, as the threonine side chain is (Figure 2). Finally, a glutamine to arginine
mutation between the two key cysteines in zinc finger 9 introduced a positive charge, which
could potentially have decreased the ability of the cysteine residues to bind zinc, by
electrostatic repulsion of the positively charged ion. Among all of the KS1 zinc fingers, there
is a strong tendency toward neutral and negatively charged amino acids between the two key
cysteines. In future, KS1-zf1 will be re-cloned to eliminate these mutations.
Filter binding assays
All FBAs, performed with either KS1-zf1 or -zf2, showed a general lack of DNA
sequence specificity under the assay conditions. However, these results were not entirely
believable, as the two peptides bound with surprisingly low affinity to their consensus DNA
binding sequence, KSE: the apparent Kd for KS1-zf1 was 114 ± 10 nM, and that of KS1-zf2
was 133 ± 13 nM (Table 6). Artificial 6-zinc finger peptides have been shown to bind to 18-
base pair DNA sites with femtomolar dissociation constants (Kim and Pabo, 1998). Seeing as
44
KS1 is thought to require 9 of its 10 zinc fingers to bind a 27-base pair minimum site
(Gebelein and Urrutia, 2001), very strong binding to the DNA consensus site was expected.
However, the caveat to the extreme binding capacity of artificial zinc finger proteins is that
groups of three tandem zinc fingers had to be separated by longer, noncanonical linker
peptides of eleven amino acids as opposed to the usual five in the canonical linker (Kim and
Pabo, 1998). Without this added flexibility, the strain of the polyzinc finger protein upon the
DNA, which must unwind slightly even for proteins with 3 zinc fingers (Peisach and Pabo,
2003), is so great that binding is hindered. Since the nine tandem zinc fingers in KS1 are
separated by canonical linkers (Luo et al., 2002), femtomolar Kd values were not expected
for this protein. Nevertheless, the dissociation constants of natural polyzinc finger proteins
are typically within the nanomolar range, and so the values observed in standard FBAs were
thought to be questionable.
It was also rather unsettling that the KS1 peptides displayed little or no specificity for
the consensus KSE compared to the mutants, or even the unrelated DNA oligonucleotide,
ZFY4R. This is particularly apparent for the KS1-zf1 peptide, which bound with slightly
higher affinity to scanning mutants M1 through 4, and with only moderately reduced affinity
to ZFY4R, the intended negative control. KS1-zf2 also showed only moderate reductions in
affinity for the scanning mutants (Table 6). These results disagreed with the very method by
which the consensus sequence had been determined, by Gebelein and Urrutia (2001): a
random library of DNA oligonucleotides was subjected to successive rounds of selection by
KS1 binding and purification, leading to the isolation of a pool of oligonucleotides with high
sequence identity, from which a consensus sequence could be deduced. Non-specific DNA
binding would not have led to the selection of oligonucleotides with sequence identities, and
so it would have been impossible to arrive at a consensus sequence.
At this point, the most likely explanation for both the weak DNA binding and the
lack of sequence specificity was very low protein activity. In the standard FBA, 100%
protein activity is assumed; however, expressing and storing the proteins in denaturing
conditions may reduce protein activity, due to improper refolding. When making
comparisons between DNA mutants, decreased activity is usually not a problem, as relative
affinities are determined as the ratio of mutant DNA binding to wild type DNA binding for
trials done in parallel, using the same refolded protein. In this way, the protein activity is
45
essentially “divided out” of the equation. (It follows from this that the Kd values for KS1-zf1
and -zf2 reported above cannot be reliably compared, since the activities of the two protein
preparations are unknown, and likely different.) Nevertheless, when considering the relative
affinity values of any one protein, the activity of the refolded protein may become
problematic if it is extremely low, to the point that the lower range of the protein dilution
series contains virtually no active protein. Indeed, an unusually low level of binding was
observed in the lower range of the protein dilution series; only the first three protein dilutions
(720 nM, 360 nM, 180 nM) showed significant DNA binding. This seemed to confirm that
protein activity was at fault; however, later evidence that poly(d[IC]) was interfering with
specific binding would disprove this hypothesis.
