volume u Number 5 1986

Nucleic Acids Research

Mutations induced by DNA polymerase a upon in vitro replication of M13mp8{+) DNA

Bernd Reckmann and Gerhard Krauss*

Laboratorium fQr Biochemie, Universitat Bayreuth, Postfach 3008, D-8580 Bayreuth, FRG

Received 22 October 1985; Revised and Accepted 13 February 1986

ABSTRACT The forward mutation of the lacZ part of the bacteriophage M13mp8 has been used to study the fidelity of the 9S DNA polymerase a from calf thyraus during in vitro replication of single-stranded DNA. Errors leading to a loss of ot-complementation were identified by DNA sequencing. The overall mutation rate of the lacZ target sequence was in the range of 1:300 - 1:1000 which is more than one order of magnitude higher than the spontaneous mutation rate. In a mutL host the mutation rate was nearly threefold higher as compared to the wildtype host. Base substitutions comprise 86 % of the errors whereas base deletions amount to 12 %. The addition of a base was detected only in one mutant out of 71 sequenced ones. The frameshift mutations occurred predominantly in runs of the same base. The frequencies of individual base substitution are in the order of 2 x 10 - 4 x 10 for most of the mismatches. Mutations involving dCTP:T and dGTP:T mismatches are observed with a lower frequency, those involving dTTP:C mismatches with a higher frequency. INTRODUCTION The fidelity of DNA polymerases in copying natural DNA can be studied in two ways: by backward mutation or by forward mutation of specific sequences. The backward mutation has been applied successfully to amber codons of *X174 DNA where reversions as a consequence of errors produced during in vitro replication can be easily scored. By this assay the fidelity of various isolated replication systems has been studied in detail (for a review see 1 ) . The backward mutation of amber codons also allows one to determine the frequency of specific mispairs formed during in vitro replication (2,3). Since backward mutation of amber sites is necessarily restricted to the TAG sequence information about sequence specificity and sequence contexts of error production cannot be obtained from this

© IRL Press Limited, Oxford, England.

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Nucleic Acids Research assay. Sequence contexts have been inferred e.g. from the reversion frequencies at different amber codons following in vitro replication by a T4 replication complex (4). The forward mutation of the lacZ part of M13mp8 phages has been introduced by Le Clerc and Istock (5) as a means for analyzing the mutational specificity of UV damage. Very recently this approach has been applied in a study on the error production by DNA polymerase B (6). The data obtained up to now show that the forward mutation has the advantage of detecting a broad spectrum of different lesions in DNA. A large number of base substitutions and frameshifts has been scored in these studies thus providing a rather detailed picture of those sequence positions where base changes can be detected in the forward mutational assay. In the present study we have investigated the mutational specificity of DNA polymerase a during in vitro replication of the lacZ part the of phage M13mp8. From the spectrum of mutations and from data of the literature we can calculate normalized frequencies of base substitutions for all 12 possible base mismatches. MATERIALS AND METHODS The bacteriophage M13mp8 and the E. coli host JM 103 ( A(lac,pro) , supE, thi, strA, SBCB15, hs -35 promotor, Q) —10 promotor, Q ) ribosome inding site, ^D promotor sequence, (?) operator sequence. 2372

