Chemically altered apurinic sites in ΦX174 DNA give increased mutagenesis in SOS-induced E. coli

Mutation Research, 288 (1993) 207-214 (C:;1993 Elsevier Science Publishers B.V. All rights reserved 0027-5107/93/$06.00

207

MUT 05255

Chemically altered apurinic sites in 4~X174 DNA give increased mutagenesis in SOS-induced Eo coli R. Bockrath, YoWo Kow and S.S. Wallace Department of Microbiology and Molecular Genetics, The Markey Centerfor Molecular Genetics, Unicersity of Vermont, Burlington, I/T 0540£ USA (Received 27 October 1992) (Accepted 6 January. 1993)

Keywords: Apurinic DNA damage; SOS mutagenesis; Translesion synthesis; Electroporation

Summary Single-strand DNA from bacteriophage q~X174 am3 is treated with mild acid and heat to produce increasing numbers of apurinic sites per molecule. Samples are assayed, either directly or after additional chemical reactions, by electroporation into the recipient E. coli strain HF4714(su°l +). Modified apurinic sites are produced by reactions with O-methyl- or O-benzyl-hydroxylamine, and reduced apurinic sites by reactions with sodium borohydride. Reversion mutation frequencies are significant only if the recipient strain is SOS-induced (by growth after UV irradiation). A simple apurinic site at the target gives rise to mutation (a transversion) with a probability of 0.07, while the modified or reduced apurinic site has a mutagenic efficiency of 0.22-0.27 or 0.29, respectively. The open ring form of deoxyribose may account for the 3-4-fold increased mutagenicity with altered apurinic lesions. Also considered are effects by temperature and cyclobutane pyrimidine dimers on mutagenicity and the relatively invariant survival curves that obtain regardless of chemical alterations at the apurinic sites a n d / o r SOS induction.

Apurinic sites arise in biological DNA more frequently than several other forms of spontaneous degradation and commonly follow alkylation or certain treatments that affect purines (Lindahl and Nyberg, 1972; Drinkwater et al., 1980; Schaaper et al., 1982a). Individual purines are released by hydrolysis of the glycosylic bond to C-1' of the deoxyribose moiety in the sugarphosphate backbone of DNA. The resulting abaCorrespondence (present address): Dr. R. Bockrath, Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202-5120, USA. Tel. 317/274 2235; Fax 317/274 4090.

sic site has been studied as a model nonocoding but potent mutagenic lesion (Strauss et al., 1982; Boiteux and Laval, 1982; Kunkel, 1984). Using DNA from bacteriophage 4~X174 am3, Loeb and colleagues demonstrated reversion mutation at apurinic sites. The mutation frequency response was dependent on transfection into an SOS-induced host (Schaaper and Loeb, 1981; Schaaper et al., 1982b) and was most often the result of an A-to-T transversion at the middle position of the nonsense codon -T-AssT-G- in the E gene (Schaaper et al., 1983). The base at 587 encodes the third position of a valine codon in the overlapping D gene (Sanger et al., 1978),

208

where substitutions would be ineffective because of 4-fold coding degeneracy for valine. Appreciation of severe DNA lesions like apurinic sites or cyclobutane pyrimidine dimers, by whole cells or specific enzymatic activities, has recently been examined in some detail and found to be more complex than initially assumed. For example, from the perspective of normal hydro° gen bonding a lesion might be presumed "non-instructive". Yet Lawrence and coworkers have compared cis-syn and trans-syn isomers of cyclobutane dimers and found sharp contrasts in mutagenesis and inactivation (Lawrence et aL, 1990a; Banerjee, 1990). Previous in vitro results from this laboratory have shown that alkoxyamine modifications of apurinic sites are recognized with varying efficiencies by the E. coli UvrABC pro= teins (Snowden et al., 1990, includes reduced apurinic sites) and by several AP endonucleases (Wallace et al., 1988; Kow, 1989). Here we show that similar alterations are recognized in vivo with different mutagenic consequences. Using the q~X174 am3 system, single-strand DNA prepared from the mature phage was treated in vitro and then introduced by electroporation into Escherichia coll. We find mutagenesis at simple apurinic sites, as previously shown, and at the altered apurinic sites to be strongly dependent on induced SOS functions. Calculations from the mutation frequency responses show marked increases in transversion at the targeting lesion when the oxygen at the C-I' of the apurinic site is replaced by O-methyl- or O-benzyl=hydroxylamine or is reduced to stabilize the open ring structure. Materials and methods

