The effect of the 3′ → 5′ exonuclease of T7 DNA polymerase on frameshifts and deletions

Mutation Research, 301 (1993) 235-241 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-7992/93/$06.00

235

MUTLET 0773

The effect of the 3' ~ 5' exonuclease of T7 D N A polymerase on frameshifts and deletions Warren Masker and Mary Ann Crissey Department of Biochemistry and Molecular Biology and Fels Institute of Cancer Research and Molecular Biology Temple University School of Medicine, Philadelphia, PA 19140, USA (Received 25 September 1992) (Revision received 20 November 1992) (Accepted 1 December 1992)

Keywords: Frameshift; Deletion; T7; DNA polymerase; 3' ~ 5' Exonuclease

Summary Both spontaneous frameshift mutation and deletion mutation were measured in a T7 phage deficient in the 3' ~ 5' exonuclease of T7 D N A polymerase. It was found that the absence of this exonuclease caused a marked increase in the reversion of both plus one and minus one mutations. The exonuclease deficiency caused essentially no effect on the frequency of deletion between 10-bp direct repeats even when the segment between the direct repeats contained a 25-bp palindrome.

The 3' ~ 5' exonuclease activity of D N A polymerases plays a major function in improving the fidelity of D N A synthesis. By removing mispaired nucleotides from a growing polymer chain the polymerase is able to edit newly synthesized D N A immediately after incorporation of a wrong nucleotide and thereby avoid mutation. Spontaneous mutagenesis is higher in mutants of bacteriophage T4 deficient in the 3'--, 5' exonuclease activity (Muzyczka et al., 1972). Similarly, Escherichia coli mutant in the epsilon subunit of the DNA polymerase III holoenzyme (dnaQ = m u t D ) are hypermutable (Scheurmann et al., 1983). Mu-

Correspondence: Dr. Warren Masker, Department of Biochemistry and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA. Tel. (215)646-0315.

tants deficient in the 3' --* 5' exonucleases of T4 DNA polymerase and E. coli DNA polymerase I show increased frequency of frameshifting (Ripley and Shoemaker, 1983; Bebenek et al., 1990). Bacteriophage T7 D N A polymerase, the product of T7 gene 5 plus host thioredoxin (Modrich and Richardson, 1975), also has a 3' --* 5' exonuclease activity. Originally, T7 DNA polymerase was purified in two forms. Form I which lacks 3'--* 5' exonuclease is more processive, does not interact directly with the T7 helicase-primase, and displays a pronounced strand displacement activity. Form II has a 3' ~ 5' exonuclease activity, is able to interact with the helicase-primase to elongate a replication fork, and is less inclined to strand displace. Form I arises from Form II when iron is bound to the polymerase and causes oxidation of important amino acids at the active site of the exonuclease (Fischer and Hinkle, 1980; Engler et

236

al., 1983). Recently, mutants with the 3'--* 5' exonuclease activity of T7 D N A polymerase inactivated have become available. These mutants revert amber mutations at a frequency about one order of magnitude higher than what is typical for T7 with a wild-type DNA polymerase (Tabor and Richardson, 1989). The mutant form of DNA polymerase is more processive than the wild-type enzyme (Tabor and Richardson, 1989). A considerable body of evidence favors the interpretation that misalignments formed during DNA replication are a primary cause of frameshift mutagenesis (for reviews see Kunkle, 1990; Ripley, 1990; Drake, 1991). Thus, a polymerase deficient in the 3'---, 5' exonuclease that acts as edit function might contribute to frameshift either if the enzymatic deficiency caused more frequent misalignment or if errors due to misalignment persisted because of failure of the editing process. Two properties of the T7 3' ---, 5' exonuclease mutant prompted our interest. First, based on the amino acid sequence of the T7 DNA polymerase it is very likely that enzyme has a domain structure similar to that of E. coli DNA polymerase I (Ollis et al., 1985). This suggests that the enzyme must displace its newly synthesized primer terminus a substantial distance in order to move the primer terminus from the active site of the DNA synthetic function to the active site for the 3 ' ~ 5' exonuclease activity (Joyce and Steitz, 1987). During the editing mode, slippage between newly synthesized D N A and downstream sequence could lead to misalignments. Thus, an altered ability to edit might affect the probability of frameshift or deletion. Also, since the mutant DNA polymerase has a higher than normal processivity, the mutant enzyme might be more tolerant of loops generated during misalignment and more inclined to continue synthesis after a misalignment. This would also be likely to increase deletion. To monitor the frequency of frameshift mutation we used a construct where the non-essential gene for ligase, gene 1.3, was put out of frame by introduction of an extra adenosine (A) in a string of three A's so that a minus one frameshift could restore gene function. We also examined a one base elimination in gene 1.3 (called " A D " ) which could be most easily reverted by a base addition