Nucleic Acids Research E. coli mismatch repair system with the expression of the replication errors we have used mutL and mutS host strains as recipients for the in vitro replicated DNA. The known mutator loci mutS and mutL are generally assumed to participate in DNA mismatch repair (24). Both for mutS and mutL hosts an increased mutation rate is observed. Table I shows that for the mutL strain the mutation rate is nearly threefold higher as compared to the wildtype whereas for the mutS strain the mutation rate is only slightly increased. This behaviour is observed for substrate I and substrate II. For the mutL and mutS strain the spontaneous mutation rate is about one order of magnitude lower. As compared to the phage system, the effect of mutL on the mutation rate of E.coli DNA is much more pronounced (24). Mutational specificity The DNA from 71 mutant clones has been sequenced. Twentyeight of the mutants were derived from in vitro replication of substrate I and 43 originated from substrate II. The spectrum of mutational events (fig. 1) shows a variety of errors. Single-base substitutions represent the most frequent errors (86 % ) . All types of single-base substitutions are observed and these are distributed over the whole target sequence. The base substitutions are found predominantly at purine positions of the template with a preference for G (table II). The spectrum of mutations reflects to some extent the frequency with which a phenotypically detectable mutation can occur upon forming a specific misincorporation at a specific site. From our data and the already published data on base substitutions in the lacZ regulatory and coding region (5,6,18,19) it can be calculated that about 40 % of the phenotypically detectable mutations are found at G positions of the template (table II) which provides an explanation for the frequent mutations at G sites observed in the present study. Stop codons were created by nine of the base substitutions. Two mutants containing a TAG codon in the cloning insert were identified although the host JM103 is an amber suppressor strain. These mutants were obtained from light blue plaques which indicates that suppression was incomplete. Deletions of one base comprise 12 % of the observed mutations.

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Nucleic Acids Research Table

II:

template base

Specificity total nutation

no.

of

t)«BO aubstitutiona

template

m Jtation be ]e no.

mismatch during in vitro replication

frequency of

known

normalized

specific base

phenotypical

frequency of

substitution

mutationa1'

specific baae substitution

A

T

G

C

14

7

25

15

dATP

A

2.5 x 10"*

13

2.4 x 10"*

dCTP

A

3.0 x 10" 4

16

2.4 x 10"4

dGTP

A

1.5 x 10" 4

6

3.2 x 10"*

3 1

dTTP dCTP

T T

5

3

dGTP

A T C

9 10 6

dTTP dATP

A T

5 7

dTTP

G

3

dCTP

T G C

5 6 3

A G C

dGTP

dATP

1.5 x 10"*

9

2.1 x 1 0 " 4

x 10" 5

10

6.3 x 1 0 " 5

T

1.5 x 10" 4

19

1

G G G

4.4 x 10" 4

16

3.4 x 1 0 " *

4.2 x 10"*

20

2.7 x 1 0 " *

3

11

3.4 x 1 0 " 4

C C C

2.5 x 10" 4

6

3.4 x 10" 4

21

1,5 x 10" 4

5

x 10"*

x 10"*

5.2 x 1 0 " * 2

x 10"*

3.8 x 1 0 " 4

data from the praaent study and from to Clerc 4 Istock (5), Kunkel (6,IB) and Le Clerc et al. (19). cf. Methods section

Only in one mutant the addition of one base was observed. The frameshift mutations are found mostly in runs of the same base. Kunkel (6) reported a pronounced clustering of frameshift mutations in his study on the error production by DNA polymerase 3. Although we find most of our frameshift mutations at the same sites, we do not observe a similar clustering. The ratio of base substitutions to frameshift mutations is about 7:1. This value overestimates the proportion of frameshlfts since all ±1 frameshifts will change the phenotype whereas only part of the base substitutions will lead to a phenotypic mutation. Thus the real proportion of frameshifts will be lower. The mutated sites are clearly clustered at specific sequences of the lacZ region (fig. 1 ) . Similar hotspots of mutation have been reported also in the earlier forward rautational studies on the lacZ gene (5,6,19). The presence of hotspots reflects the forward mutational character of the assay system. In the promotor region of the lacZ insert, mutations are found at the binding site for the cap protein at positions 6121-6126 and at the -35 and -10 position of the promotor sequence. The mutations at positions 6206-6208 affect

