li & kelly sv40 dna replication in vitro. pnas (1984) 81:6973
DESCRIPTION
Li & Kelly SV40 DNA replication In vitro. PNAS (1984) 81:6973. What else could they do to validate their system?. Fractionation to homogeneity. ELONGATION FACTORS ARE CONSERVED. RF-C. RF-C. RF-C is a five-subunit complex All subunits are related in sequence and have ATP binding motifs - PowerPoint PPT PresentationTRANSCRIPT
COS cells (infected with SV40)(expresses T Ag)
+ plasmid + dNTP + rNTP+ 32PdATP
37o C, 60 min
Analyze products by electrophoresis and autoradiography
SV40 ori
SV40 - + ori - +
Li & KellySV40 DNA replicationIn vitro.PNAS (1984) 81:6973
What else could they doto validate their system?
Fractionation to homogeneity
COS cells (infected with SV40)(expresses T Ag)
T Antigen frombaculovirus expression system
Human HeLa or 293 cells
cell lysate
PC
0.2 M KCl 0.66 M KCl
QS
0.2 M KCl 0.4 M KCl (PCNA)
ssDNA
0.6 M KCl 1 M KClRPA
+
(RF-C, pol + pol )
Prokaryotic Function Eukaryotic
SSB ss DNA coating RPA
PRIMASE RNA primer synth. Primase subunitof DNA POL ,
5'-3' polymerase DNA POL ,DNA POL
POL III CORE 3'-5' exonuclease DNA POL 5'-3' exonuclease Fen1
COMPLEX ATP dependentclamploader
RF-C
CLAMP processivity factor PCNA
LIGASE seal nicks Lig1
DnaB Helicase ?
ELONGATION FACTORS ARE CONSERVED
Subunit
Gene Bacterial Function Eukaryotic
dnaE | 5'-3'polymerase
DNA POL
dnaQ(mutD)
| POL IIICORE
3'-5'exonuclease
DNA POL
| 5'-3'exonuclease
Fen1
dnaX
dnaX | | ATP' |
COMPLEXdependentclamploader
RF-C
| |
dnaN CLAMP processivityfactor
PCNA
CONSERVATION FROM PROKARYOTES TOEUKARYOTES
RF-C is a five-subunit complexAll subunits are related in sequence and have ATP binding motifsATP hydrolysis by RF-C is associated with the loading of PCNARF-C is the functional homolog of the clamp-loader complex
RF-C
RF-C
Polymerase switching occurs even on lagging strands; pol does most of DNA synthesis
Dna2 endonuclease is also necessary for this step
How do you think the PCNA is removed after ligation?
PCNA interacts with RF-C, pol , Fen1, DNA ligase,CAF1 and MCMT
Several of these have a common motifused in the interaction: Q-X-X-L/I/M-X-X-F-F/Y
p21/CIP1/WAF1, a protein induced by the tumor suppressor p53 uses the same motifto interact with PCNA
What effect is p21 expected to have on DNA replication/repair?
FEN1 interacts with PCNA
Prokaryotic Function Eukaryotic
SSB ss DNA coating RPA
PRIMASE RNA primer synth. Primase subunitof DNA POL ,
5'-3' polymerase DNA POL ,DNA POL
POL III CORE 3'-5' exonuclease DNA POL 5'-3' exonuclease Fen1
COMPLEX ATP dependentclamploader
RF-C
CLAMP processivity factor PCNA
LIGASE seal nicks Lig1
DnaB Helicase ?
ELONGATION FACTORS ARE CONSERVED
DNA Helicase
* Helicase binds ss DNA
* Hydrolyzes ATP to move alongssDNA and peel of substrate DNA
* Can move 5‘ to 3’ or 3‘to 5’ or in both directions on ssDNAand is classified as such
* Dimers or hexamers
P P
5‘ 3‘
3‘ 5‘ 5‘3‘
Substra
te
Boiled su
bstra
te
+he
licas
e
+helic
ase+
ATP
15mer 30mer
15mer30mer
Conserved helicase motifs:
Includes the following for ATPbinding and hydrolysisI = Walker A motif GxGxGKTII = Walker B motif DEad
Binds ATP
Tight conformation
Hydrolyzes ATP
Relaxed conformation
Helicases are ATP driven molecular motors
Mutation in a yeast gene that causes a failure in MiniChromosome Maintenance (MCM genes)
Yeast containing Leu2 on a plasmid
Plate yeast on Leucine minus plates to estimate rate of plasmid loss
Grow under non-selective conditions
MCM8
MCM9
Form hexamer
Associates with MCM2-7
Newbies of unknown function
MCM homolog fromM. thermoautotrophicum(an archaebacterium)has helicase activity.
What direction?
Mt MCM formsdouble hexamers
T antigen helicase domain: XS Chen, Nature, 2003;423:512-8
MtMCM-N terminal (non-helicase) domain: XS Chen, Nature Str. Biol. 2003;10:160-7
Model of replicative double-hexameric helicase action : XS Chen, Nature, 2003;423:512-8
Formation of pre-Replicative complex
Annual Review of Biochemistry2002. Bell and Dutta
G2M
ORC
MCM MCM
CDC6 +Cdt1
Replication Machinery
“Pre-Replicative Complex”
Replication complexes during the cell cycle
ORC
CDC6+Cdt1
Slate G1
early G1
Eukaryotic initiation complex
ORC : A six subunit protein complex which has been implicated as being the eukaryotic DNA replication initiator protein.Subunits are named according to their size, with ORC1 being the largest and ORC6 being the smallest subunit.Yeast ORC specifically binds to replication origins in an ATP dependent manner and has been shown to possess ATPase activity.