There is no direct assay for the activity level of a refolded zinc finger protein. In
previous studies, the activity of zinc finger proteins, such as TFIIIA, has been assessed using
the Scatchard analysis. This method consists of a binding assay in which the protein
concentration is held constant, while the concentration of labelled nucleic acid is varied
(Romaniuk, 1985). The slope of the resulting line indicates the apparent Ka, and if this value
is in agreement with the Ka obtained by standard FBAs, then the protein can be assumed to
be 100% active. An important feature of the Scatchard analysis is that the protein
concentration is held constant in each well; it is this feature of the assay which allows protein
activity to be disregarded in Ka calculations.
The DNA excess FBA makes use of the same principle as the Scatchard analysis in
order to eliminate the protein activity variable: the protein concentration as well as the
labelled oligonucleotide concentration are held constant, and instead, unlabelled competitor
DNA with the same sequence as the labelled DNA is used to titrate the protein of interest.
Thus, the Kd in a DNA excess FBA is defined as the DNA concentration at 50% saturation
(Equation 4), instead of being defined in terms of protein concentration as in the standard
FBA (Equation 3). Therefore, I used this assay, which would not be affected by protein
activity, in an attempt to detect specific DNA binding by KS1.
The dissociation constants determined by DNA excess FBAs fell within the expected
nanomolar range: 6.18 ± 3.91 nM and 3.76 ± 1.30 nM Kd values were obtained for KS1-zf1
and -zf2, respectively. This seemed to indicate that low protein activity was at fault for the
weak binding observed in standard FBAs. The DNA excess FBA protocol uses a constant
46
protein concentration of 200 nM, while the standard FBA involves a protein dilution series
from 720 to 0.36 nM. Within the standard FBA protein dilution series, only the first two
points fell above 200 nM; if protein activity was so low that concentrations below this level
contained virtually no active protein, then this would explain why many data points showed
little to no protein-DNA binding. For DNA excess FBAs conducted with KS1-zf2, it was
necessary to increase the protein concentration to 400 nM in order to observe binding. This
requirement for higher overall protein concentration was, at the time, attributed to an even
lower activity of the KS1-zf2 protein preparation than of the KS1-zf1 preparation. Once
again, later evidence of poly(d[IC]) interference disproved these hypotheses about protein
activity, as will be discussed.
The DNA excess FBA protocol did not resolve the issue of sequence specificity that
was observed in standard FBAs. Notably, both KS1-zf1 and -zf2 appeared to bind to the
negative control oligonucleotides, ZFY4R and NRSE, with slightly higher affinity than the
consensus KSE (Table 7). Also, both proteins appeared to bind to the total mutant MX, in
which the entire 27-base pair KSE sequence was mutated. These key results rendered the
results obtained for the remaining mutants inconsequential; that is, the binding of both KS1
peptides was not sequence specific, and so any variability observed between the mutants
could only be attributed to experimental error. Indeed, the margin of error in the data was
relatively high.
The standard and DNA excess FBA protocols were modified, to determine whether
DNA binding by the two KS1 constructs was non-specific in both cases. First, the
concentration of KCl was increased over two-fold in the DNA excess assay, to assess the
contribution to binding of electrostatic contacts between positively charged amino acid side
chains and the DNA backbone. Binding of both peptides to KSE or MX was abolished in the
presence of 250 mM KCl, indicating that the binding constants observed in the normal DNA
excess assay were entirely non-specific electrostatic contacts. Backbone contacts are
common among zinc finger proteins, and serve to enhance the free energy of binding. For
example, the extensively characterized zinc finger protein, Zif268, makes 7 or 8 contacts
with the DNA backbone (Hamilton et al., 1998). However, numerous specific interactions
with the DNA major groove are normally present in conjunction with these backbone
contacts; in the case of Zif268, each finger forms hydrogen bonds with three or four DNA
47
bases (Elrod-Erickson et al., 1996; Isalan et al., 1997). Although the effect of increasing the
KCl concentration suggested that KS1 was incapable of forming specific hydrogen bonding
interactions with DNA, this result could not be taken at face value. As mentioned previously,
a consensus binding site could not have been identified if KS1 did not bind specifically to
DNA under certain conditions; therefore, the conditions of the FBAs had to be disrupting
specific binding interactions. The two most likely candidates were improper protein folding
in the TMK buffer, or interference of poly(d[IC]).