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Nucleic Acids Research the ribosome binding site of the transcribed m-RNA. In the coding region a clustering of mutations is noticed at those amino acids for which only one or two codons exist. The clustering of mutations at the amino acids 15-18 and 46-38 points to an essential function of these amino acids for the complementation of the fi-galactosidase activity. Frequencies of specific base substitutions Our data and the large number of already known base substitutions (5,6,18,19) provide a rather detailed picture of those positions of the target sequence where base substitutions are accompanied by a change in phenotype. In total, 152 base substitutions within the 265 nucleotide target sequence have been already shown to change the lacZ phenotype. Table II shows the frequencies of specific base substitutions calculated from the present data. In addition we have normalized these values for the unequal distribution of individual base substitutions in the spectrum of known substitutions events within the target sequence. The normalized mutation frequencies obtained this way can be considered to reflect fairly well the specificity of error production during in vitro replication. The only uncertainty comes from the incomplete knowledge of the total spectrum of phenotypically relevant base substitutions. How ever.it can be estimated that this uncertainty amounts to not more than a factor of 2. From the 795 possible base substitutions in the target sequence, at least two third are estimated to be silent mutations (18). From the remaining base substitutions, 152 have been considered in the calculation of the normalized mutation frequencies. The frequencies for the complete list of base substitutions are in the range of 2-4 x 10 , with larger deviations for substitutions involving dCTP:T, dGTP:T and dTTP:C mispairs. In order to convert the normalized mutation frequencies into error rates one would have to consider the number of template bases replicated and the contribution of repair processes to the expression of errors (3,29). Data on the expression rate of specific errors are however not available. Our earlier work on the error production by DNA polyraerase a showed a much greater diversity of individual error rates as is

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Nucleic Acids Research observed in the present work. However, in the earlier work, the frequencies of individual mispairs refer only to one position of the TAG sequence. In contrast, mutation frequencies from the present work represent the averages for several positions. We conclude from the much lesser diversity of specific mutation frequencies observed in the forward mutation assay that a sequence context for mispair formation exists. DISCUSSION We have probed the fidelity of DNA polymerase a from calf thymus by forward mutation of the lacZ insert of the phage Ml3mp8. Following in vitro replication of M13mp8(+)DNA by the DNA polyroerase a and transfection of the replication products into E. coli, we have isolated and sequenced 71 mutants. About 86 % of the mutations were due to base substitutions. The remaining 14 % arose from frameshift mutations with a strong preference for base deletions vs base additions. Since frameshift mutations will lead with a higher probability to a change in phenotype than do base substitutions, the proportion of frameshift mutations will be overestimated in our assay. The mechanism by which the frameshift mutations are produced during in vitro replication is not fully understood. Most of the deletions are found in runs of the same base which suggests that these errors may be caused by a slippage of the primer terminus during elongation (30). Recently the mutational specificity of DNA polymerase a has been investigated in the same assay system (6). As compared to the results reported in this work the overall mutation frequency of the lacZ part is considerably lower for the DNA polymerase a. Furthermore, DNA polymerase a produces frameshift mutations at a strikingly lower frequency. The frameshifts are not clustered at hotspots as has been observed for the DNA polymerase B. We find frameshifts at the same sites, however without clustering. The lower proportion of frameshift mutations found for the DNA polymerase a may be explained by a higher affinity of the enzyme to the primer terminus and/or differences in processivity. In the same study DNA polymerase 8 has been reported to produce template T •* G and T •* C mutations at a high frequency. A similar