CDC6/Cdc18 : An essential factor for the assembly of the pre-replicative complexes that co-operates with Cdt1 to load MCM2-7Proteolyzed in yeasts or exported out of the nucleus in mammalian cells at the G1- S transition.Overexpression of Cdc6 in yeast causes multiple rounds of DNA replication without intervening mitosis, making it a critical regulator of DNA replication.
MCM2-7 : A family of six related proteins (MCM2-MCM7) which seem to function together in a large multi-subunit protein complex.MCM 2-7 is most likely the replicative DNA helicase.
A/T DUEOREAuxiliaryelements
Auxiliaryelements
Origin recognitionproteins
TranscriptionFactors
TranscriptionFactors
Leading str
Leading str
Lagging str
Lagging str
Core origin
Origins are multi-partite
The Structure of ARS1(S. cerevisiae)
B1 ACSB2B3
ABF1
ORC
“Post-RC” protection
“Pre-RC” protection
13
456
4 1
22
ORC uses different strategies for binding DNAin different species
Eukaryotic initiation complex
ORC : A six subunit protein complex which has been implicated as being the eukaryotic DNA replication initiator protein.Subunits are named according to their size, with ORC1 being the largest and ORC6 being the smallest subunit.Yeast ORC specifically binds to replication origins in an ATP dependent manner and has been shown to possess ATPase activity.
CDC6/Cdc18 : An essential factor for the assembly of the pre-replicative complexes that co-operates with Cdt1 to load MCM2-7Proteolyzed in yeasts or exported out of the nucleus in mammalian cells at the G1- S transition.Overexpression of Cdc6 in yeast causes multiple rounds of DNA replication without intervening mitosis, making it a critical regulator of DNA replication.
MCM2-7 : A family of six related proteins (MCM2-MCM7) which seem to function together in a large multi-subunit protein complex.MCM 2-7 is most likely the replicative DNA helicase.
The Xenopus Cdc6 Protein Is Essential for the Initiation of a Single Round of DNA Replication in Cell-Free Extracts Made
from Xenopus egg extracts
Rxn Time:0-30 min30-60 min60-90 min
+ + +
+ + +
+ + +
+ + +
32P labeledDNAreplicationproduct
Replication initiation required ondouble-stranded DNA substrate
Only elongation required onsingle-stranded DNA substrate
Orc is required to load CDC6;CDC6 is required to load MCM
Immunostaining for CDC6 can pick outproliferating cells in Pap smears
Eukaryotic initiation complex
ORC : A six subunit protein complex which has been implicated as being the eukaryotic DNA replication initiator protein.Subunits are named according to their size, with ORC1 being the largest and ORC6 being the smallest subunit.Yeast ORC specifically binds to replication origins in an ATP dependent manner and has been shown to possess ATPase activity.
CDC6/Cdc18 : An essential factor for the assembly of the pre-replicative complexes that co-operates with Cdt1 to load MCM2-7Proteolyzed in yeasts or exported out of the nucleus in mammalian cells at the G1- S transition.Overexpression of Cdc6 in yeast causes multiple rounds of DNA replication without intervening mitosis, making it a critical regulator of DNA replication.
MCM2-7 : A family of six related proteins (MCM2-MCM7) which seem to function together in a large multi-subunit protein complex.MCM 2-7 is most likely the replicative DNA helicase.
Mutation in a yeast gene that causes a failure in MiniChromosome Maintenance (MCM genes)
Yeast containing Leu2 on a plasmid
Plate yeast on Leucine minus plates to estimate rate of plasmid loss
Grow under non-selective conditions
MCM8
MCM9
Form hexamer
Associates with MCM2-7
Newbies of unknown function
Propidium iodide staining
No
. o
f ce
lls
Fluorescence analysis and cell sorting (FACS)
DNA in cells stained with propidium iodide.Intensity of staining in each cell is proportional to amount of DNA in the cell.
Cross-link protein-DNA I.P. with anti MCM antibody
Reverse cross-link Purify DNA in I.P.
PCR to detect if your sequence was I.P.ed
MCM
anti-MCM Ab
Chromatinimmunoprecipitation
(ChIP) tests whether a given DNA sequence is bound by a protein
in vivo
Role of Pre-RC in mammalian cells
ORC :CDC6/Cdc18 : Cdt1:MCM2-7 :
Human ORC
Quintana, 1997, 1998Pinto/Quintana, 1999Thome 2000Dhar, 2000, 2001
ORC4
Wild type cells /- cells
Mammalian cells can survive with 0.1x the normal ORC2 level : WT vs cells
Cell proliferation of ORC2 and cells measured by MTT assay
oriP
EB
NA
1
EBNA1 protein
Replication from an origin in Epstein-Barr virus:a plasmid expressing EBNA1 and containing oriP will
replicate and be maintained as an episome inmammalian cells
Hygromycin-Resistance
gene
Establishment of drug-resistant coloniesafter transfection of EBV-based plasmid
m
m
mm
m
m
DpnI
DpnI
DpnI
DpnI
m
m
m
DpnI
DpnI
X
X
X
DpnI
DpnI
DpnIX
X
X
Bacterial plasmidMethylatedDpnI susceptible
Replicated once inhuman cellsHemi-methylatedDpnI resistant
Replicated twice inhuman cellsUnmethylatedDpnI resistant
DpnI-resistance assays episome replication in mammalian cells
1)EBV-based plasmid replication is decreased in /- cells. 2) Rescued by plasmid expressing ORC2
+ +
Some cancers have an Achilles Heel
Viral episomes that carry viral oncogenesAmplicons of cellular oncogenes, or of drug resistance genes
Normal Cancer