(Pierce and Masker, 1992). A mutation called "A2" which inactivates the 3' ~ 5' exonuclease of T7 DNA polymerase was combined with these gene 1.3 mutants and the reversion to normal gene 1.3 activity was compared with identical measurements using phage with normal DNA polymerase. To monitor the frequency of deletion we used constructs (Pierce and Masker, 1989) where gene 1.3 was interrupted by an insert of synthetic DNA that was bracketed by direct repeats 10 bp long. Deletion between the direct repeats restores the function of T7's ligase gene. The results of those measurements, presented below, indicate a deficiency in the 3'---, 5' exonuclease activity of T7 DNA polymerase substantially increases the frequency of frameshifting but has little effect on deletion of either 29- or 76-bp segments of DNA bracketed by 10-bp direct repeats. Materials and methods

Bacteria and bacteriophage strains. Bacteria used in this study were Escherichia coli strains W3110 (wild-type) and N2668 (ligts7). Wild-type bacteriophage T7 was from Dr. F.W. Studier (Studier, 1969). Construction of bacteriophage T7A + 1, which has non-essential gene 1.3 (T7 ligase) inactivated by an additional adenine residue at position 7264 (Dunn and Studier, 1983), has been described (Pierce and Masker, 1992). T7 AD is a spontaneous frameshift mutant with gene 1.3 inactivated by loss of an adenine residue at position 7186 (Pierce and Masker, 1992). T7X76, T7X29 and T7X76EP all of which have gene 1.3 inactivated by insertions of D N A at position 6663, have been described (Pierce and Masker, 1989; Scearce et al., 1991; Pierce et al., 1991). These phage have inserts of synthetic D N A bracketed by 10-bp direct repeats. The insert in T7X29 is 29 bp long while those in T7X76 and T7X76EP are both 76 bp in length. The insert in T7X76EP has a perfect 25-bp palindrome within the insert; but, there are no palindromic sequences in T7X76. T7 A2, a gift from Dr. Charles Richardson, has the 3 ' ~ 5' exonuclease activity of T7 DNA polymerase inactivated by a two amino acid deletion in gene 5 (Tabor and Richardson, 1989). This deletion generates a new NarI recognition site at

237 position 14713. Bacteria were grown in L-broth and phage plated on T-agar plates with T-soft agar as described by Miller (1972).

Construction of new T7 strains. T7 with combinations of the A2 mutation in gene 5 and various frameshift and insertion mutations in gene 1.3 were constructed by ligating restriction fragments of D N A carrying each of the desired mutations. More specifically, T7 D N A with a mutation in gene 1.3 was treated with MluI which cuts only once at position 9489 and with X b a I which cuts at positions 12830, 22924 and 34293. This produced a 9489 MluI fragment with a gene 1.3 mutation and effectively precluded formation of a functional genome from the 1.3 D N A due to the multiple XbaI cuts. T7 A2 D N A was cut with MluI and with BstNI which cuts at positions 2366 and 8188. The Mlul fragments were ligated together with T4 ligase as described by Sambrook et al. (1989). After p h e n o l - c h l o r o f o r m extraction and ethanol precipitation, the D N A was packaged in vitro as described (Kuemmerle and Masker, 1977). D N A was extracted from phage identified as gene 1.3 mutants by inability to grow on the ligase deficient host N2668 and tested for the presence of a NarI site that is diagnostic of the gene 5 A2 mutation. Assays for deletion and frameshift mutagenesis in gene 1.3. Two methods were used to measure the frequency of restoration of gene 1.3 function. Both depend upon the inability of ligase deficient T7 to form plaques on E. coli t e m p e r a t u r e sensitive for ligase (Masumune et al., 1971). Phage that have restored function of gene 1.3 by deletion of an insert or, in the case of T7 A + 1 or T7 AD, a compensatory frameshift mutation are referred to as "pseudo-wild-type". To measure the number of pseudo-wild-type phage that accumulate during phage growth, cultures of early exponentially growing E. coli were infected with a single T7 phage particle, growth was continued until lysis, and the total number of phage and pseudo-wild-type phage in the lysate were measured. The median value from several (usually at least 24) such measurements provided an estimate of the probability of deletion (Pierce and Masker, 1989). In this assay the total number of

phage equals the number of replication events. Since the accumulation of revertants is measured, the assay may be affected by differential burst sizes between the gene 1.3- mutant and pseudowild-type. The second method of analysis is the fluctuations test described by Luria and Delbriick (1943). In this assay, cultures of strain W3110 were infected with T7 at a multiplicity of infection of 0.1; the phage-infected cells were further diluted and dispersed into a large number of cultures. After lysis the total phage yield was measured in a representative sample of the cultures and the entire contents of each culture was plated on the selective strain N2668. The reversion rate to pseudo-wild-type (d) was determined as P ( 0 ) = e aN, where N equals the average phage titre in a culture, and P(0) is the fraction of cultures that contain no pseudo-wild-type phage. The dilution of phage infected cells is arranged by trial and error so that approximately one-half the cultures do not produce a single pseudo-wild-type phage. The assay measures the size to which a culture can grow before there is an approximately 50% probability that a deletion or frameshift event will occur. This analysis is insensitive to problems caused by differential growth between 1.3- and pseudo-wild-type phage. Results

