an in vitro assay for frameshift mutations: hotspots for ...complex frameshift mutations make up...

Copyright 0 1988 by the Genetics Society for America

An in Vitro Assay for Frameshift Mutations: Hotspots for Deletions of 1 bp by Klenow-Fragment Polymerase Share a Consensus DNA Sequence

Johan G. de Boer*.$ and Lynn S. Rip1eTF.T'

*Department of Biology, York University, Downsview, Ontario M3J I V6, Canada, fDepartment of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 071 03 and $Laboratory of Genetics, National

Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Manuscript received September 15, 1987

Accepted November 2, 1987

ABSTRACT The fidelity of in vitro DNA synthesis catalyzed by the large fragment of DNA polymerase I was

examined. The templates, specifically designed to detect shifts to the + 1 or to the - 1 reading frame, are composites of M13mp8 and bacteriophage T4 rlIB DNA and were designed to assist in the identification of the types of frameshifts that are the specific consequence of DNA polymerization errors. In vitro polymerization by the Klenow fragment produced only deletions, rather than the mixture of duplications and deletions characteristic of in vivo frameshifts. The most frequent frameshifts were deletions of 1 bp opposite a template purine base. Hotspots for these deletions occurred when the template purine immediately preceded the template sequence TT. The highest mutation frequencies were seen when the TTPu consensus sequence was adjacent to G:C rich sequences in the 3' direction. The nature of the consensus sequence itself distinguishes this 1-bp deletion mechanism from those operating in DNA repeats and attributed to the misalignment of DNA primers during synthesis. Deletions that were larger than 1 or 2 bp isolated after in vitro replication were consistent with the misalignment of the primer. Deletions of 2 bp and complex frameshifts (the replacement of AA by C) were also found. Mechanisms that may account for these mutations are discussed.

T HE specific molecular details of the events that underlie the production of most mutations are

still unknown. The DNA sequence of mutants and the DNA context of the sequence change can be used to probe those details. This can be a particularly powerful tool when the DNA sequence changes appear to be related to the context in a consistent manner and thus implicate a single mechanism. A probe of the details of in vivo mutation may also be provided by studying mutational mechanisms in vitro. We have combined these approaches using the specificity of mutations arising during in vitro DNA replication to evaluate the potential importance of polymerization errors to spontaneous in vivo mutagenesis.

The DNA polymerase of T4 is a major determinant of both the frequency and specificity of spontaneous frameshift mutation in vivo (RIPLEY and SHOEMAKER, 1983; RIPLEY, GLICKMAN and SHOEMAKER 1983). For example, one polymerase allele (tsLZ4I ) decreases the frequencies of some frameshifts while increasing the frequencies of other frameshifts by more than 100-fold. It is thus likely that DNA polymerases play multiple roles in spontaneous frameshift mutagenesis. Some of these roles may be more readily defined by examining in vitro DNA replication in the absence

' To whom communications should he sent at the New Jersey address.

Genetics 118: 181-191 (February, 1988)

of other DNA metabolic events such as recombination and repair.

The variety of DNA sequence changes and their contexts among spontaneous frameshifts in the bacteriophage T4 r1ZB gene implicate quantitatively sig- nificant contributions by multiple frameshift mechanisms to spontaneous frameshift mutagenesis (RIPLEY, CLARK and DE BOER 1986). Classes of frameshift mutations that occur spontaneously in T4 which are particularly relevant to the frameshift mutations described in this report include: deletions between directly repeated DNA sequences, consistent with the base paired misalignments of DNA proposed by STREISINGER et al. (1966); deletions of 1 or of 2 bp that occur in DNA contexts that are inconsistent with the misalignment model of STREISINGER (RIPLEY, CLARK and DE BOER 1986); and complex frameshifts having changes in both the number of bases and the base sequence (DE BOER and RIPLEY 1984; RIPLEY, CLARK and DE BOER 1986).

The DNA contexts of frameshift hotspots have often been correlated to sequences capable of misalignment. Misalignments based on Watson-Crick complementarity between two DNA strands were originally proposed as frameshift intermediates by STREISINGER et al. (1966). In monotonous runs of a base, the model predicts deletions or duplications of the repeated base. Frameshift hotspots are often in

182 J. G. de Boer and L. S. Ripley

such runs (CALOS and MILLER 1980; PRIBNOW et al. 1981; SKOPEK and HUTCHINSON 1984; STREISINGER and OWEN 1985). Frameshift hotspots are also found at other repeated sequences (STEWART and SHERMAN 1974; FARABAUGH et al. 1978; PRIBNOW et al. 1981). It is not known whether misalignments that could mediate such mutations occur during DNA polymerization. Studies of the lad and rIIB hotspot sites suggest that features of the DNA sequence in addition to the repeat (SCHAAPER, DANFORTH and GLICKMAN 1986) or the occurrence of DNA nicks (RIPLEY, CLARK and DE BOER 1986) may influence the in uiuo spontaneous frameshift rates in repeated DNA sequences.

Despite the strong correlation between frameshift hotspots and repeated DNA sequences, approximately half of the spontaneous single base deletions and duplications in the rIIB gene depend upon other mechanisms (RIPLEY, CLARK and DE BOER 1986). Complex frameshift mutations make up about 8% of spontaneous frameshifts in T4, most of these can not yet be explained. We have considered the possibility that complex frameshifts sometimes occur as direct DNA polymerization errors (RIPLEY, CLARK and DE BOER 1986).