Strains and growth media. E. coli strains HF4714(su-1 +) and HF4704(su ) were used to indicate plaques of parental 4~X174 am3 and of revertants, respectively (Schaaper and Loeb, 1981). HF4714(su-1 +) also was used as the recipient in transfection by electroporation. All cultures were grown in YT medium (Difco Tryptone, 10 g, and yeast extract, 5 g, and 5 g of NaC1 per 1) at 37°C in gyrating shakers, to stationary phase for indicator and late exponential for recipient. SOC medium contained 2% Bactotryptone, 0.5%

Bacto yeast extract, 10 mM NaC1, 2.5 mM KCI, 10 mM MgCl2, 10 mM MgSO 4 and 20 mM glu~ cose. UV solution contained 10 mM MgSO 4 and 0.85% NaC1. YT agar plates and top-agar con~ tained Difco°Bacto agar at 1.5 and 1.0%, respec° tively.

Transfection by electroporation. Samples of DNA (3 /A) and cells (40 tzl) were mixed and transferred to electroporation cuvettes (0.2 cm)~ After one pulse with a Bio-Rad Gene Pulser and Controller (2500 V, 25 /~FD and 200 ohms) pro° ducing a time constant of 4.6-4.7 msec, 1 ml of iced SOC medium was immediately added and the cuvettes were held on ice (procedures adapted from Bio-Rad manual, Version 2-89). Singleostrand DNA of q~X174 am3 (ca~ 0A mg/ml, see Pagano and Hutchison, 1971) in 30 mM KC1, 10 mM sodium citrate (pH 5.0) (Schaaper and Loeb, 1981) was incubated at 65°C for 0-30 min to produce 0-3 apurinic sites per molecule. The depurination rate at 65°C was estimated to be one-half that in the previous studies (Lindahl and Nyberg, 1972). The aliquots were each dialized against either 5 mM Tris, 1 mM EDTA (pH 7) or 5 mM Hepes, 1 mM EDTA (pH 7) and kept on ice (in a few instances, after a period frozen). For chemical modifications, 3 /xl of 10 mM methoxyamine or O-benzyl-hydroxylamine were added to each 3=/xl DNA sample° After 3 h ambient incubation, the reacted samples were moved to ice and then mixed with samples of prepared cells (final volumes of 46 rather than 43 /xl). Reduced apurinic sites were prepared with larger amounts of each DNA sample in the Hepes buffer, treated with sodium borohydride and again dialized to the Hepes buffer. Sodium borohydride was dissolved in water previously adjusted to about pH 12.3 (0.189 g into 50 ml). This was diluted 10-fold into each DNA sample (final conc. of 10 mM). After 1 h ambient incubation, the samples were transferred to ice, a small amount of 0.1 N HC1 (0.12 volume) was added to each to approximately neutralize the solutions and purge them of hydrogen, and the samples were dialized overnight (ca. 8°C). Cells were grown in YT medium (200 ml in a 1-1 flask) from a 2-ml inoculum of an overnight culture also in YT. When a concentration of

209

2-6 × / 0 s viable cells per ml was reached (about 3 h ir~ct~bation), the cultures typically were divided into two equal portions, transferred to UV buffer by centrifugation and one was exposed to germicidal UV, 80 J / m < Both were returned to YT (saved from the original culture) by centrifugation and incubated for 45 min. Each then was chilled on ice for 30 min and sedimented for subsequent rinses after the recommended BioRad procedure. Nalgene bottles (250 ml) in a GSA head, run at 6000 rpm for 9-10 min, or Corex tubes (30 ml) in an SS-34 head for volumes 10 ml and less, run at 7500 rpm for 7 - 8 min, were used to produce cell pellets. Cells were rinsed between rounds of centrifugation by vigorous mixing: once into an equal volume of cold sterile water, twice into cold sterile 10% glycerol (10 and then 1 ml). The final suspensions, in 10% glycerol (0.2-0.4 ml), were kept on ice until use (0.25-1.5 h). Reuersion assay. Samples from the cuvettes after electroporation were added directly (0.02 or 0.1 ml) or diluted in YT and then added to top-agar (2.5 ml) at 45°C in pour tubes to determine revertant plaques or total plaques, respectively. The smaller amounts were used for rever= sion assay when the titers of total plaque-forming units exceeded 3 - 4 × 107 per ml. Each tube was mixed after adding 0.3 ml of the appropriate indicator host (overnight culture diluted 2-fold in YT and incubated about 2 h) and poured over YT agar. The plates were immediately incubated at 37°C for 110-120 min (distributed as single plates) and then removed for ambient incubation overnight (see Results). Results and discussion