Fortuitously, I next investigated the effect of the poly(d[IC]) carrier DNA, which was
found to be at the root of the problem. In these trials, the binding of KS1-zf1 and -zf2 to
KSE, MX, or ZFY4R was measured by standard FBAs, using either poly(d[IC]), polyA, or
no carrier. Poly(d[IC]) is the non-specific carrier DNA that was used in all standard and
DNA excess FBAs, and it was included in these trials at the standard concentration. PolyA
RNA was used as an alternate carrier, to assess whether single stranded nucleic acid of a
different sequence could interfere with specific DNA binding. The standard FBA protocol
was chosen for this purpose, because all of the DNA oligonucleotides of interest are labelled,
while poly(d[IC]) or polyA is unlabelled. Thus any reduction in apparent Ka could be
attributed solely to poly(d[IC]) or polyA, unlike a DNA excess FBA, in which a fraction of
the oligonucleotides of interest would also be unlabelled.
The results of this trial represented a turning point in my project, as they strongly
suggested that specific DNA binding can be achieved using polyA carrier. In the absence of
carrier, the Ka values for KS1-zf1 or -zf2 binding to all three labelled DNA oligonucleotides
were approximately 1000-fold greater than values obtained in the presence of poly(d[IC])
(Figure 12). These findings would have implied that the KS1 peptides were binding to
poly(d[IC]) DNA, which shares no sequence identity with any human DNA. On its own, this
result would have implied once again that binding was non-specific. However, when polyA
was used as a carrier, KS1-zf1 bound to KSE with an apparent Kd of 7.14 ± 0.70 nM, and
did not show significant binding to non-cognate DNA (micromolar Kd values). KS1-zf2, on
the other hand, did not display specific DNA binding when polyA was used as the carrier;
instead, it bound weakly to all three labelled DNA oligonucleotides. Binding to KSE was
only approximately 5 times stronger than to MX or ZFY4R, and even so, the apparent Kd was
48
in the micromolar range. Thus, KS1-zf1 binds specifically to the KSE consensus sequence in
the presence of polyA carrier, but KS1-zf2 does not.
One possible explanation for the interference of poly(d[IC]) with the specific binding
of KS1-zf1 is that the repetitive inosine-cytosine sequence shares common chemical features
with critical residues of the KSE site. This seems unlikely, since inosine is not typically
found in DNA; however, an IC base pair displays the same set of hydrogen bond donors and
acceptors at the major groove as a GC base pair (Figure 15). It is only at the minor groove
that the IC and CG base pairs differ, as guanine has an additional amino group which inosine
lacks. Zinc finger proteins bind to DNA by wrapping around the major groove, and form
sparse contacts with the minor groove. Usually, zinc fingers only contact the minor groove if
they are acting as a spacer to traverse a region of minor groove separating two high-affinity
major groove binding sites, as is the case for TFIIIA zinc fingers 4 and 6 (Nolte et al., 1998).
Since KS1 is thought to wrap continuously around the major groove, it could imaginably
form the same specific hydrogen bonding interactions with an IC base pair of poly(d[IC])
carrier DNA as it would with a GC base pair of KSE, thereby essentially acting as a
competitive inhibitor of specific DNA binding. If these poly(d[IC])-KS1 complexes could
form electrostatic interactions with the DNA backbone, or if the refolded KS1 protein
preparation contained any misfolded molecules capable of forming electrostatic interactions
with DNA, then the non-specific binding of these species to labelled oligonucleotide would
have been detected in FBAs.
However, this model begs the question: when a GC box oligonucleotide was later
used as a negative control, why did this not show specific binding (Figure 14, Table 10)? A
notable feature of the GC box is that it is only 22 base pairs long; if the minimum binding site
of KS1 is 27 base pairs in length, as determined by Gebelein and Urrutia (2001), then the GC
box may have been too short to bind to KS1. Indeed, a truncated consensus site (KBE del 2),
which was also only 22 base pairs long, also failed to bind KS1 (Figure 14, Table 10).