Previous in vitro studies of frameshift mechanisms examined the ability of DNA polymerases to maintain the proper register during DNA replication on alter- nating DNA polymers. By means of nearest neighbor analysis, DNA sequences consistent with frameshift mutation have been seen (SHEARMAN and LOEB 1983; SHEARMAN, FOGETTE and LOEB 1983; TOPAL 1984). However, the nature of the sequences and nearest neighbor analysis preclude the ability of these measurements to distinguish deletions from duplications, and from some base substitutions. In other studies of the fidelity of in vitro polymerization carried out by eucaryotic a, P, and y DNA polymerases, frameshifts as well as base substitutions were identified among the mutants leading to decreased P-galactosidase complementing activity in M 13mp2. The frameshifts produced by (Y and P polymerases differ from one another; gamma polymerase was not shown to produce a detectible increase in frameshift error frequency (KC'NKEL 1985 a, b).

This paper describes an assay for frameshifts in 77 bp of wild-type T 4 rIIB DNA sequence that is a portion of the DNA sequence in which we have previously sequenced spontaneous in uiuo frameshifts (RIPLEY, CLARK and DE BOER 1986). T h e DNA templates (P74 and M74) are derivatives of M13mp8 in which a piece of T 4 rIZB sequence has been inserted to produce a frameshift in the P-galactosidase complementing fragment. In P74 the wild type reading frame is restored by a shift to the + 1 reading frame. In M74 the wild type reading frame is restored by a shift to the - 1 reading frame. In both P74 and M74, frameshifts within the rIIB insert or in nearby P-

galactosidase sequences that restore the wild type reading frame restore P-galactosidase complementing activity and can be detected by a blue plaque phenotype in the presence of X-gal.* We report here the sequences of frameshift mutations produced after the DNA polymerization of the P74 and M74 templates by the large fragment of Escherichia coli DNA polymerase I.

MATERIALS AND METHODS

Enzymes and DNA: Restriction enzymes were purchased from IBI and New England Biolabs. E. coli Pol I large fragment was from Boehringer Mannheim. T4 DNA ligase and M13 sequencing primer (15 bp) were from Bethesda Research Labs. M13mp8RF DNA was from New England Biolabs. [(U'~P]~ATP was from Amersham.

Phage and Plasmids: M13mp8 phage were grown in E. coli JM 101; both were from Bethesda Research Labs. Plasmid HH879, a pBR322 derivative bearing a HindIII fragment from the N terminus of the rIIB gene (PRIBNOW et al. 1981), was used as a source of rIIB sequence for creating M74.

Construction of M74: The M74 construction was derived from P74, and the sequences of both in the N terminus of the P-galactosidase gene are shown in Figure 1. Both derivatives contain an insertion of a 74-bp AluI-HincII restriction fragment from the N terminus of rIIB (nucleotide 471-544 reported by PRIBNOW et al. 1981) into the HincII site of M13mp8. Both derivatives have 77 bp of perfect homology to a region of the rIZB gene.

M74 differs from P74 by 2 deletions of 2 bp each. The construction was accomplished by substituting the BamHI- HindIII fragment (26-1 19) from a P74 derivative carrying an AG deletion at 114-1 16 for the BamHI-Hind111 fragment of a P74 derivative deleted for GG at 23-24. Each of the P74 deletion derivatives had a blue plaque phenotype; the successful substitution of the alternative fragment produced a colorless plaque phenotype. The construction was confirmed by DNA sequencing.

Assays for frameshift mutant frequency: All stocks were grown in Y T medium (5 g Bacto yeast extract, 8 g Bacto tryptone, and 5 g NaCl per liter). Plating was carried out on minimal agar plates (10 g K2HP04, 1.5 g Na2HP04 . 2Hz0, 2 g anhydrous citric acid, 0.2 g MgS04 . 7H20, 4 g glucose, 5 kg thiamine * HCl, and 16 g Bacto agar per her) with 2.5 ml soft agar overlays (8 g Bacto agar, 9 g NaCVliter). X-gal from Research Organics and IPTG from Sigma were added to the soft agar for the detection of P- galactosidase activity (25 p.1 of 4% X-gal in dimethylfor- mamide and 10 p.1 of 100 mM IPTG in water were added to each plate). Plates were incubated overnight at 37°C and permitted to sit at room temperature for an additional 2 days to develop full blue color.

Blue plaques were isolated from plates having 2 x IO4 or fewer total plaques. The spontaneous frequency of reversion of M74 and P74 was determined in phage stocks to be less than 1O"j (L. RIPLEY, unpublished data). Trans- fection of M74 and P74 DNA before in vitro DNA replication produced a spontaneous reversion frequency of less than lop6.

Isolation and preparation of DNA templates and primers: Single-stranded DNA was prepared from over-

' The abbreviations used are: X-gal, 5-brorno-4-chloro-3-indolyl p-D- galactoside: bp, base pairs; RF, double-stranded replicative form; IPTG, isopropyl-p-o-thiogalactopyranoside.

In Vitro Replication Frameshifts 183

night cultures of P74 or M74 grown in E. coZi JM101. Phage particles were precipitated in 5% polyethylene glycol 8000 and 0.5 M NaCl. DNA was extracted with phenol/chloro- form, precipitated with ethanol and resuspended in IO mM Tris-HC1 (pH 7.5) in 1 mM EDTA.

M13mp8C4 RF DNA was prepared by the alkaline lysis method of BIRNBOIM and DOLY (1979). The RF DNA was purified in an ethidium bromide-CsC1 gradient. The primer for gap synthesis was prepared by digesting RF with HindIII and EcoRI. The resulting 29-bp fragment was separated from the large primer fragment by agarose gel electrophoresis. The M13mp8C4 DNA differs from M13mp8 by a deletion of a G:C pair at position 6250 (the fourth base of the HincII site of the polylinker region); it has no T4 711 DNA sequences. The one bp deletion is in the small fragment created by EcoRI and HindIII digestion.

In vitro DNA synthesis: DNA synthesis was carried out on a gapped DNA template that was prepared by annealing the large fragment M13mp8C4 RF DNA digested with EcoRI and HindIII to M74 or P74. The gap to be filled extends from bp 125 to bp 15 in Figure 1.