The previous studies of mutagenesis by apurinic damage in single-strand D N A of q)X174 drew most of their conclusions from progeny analyses. The transfected recipient complexes, as spheroplasts, were allowed to produce phage and these were assayed to measure mutation frequencies. We developed an alternative protocol using electroporation to transfect whole cells, which then were assayed directly in conventional fashion on two different indicator hosts. Total plaque-forming units were determined on a sup-

pressor-containing host and revertants of the am3 defect were measured on a host lacking the required suppressor. Typically six DNA samples were assayed representing zero to 2.5 or 3 depurinations per molecule. The recipient cells were prepared for eiectroporation either without SOS induction or with SOS functions induced by growth after exposure to ultraviolet light (see Methods). As shown in the previous studies, in the absence of SOS induction, mutation frequency responses were small or non-existent (see Table 1). Accordingly, all the experiments described below were with SOS-induced recipient cells unless noted otherwise. Mutation frequencies Fig. 1 shows mutation frequency responses with depurinated D N A modified by addition of Omethyl- or O-benzyl-hydroxylamine (panels A and B) compared to responses with D N A containing simple or reduced apurinic sites (panel C). Modified or reduced apurinic sites produced mutation frequency responses at least 3-fold greater than that produced by simple apurinic sites. In our assay, it was essential to incubate the assay plates for about 2 h at 37°C and then at ambient for complete development of the plaques (see Methods). If the plates were incubated continuously at 37°C for plaque formation, the lawn deteriorated greatly because of the large number of suppressor-containing cells and infected complexes added to the indicator lawn. Revertant plaques could not be discerned. If the plates were incubated only at a lower ambient temperature (over night), the plaques were clearly defined (presumably suppressor function was less efficient), but a large number of revertant plaques arose as plate mutants and the mutation frequency response was elevated. This effect could be demonstrated after continuous incubation at 25°C. Plate mutants were indicated in the nonzero intercept, and the slope of the mutation frequency response was greater (Fig. 2). In some instances, the mutation frequency response measured with the direct assay was compared to that found by analysis of progeny. The infected complexes were diluted into YT medium and incubated 75 min at 37°C to produce free

210 TABLE 1

o

MUTAGENIC CENTS ~ Expt.

AP

2/22

6.4 (6.7) i7 (4.0) 5.9

2/28 3/4

(5.3) 7/24 7/24

7.8 12

3/!2

3.6 (6.0)

6/23 3/7 3/20

EFFICiENCIES

(P) GIVEN

MA

5/25 6/15

2.7

5/8

5.7

([o.07]) 1

([o.lo]) [ < 0.05] [0.48] 26 (26) 27 [028] 29 25 (23) 20 [2.5] 22 26 31 [1.8] 16 30 45 25 3O

2.7

7.0

2

0

1

2

0

I

2

Apurinic Sites per Genorne

7/15 Averages

o

rAP

[ < 0.20] (N.D.) [ < o.o8]

5/14 6/20 7/9 7/14 7/15

o

PER-

[ < 0.08]

7.9 57 (4.2)

BA

AS

22

27

Fig. 1. Individual examples of mutation frequency responses to simple and chemically altered apurinic sites. Samples of single strand q)X174 am3 DNA containing 0-2.5 apurinic sites per genome (average of 1 site per 10-rain depurination time) were assayed by transfection. Revertant and total plaque-forming units were determined to give mutation frequencies (see Methods), which were plotted against apurinic damage. The three panels show: (A) modified with O-methylhydroxylamine; (B) modified with O-benzyl-hydroxylamine; and C, reduced with sodium borohydride or simple apurinic (triangles). Lines from the origin were fitted by regression (slope = ~Txy/Xx2), and each slope determined mutation per apurinic site or the mutagenic efficiency P once multiply by 2500 (see text). These and other results appear in Table 1.