49
Figure 15: Comparison of IC and GC base pairs. Both present identical groups at the major
groove. D denotes a hydrogen bond donor, and A denotes a hydrogen bond acceptor.
An alternate explanation is that KS1 bound non-specifically to the poly(d[IC])
backbone, but the concentration of poly(d[IC]) was too great. Although poly(d[IC])
concentrations as high as 5 µg/ml have been used in FBAs (Romaniuk, 1990; Hamilton et al.,
1998), it is possible that KS1 is more sensitive to the presence of non-specific carrier DNA
than are other zinc finger proteins. In this model, KS1 forms strong bonds with the DNA
backbone of any 27-base pair site. If a poly(d[IC]) fragment is taken to be approximately
1000 base pairs long, then each fragment contains 37 potential KS1 binding sites. Thus, KS1
is easily saturated by poly(d[IC]), and it is unable to bind to labelled, specific
oligonucleotides except at high protein concentration. The results of FBAs conducted without
carrier support this model: both the KS1-zf1 and -zf2 peptides bound to the non-cognate
DNA molecules, MX and ZFY4R, with Kd’s in the nanomolar range (Figure 12). From this,
it follows that the peptides may too have bound to each 27 base pair site along the
poly(d[IC]) backbone with a Kd in the nanomolar range. This problem could potentially be
resolved by decreasing the poly(d[IC]) concentration. For the sake of thoroughness, the effect
of reducing the concentration of polyA should also be studied, to ensure that the observed Kd
values represent as accurately as possible the affinity of KS1 for the DNA oligonucleotide in
question.
Unlike poly(d[IC]), 1 µg/ml polyA did not interfere with the specific binding of KS1-
zf1 to KSE, while still preventing non-specific interactions with MX and ZFY4R (Figure 12).
As a single stranded RNA species, polyA neither presents a pattern of hydrogen bonding
Major groove side
Minor groove side
D A A D A A
50
groups in the context of a major groove, nor does it possess a double-helical backbone. DNA-
binding zinc fingers form bonds with both strands of the DNA double helix (Isalan et al.,
1997), and so binding to a single stranded nucleic acid molecule would be much less efficient
than to double stranded DNA. Although some zinc finger proteins, such as TFIIIA, are
capable of binding to both DNA and RNA, they do so using different subsets of zinc fingers
(Romaniuk, 1985; 1990). Thus, a zinc finger that binds specifically to DNA will not show
significant RNA binding, and vice versa. Since KS1 is a DNA-binding zinc finger protein, it
would not be expected to form strong interactions with RNA; therefore, the polyA RNA
carrier acted as a suitably weak competitor to disrupt non-specific binding, while leaving
specific protein-DNA interactions intact. Further research is needed to confirm that KS1 is
not capable of binding specific RNA sequences; however, for the purposes of this study, it is
sufficient to state that KS1 does not bind specifically polyA RNA.
An important conclusion came out of the success of FBAs with the polyA carrier: my
prior hypothesis that the activity of the refolded KS1-zf1 protein preparation was extremely
low was disproved. The same cannot be said for the KS1-zf2 preparation, as this protein did
not show specific binding to KSE when polyA was used as carrier. However, this may
instead be evidence that zinc finger 1 and/or the 56-amino acid spacer region of the KS1 zinc
finger domain is required for specific DNA binding. Although a study by Gebelein and
Urrutia (2001) indicated that these regions do not participate directly in DNA binding, they
may be required to stabilize protein folding. Krüppel-type zinc fingers undergo a
conformational change from a flexible state to a rigid state upon DNA binding, due to C-
capping and N-capping interactions that form between zinc finger α-helices and surrounding
residues (Laity et al., 2000). It is conceivable that certain amino acids in the spacer region
between zinc fingers 1 and 2 could adopt a similar role in stabilizing zinc finger 2. However,
further research is required to determine the minimal KS1 peptide that is capable of specific
DNA binding. At this time, it can only be concluded that zinc fingers 2 to 10 do not appear to
be sufficient for specific DNA binding.