Primer-template annealing was carried out at a template concentration of approximately 60-70 Fg/ml in 7 mM Tris- HCI (pH 7.5), 7 mM MgC12, and 50 mM NaCl. The molar ratio of primer to template was 2. The double stranded DNA fragment used to prime synthesis was heated at 95°C for 3 min before adding template. After annealing, template concentration was adjusted to a concentration of 20 pg/ml in Klenow buffer (50 mM Tris-HC1 (pH 7.6), 10 mM MgC12, 1 mM dithiotheitol, and 400 p,g/ml bovine serum albumin), 250 p , ~ of each dNTP, and the synthesis was started upon the addition of Klenow polymerase to a concentration of approximately 0.2 units/p,l (approximately a 20-fold molar excess over DNA template). Synthesis was permitted to proceed for 40 min at 22" and was stopped by the addition of 0.2 M EDTA to a final concentration of 10 mM. Gel analysis of the product indicated substantially complete filling of the gap.

Assay for in v i t ro mutation: Competent cells were prepared using the CaC12 method (see DE BOER and RIPLEY 1984). One milliliter of CaClz-treated cells and 150 ng template DNA were added to each minimal plate. Blue plaques were isolated after incubation at 37", were purified by restreaking, and DNA was isolated and sequenced by the dideoxy chain terminating method (see DE BOER and RIPLEY 1984). Approximately 20,000 plaques per plate were recovered. The frequency of blue plaques was found to be between 7 and 8 X for M74 and 1 X for P74.

Theoretical DNA-folding: Using the RNA2 program of ZUKER and SANKOV (1984) we examined the patterns of predicted DNA folding in the N-terminal region of the p- galactosidase constructs of M74 and P74. No attempt was made to examine long range folding throughout the approximately 7000 nucleotides present in these substrates. The DNA during the experiment is single stranded in the region between bp 15 and 125, but is otherwise double stranded. Thus, we suspect that DNA secondary structures involving primarily this single stranded region should dom- inate the population of molecules participating in replication. The energy estimates from the folding program are based on values estimated from experiments in RNA and thus may differ from values in DNA.

RESULTS

T h e sequences of the P74 and "74 DNA templates are illustrated in Figure 1. DNA synthesis was primed

from a restriction fragment annealed to the template leaving a gap between positions 125 and 15. Thus, DNA synthesis copies the illustrated strand beginning at position 124. T h e 77 bp of DNA that are found in the rZZB gene of T4 a re between positions 34 and 1 10. In the P74 template a shift of + 1 (+- 372) restores the wild-type reading frame. In M74, a shift of - 1 (k 3n) restores the wild-type reading frame. The four bases deleted from the poly-linker region to create the M74 template from P74 are shown as spaces at positions 25-26 and 115-1 16.

The mutants are identified by their ability to produce p-galactosidase-complementing activity detected as a blue plaque phenotype in the presence of inducer IPTG and the indicator dye X-gal. DNA synthesis was catalyzed by the large fragment of E . coli polymerase I. The synthetic product after transfection into JM 101 cells resulted in a blue plaque frequency of 7-8 x l op4 for M74 and 1 X for P74. T h e frequency of mutation in templates that were not replicated was or less. The frequency of spontaneous blue plaque mutations in the P74 and M74 phage is between lop6 and lo-?.

Spectrum of frameshifts in M74: The DNA from 137 blue plaques was isolated and sequenced in the N-terminal region of the P-galactosidase gene. A total of 136 mutants had a DNA sequence change between position 42 and 13 1. T h e theoretical downstream limit for mutation is defined by an out-of-frame termination codon (UGA) at 152. Some of the mutants lie between positions 124 and 13 1. The production of these mutants would require either en- largement of the gap through the 3'+5' exonuclease activity of the polymerase or by synthesis all the way around the M13 molecule. Mutations arising at position 125 occurred at similar frequency when synthesis was primed by an oligonucleotide having a 3' terminus at nucleotide 142 instead of the gapped substrate (results not shown).

Figure 1 shows the positions of 107 of these sequenced mutations. Note that the illustrated strand is the template strand, so that deletions occur during the synthesis of the complementary strand. A total of 102 of these mutations are l-bp deletions; 5 are complex (base substitution-frameshift) frameshifts. All 5 complex mutants occurred at position 73-74 and result in the replacement of two As by a C in the template.

Figure 2 shows the sequence of a deletion extend- ing from position 55 to 90. This mutation was found twice. The endpoints of the deletion reside within a directly repeated 4-bp sequence which can be extended to a 7 out of 9 match by permitting an interruption. This mutation parallels many spontaneous deletions and can be predicted by slipped pairing of the primer during synthesis. These mutations predict the misalignment of the CAAG se-

184 J. G . de Boer and L. S. Ripley

1 15

I I 1 0 I 30

1374 A T G A C C A T G A T T A C G A A T T C C C G G G G G A T C C G T C M74 A T G A C C A T G A T T A C G A A T T C C C G G A T C C G T C

40 I U 1 I 2 - 0

C T G A A A T T G T T A A A C T G T A T T C A A G T G G T A - A T T C T G A A A T T G T T A A A C T G T A T T C A A G T G G T A A T T

n 2

90 6

I

0 A C A C C C A A C A G G A A T T G G C T G A T T G G C A A G G T A C A C C C A A C A G G A A T T G G C T G A T T G G C A A G G T

0 0 5 3 Tcl 14

9 0 5

I " V

1 i I

I nn 130 6

1 n

U 21

150 I

U

25

G G C C G T C G T T T T A C A A C G T C G T G A C T G G G A A A G G C C G T C G T T T T A C A A C G T C G T G A C T G G G A A A 17