[

I

I

2

3

29

a Values for P were calculated from the slopes of mutation frequency responses as indicated in Fig. 1. Entries on the same line are for pairs of tests on a given day to compare SOS-induced and control ([ ]) recipient cells or differently treated DNA samples in the same SOS-induced recipient cells. Values in parentheses were determined from progeny analyses conducted in parallel with the direct assays (see text). Where no revertant plaques appeared, one plaque per plate was assumed at each sample to calculate an upper limit ( < ). Six DNA samples containing 0-2.5 or 3.0 depurinations per genome were used in each assay: AP, simple apurinic; MA, apurinic sites modified with O-methyl-hydroxylamine; BA, modified with O-benzyl-hydroxylamine; or rAP, reduced with sodium borohydride.

O

•-

3

X

°

/

C

2

tl_ e" O

0 phage, which were subsequently assayed for total and revertant plaque-forming units. Results by t h e t w o m e t h o d s w e r e s i m i l a r ( s e e T a b l e 1). The mutagenic efficiency (P) for a depurinat i o n a t r e s i d u e 587 o f t h e q ~ x 1 7 4 g e n o m e m a y b e estimated from a formula equating the mutation frequency to the probability of a genome with one d e p u r i n a t i o n ( g i v e n t h e auerage n u m b e r o f

1

Apurinic Sites per Genome Fig, 2. Mutation frequency response to simple apurinic sites with incubation at 25°C. Points are the averages of 2 Expts. (see Fig. 1 and text) where the revertant plaques were allowed to developed after electroporation by continuous incubation at 25°C (line fitted by regression). The broken line (P - 0.07) indicates the response typical of the same DNA samples and recipient ceils assayed with the initial incubation at 37°C (see Methods).

211

depurinations per genome) divided by the proportion of surviving plaque-forming units and the number of possible sites for a depurination in the gcnome (2500) (Schaaper and Loeb, 1981). The value P then becomes the proportionality constant between the mutation frequency response and the average number of depurinations per genome. Table 1 shows the values for P (given as percent) obtained from several assays, each a measure of the mutation frequency response as shown in Fig. 1. The values vary somewhat from day to day, a result probably of some variation in the prepared cells. The averages of these values, however, indicate a marked difference in the probability of the scored transversion at a simple apurinic site or at chemically altered apurinic sites~ Our measured value of P for simple apurinic sites is somewhat larger than the previous estimate for single strand @X D N A (approximately 1%) possibly because of the difference in cell preparation for transfection. Here the cells were suspended in cold distilled water in preparation for electrophoresis, whereas preparation of spheroplasts as used in the previous transfections required a hypertonic solution. Since osmoregulation in E. coli appears to involve changes in D N A supercoiling and the spectrum of genes expressed (Higgins et al., 1990), differences in metabolism may arise within the differently prepared cells that affect the mutagenesis process when metabolism is restored. In this regard, we found the efficiency of transfection to increase about 5-.fold if the cells were treated with nalidixic acid or novobiocin before preparation for electroporation (50 or 250 / , g / m l , respectively, for 15-45 min), as though inhibited supercoiling better prepared cells for distilled water (the treatments induced SOS functions for apurinic mutagenesis only poorly) (data not shown). Another study of abasic sites, which did not use spheroplasts, found mutagenic efficiencies similar to ours (Lawrence et al., 1990b). The large mutation frequency response produced by reduced apurinic sites could not be anticipated from our earlier in vitro results with UvrABC excision repair (see Introduction). The more complex SOS-dependent mutagenesis process in the cell strongly distinguishes between