The effect of an exonuclease deficiency on frameshift mutations. The mutation that inactivates the 3' --* 5' exonuclease activity of T7 D N A polymerase was introduced into T7 with a plus one frameshift in a series of three adenines in gene 1.3. Previous studies with this gene 1.3 mutation showed that it can be reverted by minus one frameshifts which are not necessarily in the same series of adenines where the inactivating base addition was originally engineered (Pierce and Masker, 1992). A comparison of phage with normal T7 D N A polymerase and with the strain deficient in 3' ~ 5' exonuclease (Table 1) shows a greater than 30-fold increase in the frequency of reversion of a plus one frameshift. The same type of experiment was done combining the 3 ' ~ 5' exonuclease mutation with a gene 1.3 mutation (AD) which could only revert via a plus one (or

238 TABLE 1 ACCUMULATION SHIFTING a

OF

REVERTANTS

BY

FRAME-

Phage

Type of mutation to be reverted

Number of lysates tested

Median revertant frequency ( X 10 6)

A+I A + 1 A2 AD AD A2

+1 frameshift + 1 frameshift - 1 frameshift - 1 frameshift

24 32 40 24

8.1 266 0.067 18

a Reversion of the plus 1 frameshift mutant, A + 1, and the minus 1 frameshift mutant, AD, was determined by measuring accumulation of phage able to form plaques on ligts7 strain N2668, as described in the text. The values for revertant frequency are the median of the number of independent measurements indicated.

perhaps a minus 2) frameshift. Again inactivation of the 3 ' - ~ 5' exonuclease activity of the D N A polymerase caused a substantial increase in the frequency of compensatory frameshift mutations (Table 1). Fluctuations tests were also used to measure reversion of the plus one frameshift caused by an extra adenine in gene 1.3. When phage with normal D N A polymerase were used the values obtained using the fluctuations test was slightly lower than what we had found using the method that measures accumulation of revertants (Table 2). When fluctuations tests were used to measure

reversion of the plus one frameshift in a phage with reduced 3' ~ 5' exonuclease activity again it was found that inactivation of the edit function increased the revertant frequency (Table 2). The same comparisons were made with regard to reversion of the minus one frameshift mutation. These data showed about an order of magnitude increase in revertant frequency as the result of inactivation of the 3 ' ~ 5' exonuclease of D N A polymerase I (Table 3).

The effect of an exonuclease deficiency on deletions. Since the data presented in Tables 1 and 2 indicate an increase in one base deletions as a result of deficiency in the T7 D N A polymerase 3 ' ~ 5' exonuclease, we considered whether the exonuclease deficiency would increase the frequency of larger deletions. To examine this phage with gene 1:3 interrupted with inserts of foreign D N A bracketed by 10-bp direct repeats were used. But, only small differences in the deletion of 29- or 76-bp inserts were found upon comparing normal T7 and T7 A2 mutants with the exonuclease inactivated (Table 4). The experiments with T7X76EP (Extended Palindrome) were especially interesting since this insert has 25-bp palindromic sequences with considerable potential for secondary structure. If cruciform structures form as a result of the inverted repeats within X76EP this would bring the 10-bp direct repeats at the flanks of the insert physically closer

TABLE 2 R E V E R S I O N OF PLUS O N E F R A M E S H I F F S M E A S U R E D BY F L U C T U A T I O N S TESTS " Phage type

Number of cultures

Average titre ( x 10- 4)

P(0)

A+ A+ A+ A+

1 1 1 1

71 72 72 72

7.4 6.4 9.8 3.0

0.61 0.58 0.46 0.83

A+ A+ A+ A+

1 A2 1 A2 1 A2 1 A2

40 71 72 72

0.73 0.88 0.99 4.50

0.60 0.58 0.49 0.35

Mutation rate ( x 10 6) 6.7 8.5 7.9 6.1 70 62 72 23

Average mutation rate (xl06) 7.3

56

a Mutation rate was determined by measuring the titre of phage in a lysate where the indicated fraction [P(0)] of individual cultures contained no phage able to form a plaque on the ligts7 host N2668.