2 FIGURE 1.-Spectra of M74 and P74 frameshifts produced by E . coli polymerase I large fragment polymerase in vitro. The template

sequences for both P74 and M74 are shown. P74 can be viewed as a - 1 frameshift mutation. Its reading frame can therefore be corrected by deletion of two bases or by addition of one base. M74 can be viewed as a + 1 frameshift mutation. Its reading frame can be corrected by deletion of one base or addition of two bases. Position 1 marks the ATG codon that begins the complementing P-galactosidase gene fragment. To simplify comparisons between shifts to the plus reading frame isolated in P74 and shifts to the minus reading frame in M74, the 4 bases present in P74 but absent in M74 are represented by spaces at positions 25-26 and 115-116, and the numbering of other positions is maintained. Mutants illustrated below the sequence were isolated in M74 and are deletions. The height of the bar over the deleted base is proportional to the number of mutants at the site in the spectrum. When the deletions occurred in a base run, that lost base is arbitrarily indicated at the 5' position of the run. The 102 single base deletions are distributed among 11 distinguishable positions. The five complex DNA sequence changes all occurred at 73-74 and are indicated by a box representing the loss of two As and the incorporation of a C. The mutant sequence read from position 70 is CCCCCAG. All of the frameshifts in P74 were deletions of 2 bp are distributed among 9 distinguishable sites and are illustrated above the sequence.

185 In Vitro Replication Frameshifts

1 40 I I

ATG ACC ATG ATT ACG AAT TCC CGG ATC CGT CCT G M A l l GTT AAA

55 92 I I

‘T CAAGTGGTAAlTACACCCAACAGGAAlTGGCTGAlTGG

m j 50 110 130

1 I CTG TAT T CA AGG TGT ATC GGT TGA CCT GCC CAA GCT TGG CAC TG ~-

E l I I

I I I 110 130

I I

I

GTG TAT CGG TTG ACC TGC CCA AGC TTG GCA CTG

El prJ FIGURE 2.-A plus frame deletion detected in M74. The deletion extends from position 55 to 92, a net loss of 38 nucleotides. The

deletion endpoints are consistent with the misalignment of DNA based on extended but imperfect homology at positions 55-63 and 93- 101. Annealing of a primer strand replicated up to position 93 to position 55 could be mediated by a 7 out of 9 bp homology of the primer terminus with the template. The positions of homology are indicated by underlining. Extension of the DNA after the misalignment would produce precisely the deletion observed. This deletion does not restore the reading frame to its wild type position, however. Instead, the deletion brings into frame a TGA termination codon at position 107. This position is labeled B6 because it is barrier 6 of the rZlB gene (BARNETT et al. 1967). Termination at B6 in T4 rZZB permits the reinitiation of rIZB protein synthesis at both reinitiation site VI (GTG) and VI1 (TTG) (NAPOLI, GOLD and SINGER 1981). Reinitiation at these sites is in the wild type reading frame for downstream p- galactosidase. The P-galactosidase sequence begins with CTG at position 130. In the M74 construct a third potential reinitiation site from the polylinker region of M13mp8 in M74, (TTG) at position 124 might also be activated by the B6 termination.

quence at 93-96 to position 55-58. In this in vitro reaction, the direction of slippage is known; had the same mutation been recovered from an in vivo experiment, slippage in the opposite DNA strand could have offered an alternative explanation.

The deletion illustrated in Figure 2 has a very light blue plaque phenotype compared to other frameshifts isolated. After DNA sequencing it became obvious that the blue plaque phenotype of this mutation requires a special explanation because it does not restore the reading frame to that of the wild- type protein. The explanation for the complementing activity of this mutant is likely to be the production of an active P-galactosidase fragment as a consequence of translational reinitiation. Other studies have shown that comparable deletions in the T4 rZZB gene lead to translational reinitiation within this sequence (NAPOLI, GOLD and SINGER 1981). For example, the deletion of 8 bp (523-530) in the rZZB gene should lead to termination at UGA (544-546). In that case reinitiation at both GUG (534) and UUG (543) have been demonstrated. In M74 the deletion

from 55 to 92 is equivalent to rZZB nucleotides (492- 529), the predicted termination is at the same UGA codon (107-109 in M74, 544-546 in T4 rZZB), and the predicted reinitiation at the GUG or UUG codons (97 or 106 in M74) is in the wild-type reading frame.

The remaining sequenced mutants (27) were iden- tical deletions that had lost the rZZ segment from 34 to 107, had a deletion of G at position 108 and had an insertion of AG at 1 15- 1 16. This sequence is equivalent to M13mp8 sequence except that it retains the 2-bp deletion at 23-24 that had been introduced into M74 and has a l-bp deletion at 108; this com- bination produces a wild type reading frame. The fact that all the rZZ sequence deletions were accom- panied by the same l-bp deletion and 2-bp insertion suggested that all of the changes occurred as a concerted process. This process is illustrated in Figure 3. It is almost certainly caused by DNA primer molecules cut only with EcoRI and not with HindIII. The primer molecules had no rZZ DNA sequence, but did have the complement of the AG sequence at 1 15- 1 16 and the deletion of bp 108. The deletion at

J. G. de Boer and L. S. Ripley 186

A

B

20 40 100 110 130 M74 T T A C G A A T T ~ C C - G G A T C C G T C C T G A A A ~ /\/th/\ ~ A T C G G T T G A ~ C T G C - C C A A G C T T G G C A C ~

c4 A A T G C T T A A - 5 ’ 3 ” A C C G T G A

(2)

2t ( 1 ) 40 100 110 130 M74 T T A C G A A T T C C C - G G A T C C G T C C T G A A A ~ AAAA ~ A T C G G T T G A ~ C T G C - C C A A G C T T G G C A C ~

( 3 ) ( 4 ) c4 A A T G C T T A A - S 3 ’ - G G G C C C C T A G G C A G T G G A C G T C G G T T C G A A C C G T G A

20 110 130

C Deletion T T A C G A A T T ~ C G G A T C C G T C DEtErEDBI\sEs A ~ C T G C A G C C A A G C T T G G C A C !