reduced and simple apurinic sites. It seems unlikely that the open deoxyribose ring associated with the reduced apurinic site (Kow, 1989) enhances miscoding because there is no residue at this position. Possibly a more pliant polymer structure allows the base immediately after the apurinic site to serve better as template in a transient misalignment to bridge the missing base, which then recovers to avoid a - 1 base deletion in a manner analogous to dislocation mutagenesis (Kunkel, 1990; Kunkel and Alexander, 1986). Since the next base is T at the arn3 codon, this mechanism could produce the required transversion. Data for the mutation spectrum in a known DNA sequence produced by reduced apurinic sites would test this idea. Alternatively, the mechanism inherent to miso incorporation at an apurinic site may benefit by the open ring structure. The shape of the sugarphosphate polymer may be as important as base pairing to facilitate reciprocating translocation of template DNA in a confining replication structure (Freemont et al., 1988; Kong et al., 1992). Hence, the trans-syn cyclobutane dimer, which is moderately acceptable in template DNA even without induced SOS functions (Banerjee et al., 1990), and the ring-opened apurinic site (also marginally mutagenic in control cells, see Table 1) both may function better in mutagenesis because of the conformation of the sugar-phosphate backbone. The mutagenic efficiencies obtained with modified apurinic sites were approximately as great as that with reduced apurinic sites (Table 1). This may reflect influences by the occupying residues (Snowden et al., 1990) or the extent to which the addition of substituted hydroxylamine to C-I' might stabilize an open deoxyribose ring (Kow, 1989).

Sur~ieal of plaque-forming units In the previous studies of single°strand @X174 DNA, plaque-forming units were inactivated by one depurination per genome (Schaaper and Loeb, 1981). Our results are consistent with this. An average of one depurination was produced per 10 rain of depurination time (see Methods) and measurements of several exponential inactivation curves indicated one lethal event in slightly

212 TABLE 2 MEAN LETHAL D E P U R I N A T I O N TIMES (MIN) F O R INACTIVATIONS OF P L A Q U E - F O R M I N G UNITS a f

l~xpt.

AP

~/11 2/12 2/22 2/28

12.2115.7] 11.2 [10.6] !1.5 [10.4] 10.3 [9.8]

3/4

11.7 [10.5]

7/24

11.4 [13.8]

3/12

11.5

6/23 3/7

13.5

3/20

16.1

5/25

6/15

14.8

5/8

1o.5

5/14 6/20

14.2

MA

BA

12.7 14.9 [12.7] 14.3 11.6 13.9 [11.5] 16.8 i3.7 11.5 [15.3] 12.9 11.3 9.7 10,8

7/9 7/14 1/15 Averages

12.4

rAP

14.1

14.0

11.7

~'Straight lines were fitted by regression to logs of the values for plaque-forming units per electrophoresis cuvette (ml) treated with D N A that was depurinated for 0 to 25-30 min (see Methods). The mean lethal times were those required for survival to be reduced by 37% along each fitted line. Zero-time values were roughly 2 6 x 1 0 7 plaque-forming ,jnits per ml (from about 0.3 /xg of DNA) and generally greater with control cells than SOS-induced cells. Open numbers are for assays in SOS-induced cells; those in [ ] are for assays in control cells.

r~ore than 10 min (Table 2). Our results also stlpport the earlier observations that inactivation efficiencies were similar whether the D N A was assayed in cells containing induced SOS functions or in uninduced cells (Schaaper and Loeb, 1981; Laspia and Wallace, 1989). The averages of 6 comparisons with D N A containing simple apurinic sites gave mean lethal times of 11.4 and ll.8 rain, respectively, in the tWO types of recipier~t cell (from Table 2). Measures of surviving plaque-forming units ~vere not as reliable as those for the mutation frequency responses because of variations in transfection efficiency (variations inherent to individual electroporation manipulations and the