239 TABLE 3 R E V E R S I O N O F M I N U S O N E F R A M E S H I F T S M E A S U R E D BY F L U C T U A T I O N S TESTS

Phage type

Number of cultures

Average titre ( x I O -5)

P(0)

_4D /1D /1D /1D AD

40 72 72 72 72

9.7 2.9 5.8 4.8 10.8

0.63 0.63 0.78 0.31 0.25

_4D/12 AD A2 _4D A2 /1D _42

40 72 72 40

0.36 0.18 0.29 0.24

TABLE 4

0.68 0.83 0.76 0.70

Mutation rate ( x I O 6) 0.48 1.60 0.43 2.4 1.3 11 10 9.5 15

Average mutation rate ( × 106 )

1.2

11.4

Phage

Number of lysates tested

Median revertant frequency (Xl06)

ciency caused essentially no effect on deletion of this insert. To be sure of this result, we performed fluctuations tests on the insert with the long palindrome. These data (Table 5) confirm that there is essentially no increase in deletion frequency as the result of a deficiency in the 3 5 exonuclease of T7 D N A polymerase.

X29 X29 -42

24 24

2.0 1.6

Discussion

X76 X76 _42

24 24

1.3 2.8

X76EP X76EP/12

32 24

2.8 2.1

ACCUMULATION OF REVERTANTS DUE TO DELETION BETWEEN DIRECT REPEATS

together and would also potentially form a partial impediment to replication fork progression. But, as seen in Table 4, the 3 ' ~ 5' exonuclease deft-

The data presented above show a substantial increase in frameshift mutagenesis in a T7 strain deficient in the 3' --* 5' exonuclease of T7 D N A polymerase. This effect is most readily explained by a failure to edit mispaired bases formed during misalignments. The increased processivity of the T7 mutant (Tabor and Richardson, 1989) may also contribute to an increase in ffameshifts by

TABLE 5 D E L E T I O N O F A N I N S E R T W I T H P A L I N D R O M I C S E Q U E N C E S M E A S U R E D BY F L U C T U A T I O N S TESTS

Phage type

Number of cultures

Average titre (X 10 -5)

P(0)

Mutation rate ( x 106)

Average mutation rate (×106 )

X76EP X76EP X76EP

40 72 72

2.3 2.5 4.2

0.58 0.72 0.75

2.4 1.3 0.68

1.5

X76EP/12 X76EP/1 2 X76EP/12

40 72 72

3.4 1.9 6.5

0.48 0.57 0.24

2.2 3.0 2.2

2.5

240

increasing the probability that the polymerase will extend a misalignment error. Previous studies from our laboratory showed that the minus one frameshift mutation, AD, is usually reverted by plus one frameshifts near the site of the original AD mutation (Pierce and Masker, 1992). The exonuclease deficiency caused a pronounced effect on reversion of the AD mutation. This was seen both when measuring the accumulation of revertants in a culture and when fluctuations tests were used to measure actual frequency of frameshift per cycle of replication. One interesting observation that we are unable to satisfactorily explain is that reversion of the AD mutation in T7 with normal D N A polymerase appears to be lower when accumulation of pseudo-wild-type is measured (Table 1) than when the revertant frequency is measured by fluctuations tests (Table 3). In all other 1.3- phage that we have examined accumulation of revertants has always been higher than the true revertant frequency. It may be that due to the nature of the AD mutation some pseudo-wild-type revertants grow more slowly than phage that still have the original AD mutation. We found essentially no effect on deletion frequency due to the exonuclease deficiency. Obviously, it must be borne in mind that these observations are restricted to specific inserts with 10-bp direct repeats at the ends. The exonuclease deficiency also had no effect on deletion of a segment with long palindromic sequences that could bring two 10-bp direct repeats immediately next to one another. Our earlier work with this insert and others like it showed that in phage with a normal D N A polymerase the palindrome had an effect on deletion frequency only when the insert was bracketed by 5-bp direct repeats causing a deletion frequency low enough to make the effects of the palindrome detectable. Thus, with 5-bp direct repeats the palindrome increased deletion frequency by two orders of magnitude (Pierce et al., 1991). But, as confirmed in Table 5, the palindrome has essentially no effect on deletion between 10-bp direct repeats with or without normal levels of the 3' ~ 5' exonuclease. The fact that this same palindrome has a marked effect on deletion between short direct repeats argues that the inverted repeats must cause secondary struc-

ture in the linear T7 genomes in vivo. Thus, the data in Tables 4 and 5 are especially interesting in light of the probability that the palindromes must cause some impediment to replication fork progression. Apparently when 10-bp direct repeats bracket the insert any effects on the replication fork caused by the palindrome do not contribute measurably to increased deletion frequency even under conditions where the mutation in the exonuclease domain leads to a more processive D N A polymerase.

Acknowledgments We thank Drs. Charles Richardson and Stanley Tabor for the gift of the T7 D N A polymerase 3'--, 5' exonuclease mutant. This work was supported by Public Health Service research grant GM-34614.

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