c4 A A T G C T T A A - 5 ’ 3 ’ - G G G C C C C T A G G C A G T G G A C G T C G G T T C G A A C C G T G A + FIGURE 3.-Deletions arising as a consequence of the C4 primer. The mutant sequence can be readily accounted for by the presence

of incompletely cut C4 primer and the action of the 3’ exonuclease of polymerase I DNA polymerase. This class of deletions is not seen when an oligonucleotide primes DNA synthesis. (A) The usual substrate created by annealing a C4 RF molecule digested with EcoRI and HindIII to the M74 single stranded template. (B) The same template annealed to a C4 RF molecule cut at EcoRI, but not at the HindIII site. In this substrate the primer differs from the template at three positions: (1) C4 does not have the 2 base deletion present in M74 at 23-24. (2) C4 has no T 4 rIIB insert (positions 34-108). (4) C4 does not have the 2 base deletion at 115-116 present in M74. The 14 3’ terminal bases of the primer are not homologous to M74 sequence beginning at position 108. However, 9 of those primer bases (3) (indicated by overlining) are precisely homologous to the template between positions 25-33 (indicated by underlining). Every nucleotide of the deletion mutant can be accounted for by a misalignment of the primer to the downstream template sequence and by the removal of 5 bp from the 3’OH end of the C4 primer, a reaction expected to be catalyzed by the 3’-exonuclease of the polymerase I enzyme, followed ultimately by resynthesis of 3 bases (Gs) and ligation. (C) The relationship between the primer and the mutant by complementarity between the mutation that occurred and the incompletely digested C4 primer.

108 had been introduced into the primer to prevent the production of blue plaques by uncut (or religated) primer DNA. Figure 3A illustrates the usual substrate in which fully cut primer is annealed to M74 template. In contrast, primer cut only at the EcoRI site is shown in Figure 3B. The primer leads to the AG insertion at 115-1 16 (4), complementarity of the overlined sequence in the primer (3) with the underlined sequence in M74 leads to the deletion of the rZZ sequence and the G deletion at 108 (2). Exonucleolytic removal of 5 bases (indicated by the arrow) followed by resynthesis and ligation leads to the retention of the 2-bp deletion at 23-24. The ability of the primer to execute all of these changes if 5 bases of the 3’ terminus are removed is illustrated in Figure 3C by showing the homology of the primer to the mutant sequence. While it is formally possible that these DNA sequence changes could have been produced after transfection (by mismatch repair or recombination), no recombinants were found among the P74 mutants. Moreover, deletions of the entire rZZ DNA sequence have not been found among mutants recovered after priming M74 with an oligonucleotide ( C . PAPANICOLAOU, unpublished data).

Spectrum of frameshifts in P74: A sample of 92 mutants were sequenced. They were all 2-bp deletions; their positions in the template sequence is illustrated in Figure 1 . The number of sites at which 2-bp deletions can be detected in our assay is unknown. A minimum estimate is 78. This estimate is based on the fact that there are 80 distinguishable 2- bp deletions possible between the most 5‘ position (base 20) and the most 3‘ position (base 116) of mutants detected in the spectrum, and that deletions at two of these positions (44-45 and 102- 103) would not be detected since they result in an in-frame UAA or UAG codon.

DNA contexts of l-bp deletions: Deletions of 1 bp occurred at 1 1 distinguishable sites (Figure 1). A conservative estimate of the number of sites at which these mutations could have been seen is 58. There are 60 distinguishable sites between positions 42 and 13 1- 132, the sequence extremes from which mutations were recovered. Deletions at two sites (opposite Cs at 55 and 68) cannot be detected because they produce in-frame termination codons.

The primary DNA sequences surrounding each mutation site are illustrated in Table 1. Each mutation

I n Vitro Replication Frameshifts 187

TABLE 1

DNA context of single base deletions

DNA template contextb

mutants' 5' Context Del site 3' Context deleted'

27 TT G ACCTGC 108 25 TT G GCACTG 125 or 126 14 TT G GCAAGG 91 or 92 13 CT G CCCAAG 113 9 TT A CACCCA 67 5 CC A ACAGGA 73 or 74 3 TT G GCTGAT 83 or 84 2 GT A AGTGGT 63 or 64 2 CT G GCCGTC 131 or 132 1 GT G TATCGG 99 1 TT G TTAAAC 42

No. of Nucleotide

- "

- "- - "

- "

- "

- - - "

- "

- "

- - - -

a A total of 102 single base deletions were sequenced. Nucleotide sequence surrounding the template base at which

single base deletions occurred. In cases where mutations fell into dinucleotide sequences, the 5' most purine is indicated as the template, but mutations occurring in the adjacent position result in the same DNA sequence change.

The nucleotide positions of the deletions indicated are shown in Figure 1.

is opposite a purine in the template, and 85% (85 of 102) occurred opposite a template G. The specificity difference between As and Gs is not due to the absense of template As in the sequence. Between positions 42 and 131, there are 15 A sites and 19 G sites. Mutations were found at 3 A sites and 8 G sites. There were no deletions opposite any of the 24 pyrimidine sites.

Every G that served as a template for a 1-bp deletion was immediately followed in the template by a T. Note that during DNA replication this T is the template for the base that must be incorporated after the deletion. In fact, until an A is incorporated opposite this T, no deletion exists. The most frequent sites of these deletions have two 5' Ts. The exceptions account for only 16 mutants. Thus, TTG is a template for deletions opposite G for 81% (69 of 85) of the G deletions.