This conclusion disagrees with the work of Gebelein and Urrutia (2001), which
demonstrated that KS1 requires only 9 of its 10 zinc fingers to bind DNA. However, in that
particular study, the DNA binding activity of KS1 was measured based on repression of a
reporter gene. The transcriptional repressor function of KS1 also relied upon the presence of
51
an N-terminal KRAB domain; in the construct containing only 9 zinc fingers, this domain
was fused to the N-terminal end of zinc finger 2. In this way, the KRAB domain may also
have acted to stabilize zinc finger 2. In the current study, the KS1-zf2 construct began with
four amino acids upstream of zinc finger 2; the first residue was mutated from glutamine to
aspartate (data not shown), and this negative charge at the protein’s N-terminus may have
destabilized the folding of zinc finger 2. This implies that zinc finger 2 plays a critical role in
specific DNA binding, if disrupting its structure is enough to abolish the function of the
entire zinc finger domain.
Having determined the assay conditions which could be used to detect specific DNA
binding by KS1-zf1, I conducted standard FBAs involving scanning mutants M1 through M4
again, this time using the polyA carrier. The assay conditions were still not ideal, as it was
necessary to suppress the data points for the two highest protein concentrations (720 and 360
nM) when fitting the equilibrium binding curves. In order for an equilibrium binding curve to
be useful in determining a Kd value, the curve must plateau at the point when saturation is
reached. Such a plateau was indeed reached in all of my equilibrium binding curves,
however, the fractions of DNA bound by 720 and 360 nM protein were considerably higher
than the plateau. In the case of mutants M1 and M4, an apparent plateau was reached at even
lower protein concentrations, and so the curves appeared biphasic even when the final two
data points were suppressed. This may be explained by an increase in retention efficiency on
the nitrocellulose filter at high concentrations of DNA-protein complex. It is generally
assumed that the retention efficiency does not change over the range of protein
concentrations used in the standard FBA; however, this assumption must be tested, and
modifications to the protocol must be made accordingly, in the case of the KS1 protein.
Despite these problems, the results of FBAs with polyA carrier were still informative,
revealing that the strongest contacts are formed at the highly conserved regions of the KSE
consensus binding site. Mutation of the A box (mutant M2) reduced the binding affinity by
approximately 10-fold, while mutation of the B box (mutant M3) reduced the affinity by
approximately 50-fold (Table 8). Alterations to the regions flanking the A and B boxes also
affected KS1 binding; however, these mutations only reduced the affinity by approximately
2-fold (Table 8). Therefore, my results indicate that the KSE B box makes the greatest
contribution to the free energy of KS1 binding, and the A box also contributes significantly.
52
These findings agree with the nature of the KBE consensus sequence derived by Gebelein
and Urrutia (2001): the strong consensus among all of the A and B boxes in the selected
oligonucleotides implies that these regions were required for high-affinity binding during the
library enrichment process. The flanking regions did not show an equally strong consensus,
except at the -2, -4, and -8 positions upstream of the A box, as well as at the +5 position
downstream of the B box (highlighted in gray in Figure 3). The importance of these
individual base pairs, as well as the free energy contribution of each base pair within the A
and B boxes must be investigated in the future with KSE point mutants.
While the findings of these FBAs agreed with the general features of the consensus
binding site for KS1, there still remained one major discrepancy between the protein-DNA
binding data reported by Gebelein and Urrutia (2001) and my own: the Gebelein group had
observed what appeared to be specific DNA binding using the poly(d[IC]) carrier, while I
had not. This previous report had used non-quantitative eletrophoretic mobility shift assays
(EMSAs) to investigate protein-DNA binding. In these experiments, protein and
radiolabelled oligonucleotides were incubated in an appropriate buffer, along with
poly(d[IC]) carrier. Reaction mixtures contained 200 ng of protein and 50 µg/ml poly(d[IC]),
whereas even the most concentrated well of the standard FBA protein dilution series
contained approximately 10 ng protein with 1 µg/ml poly(d[IC]); therefore, the specific
binding observed by the Gebelein group was not achieved by using an excess of protein.