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Nucleic Acids Research preference for this type of base substitution is not observed in the present work. Clearly the different mutational spectra reflect different chain elongation properties and different propensities for error production of the two enzyme systems. The large number of already known base substitutions that are accompanied by a change in phenotype allowed us to calculate frequencies of specific base substitutions. These values can be considered to mirror rather accurately the pattern of base mismatches formed during in vitro replication. It is one advantage of the forward mutational assay that one can obtain mutation frequencies for all 12 possible mispairs. In contrast, we had been able to measure the frequencies of only seven mispairs in the 4>X174 amber revertant system (3). Specific base substitutions are formed during in vitro replication of the lacZ part at a frequency of 2 - 4 x 10" . In accordance with experimental results on the same and other DNA polymerases obtained from the amber revertant system (3/29,31) and in agreement with theoretical considerations (32), we find a low frequency of dCTP:T mismatches. The other pyrimidine-pyrimidine mismatches are formed however at a rather high frequency. Furthermore we do not observe the frequent formation of G:G and G:T mispairs as was the case in our earlier study (3). In comparing the results from the two assay system one has to keep in mind that the mutation frequencies obtained from the forward mutation assay represent average values that have been scored from several positions of the template DNA. We ascribe the lesser variation of the mutation frequencies from the forward mutation assay to an averaging between positions of high and low error frequency in the lacZ sequence. This interpretation implies a strong sequence context for the error production by the DNA polymerase a. In order to get more insight into the nature of the sequence specificity of error production one will have to analyze a much larger number of mutants than has been done in the present work. Furthermore, silent mutations also must be considered. We think that one of the most important factors that might modulate the error rate, will be the presence of secondary structures in the template DNA. We are currently investigating the relation between mutation

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Nucleic Acids Research frequencies structures.

and

the

pausing of DNA polymerase a at secondary

In order to minimize the interference of repair processes in the host cell with the detection of errors produced during in vitro synthesis the gapped substrate has been constructed from an unmethylated (+) template and a methylated (-) strand fragment. This configuration should direct the mismatch repair to the (+) strand and should thereby allow a maximal expression of errors. The mutants analyzed were obtained by about equal parts from wildtype, mutL and mutS hosts. It is improbable that the difference in repair background puts a bias on the mutational spectrum. It has been shown recently by Kramer et al. (33) that mutS and mutL strains have lost proficiency in methyl directed mismatch repair for all the different mispairings. In the mutL host an increase of the mutation rate by nearly a factor 3 is observed although the background of spontaneous mutations in the lacZ part is still by one order of magnitude lower. This is an unexpected result since it had been shown that a methylation pattern similar to that of our gapped substrate allows the expression of 70 - 80 % of the errors (33,34). A threefold increase of the mutation rate in mutL as compared to the wildtype can thus only be explained if the expression rate of errors in the wildtype is not as high as expected from the methylation pattern. Our present work underscores the versatility of the forward mutational system for studies of the mutational specificity of isolated replication systems. From changes in the mutational spectrum as consequence of e.g. error prone conditions during in vitro DNA synthesis a better insight into the mechanisms that underly error production can be obtained. Due to the wide range of DNA lesions that are detectable in the forward mutation assay, this approach complements results obtained from the amber revertant system.

Acknowledgements The technical assistance of M. Wehsling and C. Fischer is gratefully acknowledged. This work was supported by grant KR 704/3 from the Deutsche Forschungsgemeinschaft to G.K.

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Nucleic Acids Research *To whom correspondence and reprint requests should be addressed

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Nucleic Acids Research 30. S t r e i s i n g e r , G., Okada, Y . , Emrich, J., Mewton, J., Tsugita, A., Terzaghi, E. and I n o u y e , M. ( 1 9 6 6 ) , Cold S p r i n g Harbour Symp. Q u a n t . B i o l . _3_1.» 7 7 - 8 4 31. F e r s h t , A.R., Shi, J.P. and T s u i , W.S. (1983), J . Mol. B i o l . 1§1' 655-667 3 2 . T o p a l , M.D. and F r e s c o , J . R . ( 1 9 7 6 ) , N a t u r e 26 3 , 2 8 5 - 2 8 9 3 3 . K r a m e r , B . , K r a m e r , W. and F r i t z , H.J. (1984), Cell 38, 879-887 3 4 . K r a m e r , W., S c h u g h a r t , L. and F r i t z , H.J. (1982), Nucl. A c i d s R e s . 1 0 , 6475-6485

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