Although the 3' template sequences did not dem- onstrate a single conserved sequence at the frameshift hotspots, there is a bias for G:C-richness in these sequences. Sites accounting for 95% (81 of 86) of the G-site deletions had either 4 or 5 G:C pairs among the 6 pairs immediately before the site of the deletion. Among the 3 TTG sites that were frequent deletions sites (represented by 14 to 27 mutations per site), all had 4 G:C pairs out of 6. In contrast, the TTG site (81-83) represented by only 3 deletions, had 3 G:C bp out of 6 and the TTG (39-41) site represented by 1 mutation had 1 G:C pair out of 6. Note that this is the portion of DNA that has been replicated before the mutation occurs, so is expected to be double

stranded and directly in contact with the DNA polymerase.

The context rules for deletions at template A appear to be similar to those at template G, but are based on a much smaller sample (16 mutant sequences distributed among 3 sites). The most frequent deletion opposite a template A site accounted for 9 of the 16 mutations. The DNA context of this site is like that of the most frequently mutated G sites. The template A is adjacent to a 5' TT sequence and in the 3' direction there are 4 out of 6 G:C pairs. An examination of the template sites (between 42 and 13 1) at which no mutations were seen reveals no examples of TTG sequences but did reveal one TTA (43-45) sequence. However, this TTA sequence has only one G:C pair out of 6 in its 3' context.

Of the 1-bp deletions, 85% were found opposite template Gs, 15% opposite As and none were found opposite pyrimidines. Although this bias was not predicted on the basis of the frequencies of purines and pyrimidines between positions 43 and 131 in the template, we have observed that there are 4 TTG sequences, 2 TTA sequences and no TTT or TTC sequences at which mutations could have been detected. (Deletion of C at the TTC sequence at 53-55 leads to an in-frame UAA codon.) Although the spectrum suggests that pyrimidine template sites are rarely sites of 1-bp deletion frameshifts, a rigorous demonstration of this within the most error prone context identified for the polymerase I enzyme will require altered DNA templates.

DNA contexts of 2-bp deletions: Table 2 illustrates the sequences surrounding each of the 2-bp deletions. More than 80% of the mutations occur at just three sites. At the three hotspots, deletions occur opposite template purines, just as do the 1-bp deletion hotspots. At two of the three hotspots the deletion occurs within a purine run. Thus precisely which two purines are deleted cannot be determined from the mutant sequence. If the assumption is made that at these two sites the deleted bases are at the 5' end of the run of 3 As or 4 Gs, respectively, repeated pyrimidines are found in the template adjacent to the mutant site. No common features were found in the 3' context, and G:C content did not correlate to mutant frequency. There is a notable absence of overlap between hotspots for 2-bp deletions and 1 bp deletions. There are no examples of 1 base deletions at the two most frequent 2-bp deletion sites nor are their examples of 2 base deletions at the two most frequent 1-bp deletions sites.

In the absence of an identifiable consensus sequence at the 2-bp deletion hotspot sites, we evaluated the presence or absence of other sequence elements previously correlated to frameshift sites. We reasoned that different mechanisms might contribute different 2-bp deletion hotspots to our spectrum. Two of the

188 J. G. de Boer and L. S. Ripley

TABLE 2

DNA context of 2-bp deletions

D N A template context''

mutants" No. of . . .

Position 5' Context Mutdnl sitc S' (:ontext ~

(:(:(:A'

38 45-47 7'TGTT AAA CTGTA 21 23-26 TT<:C<: GGGG ATCCC;

"

16 73-74 C:A<:C<: .4 A CAGGA * 6 67-70 TAATT ACAC CCAAC * 6 115-1 16 CCTGC A(; CCAAG * 2 63-64 (;TGGT AA TTACA 1 20-22 GAATT CCC 1

M X G A 56-57 TATTC A A ( ;m;T

1 112-113 TGAW T(; CA<;CC * A total of 92 mutants were sequenced. At sites at which more than two bases are inciicated, any two adjacent bases can be deleted t o produce the mutant sequence. The asterisks denote those sites sharing a CCCA consensus sequence in the template strand after the 2-hp deletion. At position 67-

70, this is newly formed only when the 2 bases deleted are 69 and 70. The asterisk denotes

three hotspots lie in mononucleotide repeats. Mon- onucleotide repeats are frequently hotspots for - 1 or + 1 frameshifts; however, they have not been shown to be hotspots for + 2 or - 2 bp frameshifts. In fact, in bacteriophage T4, this class of frameshifts does not occur preferentially in repeats, but does show a correlation to the bases of DNA hairpins (RIPLEY, CLARK and DE BOER 1986).

Figure 4 illustrates two alternative hairpins that place the most frequent 2-bp deletion in this spectrum immediately adjacent to a hairpin. Spontaneous 2-bp deletions arising in the T4 rZZB gene do not occur at this run of As, but neither of the hairpins in Figure 4 would form in the rZZ gene because they involve M13mp8 DNA sequences. Within the context of the gapped template substrate used in these experiments, the formation of the larger hairpin would require substantial melting of the 5' end of the primer strand, while the smaller hairpin would only require dissociation of 5 bp. Computer-predicted hairpins that placed the other two hotspot sites at the downstream terminus of substantial DNA hairpins all involved DNA sequences that would require substantial dissociation of the 5' end of the primer strand and are not illustrated.

Complex frameshift mutations consistent with misalignments of a primer during synthesis, followed by realignment to the original site have been found among spontaneous rZZB frameshifts (DE BOER and RIPLEY 1984; RIPLEY, CLARK and DE BOER 1986). The model for these mutations correlates the specific DNA sequence change seen to the presence of a nearby DNA sequence that is perfectly homologous to the mutation, and partially homologous to the starting sequence. The mutations are hypothesized to occur by misalignment based on partial homology, followed by limited DNA synthesis at the misaligned position, followed by realignment to the initial posi-

tion and thereby explaining the perfect homology between the mutant sequence and the nearby sequence. The hotspot occurring at 73-74 might be templated by such a mechanism by a sequence of CCCNTGGG. This sequence would allow at least 2 bp to mediate the misalignment and at least 2 bp to mediate the realignment-deletion. Additional homology would make the argument for the participation of the misalignment-realignment mechanism in the mutational process more attractive. However, we have noted with some interest that this same minimal sequence CCCAITGGG also describes the context of 3 other deletion sites in our spectrum. A total of 29 mutants at 4 sites have this sequence at the site of the mutation. No other site in the genetic target of P74 could give a CCCNTGGG sequence as a consequence of a 2-bp deletion. An examination of M74 for sequences that give CCCNTGGG sequences at the mutant site as a consequence of a 1-bp deletion revealed no such site. However, the complex frameshift mutant, found five times in M74, that substitutes a C for 2As creates the sequence CCCA/TGGG.