Binding was subsequently analyzed on a non-denaturing polyacrylamide gel, where bound
protein would cause a supershift in the position of the labelled oligonucleotide, due to higher
combined molecular weight and due to the globular structure of the protein. Binding
appeared to be specific in these trials, as KS1 bound only to its high affinity binding site, and
not to unrelated oligonucleotides. In addition, visual inspection implied that supershifts were
weaker (although these experiments were not quantitative) when mutations were introduced
into the high affinity binding site, and binding was abrogated by deletions of the regions
flanking the A and B boxes.
I attempted to recreate the results of Gebelein and Urrutia by using identical
oligonucleotides (Table 9) in standard FBAs with either poly(d[IC]) or polyA carrier. If the
poly(d[IC]) carrier interfered with the binding of KS1-zf1 to the same oligonucleotide
sequences that had been bound previously, it would confirm that conducting FBAs with an
53
alternate carrier was an appropriate means of measuring KS1-DNA binding. Similarly, FBAs
conducted with polyA carrier would be reconciled with the literature if KS1 was shown to
bind specifically to the same sequences that had previously been bound in EMSAs. Indeed,
binding was non-specific in the presence of poly(d[IC]) carrier, while KS1-zf1 bound
specifically to one of its high affinity binding sites, ROB1, in the presence of polyA (Figure
14 and Table 10). Deleting five base pairs at the 3’ end of the KS1 consensus binding
element (KBE del 2), which rendered it only 22 base pairs long, abolished protein-DNA
binding, as was reported in the literature. Unfortunately, this result lacks an important
positive control (demonstrating that KS1-zf1 binds to full length KBE in standard FBA
conditions), as some oligonucleotides could not be end labelled due to short 5’ overhangs.
Nevertheless, the following conclusion can be drawn: since mutation of the analogous base
pairs in KSE (mutant M4) caused only a 2-fold reduction in binding affinity, the deletion
mutant must have abolished protein-DNA binding because the oligonucleotide no longer
satisfied the minimum binding site length, and not because the deletion removed a high-
affinity binding site.
PolyA also prevented non-specific interaction with two non-cognate oligonucleotides
which had been used as negative controls by Gebelein and Urrutia: a GC box, and a mutant
GC box, in which one GC-rich tract was interrupted by two mutations to thymine (Table 9).
It is important to note, however, that these negative control oligonucleotides were only 22
base pairs long. Thus, KS1 may have failed to bind to these sequences because they did not
satisfy the minimum binding site length, and not because of sequence specificity. Gebelein
and Urrutia postulated that KS1 requires a 27 base pair minimum binding site; however,
truncated binding sites between 23 and 26 base pairs in length have not been tested to date. It
may be worthwhile to test a series of successive deletion mutants to determine the true
minimum binding site length of KS1.
54
Conclusion
Despite some difficulties and discrepancies, this study agrees with and builds upon
the current model for DNA binding by KS1. KS1 binds specifically to its full length binding
site, and not to a truncated binding site, as was reported previously. Specific binding appears
to require the full length KS1 zinc finger domain, at least to achieve correct protein folding.
Thus, at this point it is still conceivable that the entire zinc finger domain wraps around the
DNA double helix at the major groove, as was proposed previously. This study demonstrates
that protein-DNA binding, whatever the mechanism may be, has a dissociation constant of
7.10 ± 1.28 nM. The strongest interactions occur between KS1 and the B box region of the
consensus binding site; the A box also forms strong interactions with KS1, but these are
approximately 5-fold weaker than the interactions formed at the B box. The A and B boxes
together make up a 14 base pair core binding site; if each KS1 zinc finger motif binds a three
base pair subsite, then this implies that four or five zinc fingers contact this central region of
the binding site. Therefore, KS1 may circumvent the problem of strain associated with many
zinc fingers continuously wrapping around the DNA double helix by forming strong contacts
with only a subset of its zinc fingers, and forming weaker, more flexible contacts to the DNA
double helix with the rest of its zinc fingers.
55
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