A search of the entire M74 sequence for a sequence perfectly homologous to the complex frameshift at 73-74, revealed that the maximum homology was a 7-bp sequence approximately 150 bases away from the site of mutation. The sequence occurs between position - 81 and - 75 (numbering conserved from that shown in Figure 1) and is CCCCAGG in the template strand. This sequence lies between the CAP site and the - 35 promoter of the @galactosidase gene. If this sequence is indeed responsible for these complex mutations, the initial misalignment of the primer would have to depend upon 4 bp of homology to span the 150-bp distance (CAGGKCTG). We found no apparent additional homology that might stabilize this intermediate. The next step would require the incorporation of 3 Gs complementary to


5 ” C A C C C A A C A G G - 3 ’ 5 ” C A C C C A A C A G G - 3 ’ I I 1 1 1 1 1 1 1 1 1 1 1 G G - G G T C C - 5 ’ G G G - G T C C - 5 ’

OH OH

1 2 FIGURE 5,”Realignment to create complex frameshift or 2-bp

deletion. Misalignment 1 demonstrates the production of the complex sequence change. Misalignment 2 demonstrates how the same sequence could probe a 2-bp deletion at that site.

t 8 I

C C A T G A T T A C G A A A G T

T T

A C T C C C

G G A G

C G A G

A A -8 - A*T- t28

G t c ,G *C

A I ‘C G

A *T ’ ‘c c \ c

AtT’ C G T *A T *A T *A A *T A*T C*G

-28 A r T A*T +48

I I GC-A ,T**,A-A-c

G A

O C GtC CSG

G T- t28

‘ E c c \ T CSG T*A I A T*A A*T A*T

G I QG A*T

T*A , ’ T t 4 8

T- G-A , T I A , ~ - c - ~ I

FIGURE 4.-Hairpins adjacent to a 2-bp deletion hotspot. The illustrated DNA hairpins, if present in the DNA template during replication, intersect with the progression of the DNA polymerase in the run of 3 As at 45-47 that is a hotspot for - 2 bp frameshifts in P74. In hairpin 1 (left), there are 1 1 bp of perfect homology between the rIIB sequence of P74 and ZucZ operator sequence. There is some additional but imperfect homology as well. In hairpin 2 (right), the same rIIE sequence is paired to the initial portion of the modified P-galactosidase gene fragment carried by M13mp8. In the gapped DNA templates used, the formation of hairpin 1 would require dissociation of 40 bp of the 5’ end of the primer from the template; the formation of hairpin 2 would require the dissociation of only 5 bp.

the 3 Cs in the misaligned context, followed by a realignment to the site of mutation. This realignment could occur in two alternative ways as illustrated in Figure 5.

Alignment 1 would produce the observed AA+C complex sequence change. Alignment 2 would produce a deletion of 2 As. The deletion of 2 As is one of the hotspots for 2-bp deletions in P74. The frequency of these mutations are similar. If a misalignment mechanism is responsible for complex mutation, perturbation of conditions influencing misalignment should have similar effects on the P74 2-bp deletion hotspot and the complex mutation in M74.

DISCUSSION

A specific assay for frameshift mutations arising as a consequence of in vitro DNA replication is described. The assay depends upon the insertion of T 4 rIIB DNA sequences into the N-terminal portion of the P-galactosidase gene fragment of M13mp8. This assay permits the direct comparison of in vivo and in vitro mutant spectra, and is general in that any alternative DNA sequence having two open reading frames could be similarly examined. Frameshifts arising in vivo in pBR322 sequences incorporated into M13mp8 have been detected (LORENZETTI, CESARENI and CORTESE 1983). The assay detects many different kinds of frameshifts, and frameshift specificity can be readily examined without examining high frequencies of base substitutions, a major problem in many assays.

Spontaneous addition and deletion frameshifts occur at roughly equivalent frequencies in vivo (Rr- PLEY, CLARK and DE BOER 1986). Moreover, mutant polymerase alleles increase the frequency of addition frameshifts as well as deletion frameshifts in vivo (RIPLEY and SHOEMAKER 1983; RIPLEY, GLICKMAN and SHOEMAKER 1983; L. RIPLEY, unpublished data). Thus, our observation that the frameshifts arising after in vitro replication were essentially all deletions was unexpected. Deletion frameshifts rather than addition frameshifts are also the consequence of replication by eucaryotic DNA polymerases on an M13mp2 template (KUNKEL 1985a, b). This result indicates that the duplication of a particular sequence and the deletion of that same sequence may depend on very different mechanisms.

The contribution of several frameshift mechanisms to the spectra of frameshifts produced as a consequence of in vitro replication by the polymerase I enzyme is indicated by the different kinds of mutant DNA sequences produced and by the different DNA contexts in which the mutants are found. These will be individually discussed in the following sections.

The 1-bp deletions: Deletions of 1-bp occurred an order of magnitude more frequently than the other kinds of frameshifts as a consequence of replication by the large fragment of the E . coli polymerase I enzyme. These 1-bp deletions occur most frequently opposite template purines adjacent to T and having a G:C rich 3‘ context. This striking sequence speci-

190 J. G . de Boer and L. S. Ripley

ficity is likely to reflect the propensity of the consensus DNA sequence to assume an unusual structure that leads to polymerization errors, and/or an error- prone interactions between the DNA binding site of polymerase I large fragment and the consensus DNA sequence in its usual form.

Previous studies of interactions of polymerase I with damaged DNA templates in vitro have revealed polymerization characteristics that may be related to the 1-base deletion specificity. In vitro characterization of the DNA synthesized on depurinated templates (STRAUSS et al. 1982) coupled with measurements of mutagenic specificity in single stranded phage after heating (SCHAAPER, KUNKEL and LOEB 1983; KUNKEL 1984) have led to the view that E . coli polymerase I may preferentially incorporate purines, particularly A, when confronted with damaged template bases that block further extension of the primer. The DNA contexts that stimulate such incorporations are not yet well defined. However, in one instance polymerase I-mediated bypass of thymine glycol lesions has been enhanced by manganese preferentially at template base that are followed by thymine or cytosine in the 5' direction (IDE, Kow and WALLACE 1985). We find it an interesting possibility that the polymerase I frameshift consensus sequence that we identify here is a sequence prone to polymerase translocation errors ( i e . , bypass of a template base). If translocation errors by polymerase I are linked to the incorporation of A, a 5' T in the template would permit complementary pairing of the misincorporated A and support further extension, thus capturing the l-base deletion error.

The high frequency of l-bp deletions produced by the large fragment of polymerase I relative to any other mutational class and the strong dependence of their frequency on a specific DNA context suggests that most of these mutants arise by a single mechanism. There does not appear to be any role for slippage in the in vitro production of most l-bp deletions in these experiments. Fifty percent of the mutations occur at the site of a single purine sur- rounded by different neighbors. Among spontaneous frameshifts in T4, 12% of the l-bp deletions occur in such nonrepeated sequences (RIPLEY, CLARK and DE BOER 1986). The identification of a consensus sequence that clearly implicates a mechanism different from primer slippage is an important step in the ultimate identification of the mechanism responsible for single base deletions. This work implicates the interaction of particular DNA sequences with DNA polymerase as a source of frameshift mutation.

Large deletions: The DNA context for the 38-bp deletion recovered twice in the M74 spectrum and illustrated in Figure 2, predicts that this mutation is mediated by the misalignment of the primer during DNA replication. The potential for additional pairing

beyond the perfectly homologous pairs at the end points of this deletion has been seen previously among spontaneous deletions (ALBERTINI et al. 1982; RIPLEY, CLARK and DE BOER 1986). The recovery of these mutations supports the view that the in vitro replication system is subject to primer slippage mechanisms. Thus the absence of examples of large num- bers of single base deletion is mononucleotide runs simply reflects their lower frequency.

The misalignment model predicts duplications as well as deletions, but as noted above, duplications were not seen. It is perhaps surprising that we found no examples of 8-bp deletions or duplications in the sequence between 80 and 93. In T4 this sequence is a frameshift hotspot; the mutant sequences are consistent with the misaligned pairing of a 6-base sequence (ATTGGC) to its complement 8 bases away. Mutations at this site constitute 20% of the spontaneous frameshifts in the N terminus of the rZZB gene (RIPLEY, CLARK and DE BOER 1986). The absence of this mutation from any in vitro spectrum determined so far (unpublished) suggests that in vitro the frequency with which misalignments lead to deletion mutation are strongly influenced by factors other than the length of the DNA repeat involved.

Complex frameshifts: One complex sequence change (AA-C) was seen in the M74 spectrum. At least one example was found in each of the three independent experiments from which mutants were sequenced. Our previous studies of complex sequence changes in spontaneous T4 frameshift spectra, has shown that when such a change occurs often, that the change is perfectly homologous to a nearby DNA sequence that is a direct repeat or a palindrome (DE BOER and RIPLEY 1984; RIPLEY, CLARK and DE

BOER 1986). These perfect homologies led us to propose that DNA misalignments associated with local DNA synthesis followed by realignment of the DNA could account for this mutational specificity (RIPLEY 1982a; RIPLEY and GLICKMAN 1983). All five of the spontaneous frameshifts sequenced in the lacl gene were all consistant with these mechanisms (SCHAAPER, DANFORTH and GLICKMAN 1986). On the other hand, many complex frameshift mutations assay in vivo can not be accounted for by these models within known T 4 DNA sequences (RIPLEY, CLARK and DE BOER 1986). Most of these, can be described as closely linked transversion substitutions and frameshifts, as is the AA+C change seen here. The in vitro DNA replication system offers an opportunity to evaluate the possible contribution of these alternative mechanisms to complex frameshift mutagenesis.

Deletions of 2 bp: We have found no single feature that would appear to account both quantitatively and qualitatively for all of the hotspots associated with 2- bp deletions. The repeats at 4 sites (2 hotspot sites and 2 less frequent sites) might account for the


frameshifts by a slippage mechanism. However, slippage also predicts + 1 frameshifts in 3 of these 4 sequences. No + 1 frameshifts were found. At the very least, the mere presence of a repeat is not a sufficient condition for producing 2-bp deletions. For example, no 2-bp deletions occurred in the run of 3 As at 37-39, located only 5 bases away from the run of 3 As at 45-47 that is a hotspot.

The work reported here was funded in part by a Biomedical Research Support Group grant from the University of Medicine and Dentistry of New Jersey (UMDNJ) and grant AGO6171 from the National Institutes of Aging to L.S.R. The authors would like to acknowledge the technical assistance of R. WALLACE at the National Institute of Environmental Health Services and E. KA- LATZIS at UMDNJ. The authors thank C. PAPANICOLAOU for the use of data not yet published, and C. NEWLON for comments on the manuscript. GRAHAM R. CLEAVES provided the SPAC program used to examine the M74 and P74 templates for homologies to mutant sequence and adapted the folding programs of M. ZUKER and D. SANKOV to run on an HPIOOO computer.

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