concentration of DNA in each sample after dialysis). Since mutation frequency was a ratio of revertant plaques per total plaques, measured after electroporation, it would not be affected by transfection efficiency. Each mutation frequency basically depended on the average number of depurinations per genome, which was a stochastic function of a well-defined depurination period (see Methods). In addition, to maximize tile yield of revertant plaques, the extent of depurination in the D N A samples was limited. This limited the inactivation curves to a range of about 1 log m. Therefore the apparent differences in mean lethal depurination times for simple and altered apurinic sites (Table 2) were not analyzed for statistical significance. An increase in the value P may be expected to produce some decrease in the efficiency of inactivation. If a change in P from 0.07 to 0.27, for example, could be attributed entirely to a increase in the probability for translesion synthesis, then the probability that depurination lethally blocks DNA synthesis would change from 0.93 (1.0=0.07) to 0.73 (1.0=0.27), and the mean lethal depurination time would increase by a factor of 1.27 (0.93/0.73). The data in Table 2 for "AP" and " M A " are approximately consistent with this example. Moreover, an analogous calculation to estimate an increase in the mean lethal depurination time for survival of " A P " D N A in control and SOS induced cells gives 1.08 (1.00/0.93), which is 12% greater than the experimental ratio (11.4/11.8, above) and within reasonable error. However, the data in Table 2 for " A P " and " r A P " are not consistent with this line of analysis. Either the inactivation data simply are not sufficiently precise for conclusions in this regard or other aspects relating to a model must be taken into consideration: (a) P may vary not only by the extent of translesion synthesis but also by the probability of specific error incorporation and (b) inactivation may sometimes result from successful translesion synthesis that produces a lethal mutation. We are currently sequencing mutations derived from altered abasic sites to examine (a).

Mutagenic SOS functions in the recipient cell We did not attempt to improve the protocol for expressing UV-induced SOS functions since

213 previous results suggested that the parameters of fluence and p o s t - U V incubation time used here were satisfactory (Laspia and Wallace, 1989). In one trial, however, cells were exposed to U V light during the first preparative wash with water and were not incubated for 45 min to allow for expression of SOS functions. T h e y were then transo. fected with D N A after completion of the prepao ration for electroporation and assayed. No rever= rant plaques with depurinated D N A were observed (data not shown). Transfection by electro° poration, unlike the use of calcium and heat shock,, seems to both tolerate and require preliminary expression of the SOS functions (cf. Lawrence et al., 1990b). A study of s p o n t a n e o u s mutations arising in an SOS constitutive strain of E. colt attributed a number of mutations to transversions at a particular s p o n t a n e o u s apurinic site, which decreased when the cells assayed were first exposed to U V (Bockrath and Ruiz-Rubio, 1988). Using the @ x i 7 4 am system, we tested the suggestion that

O '~

©

©

X

>" 0

2 Acknowledgements

0

o

This research was s u p p o r t e d by the gracious advice and assistance of Mr. Gustavo F o n d e z and by N.I.H. grants GM21788 and CA2040. O n e of us (R.B.) thanks the others for their kind and stimulating hospitality.

LL

o

cyclobutane dimers in m e g e n o m e can have a negative effect on mutagenesis at apurinic sites. A n excision repair defective derivative of the recipient strain, HF4714uvrA, was used. After exposure to U V and growth to express SOS activities, one-half of the cells was kept as a dark control and the other was exposed to photoreactirating light for 20 min. A f t e r transfection with D N A samples containing r e d u c e d apurinic sites, a greater mutation frequency response was observed in the photoreactivated cells where dimers had been r e m o v e d (Fig. 3). A greater response was also observed with SOS-induced cells (6 J / m 2, 45 rain for expression) than with the same cells exposed to an additional small U V fluence (2 J / m 2) just before p r e p a r a t i o n for electroporation (data not shown). These results," consistent with the earlier re= sults with s p o n t a n e o u s mutations, show explicitly that mutagenesis at an apurinic site is adversely affected by cyclobutane dimers elsewhere in the cell. The simplest explanation is that one or more of the SOS-induced molecules required for mutagenesis at an apurinic site (or the r e d u c e d apurinic site) preferentially binds to a dimer lesion. This could occur before or after the e n c o u n t e r between the replication apparatus and d a m a g e in template D N A .

1

0

I

0

t

1

I

I

2

Apurinic Sites per Genome

Fig. 3. Effect of photoreactivation on SOS=dependent mutagenests at reduced apurinic sites, Recipient cells deficient in excision repair were grown after exposure to UV light for expression of SOS functions (8 J/m 2 and 45 min). One-half were exposed to photoreversing light (10 cm below 4 fluorescent, 15=W lamps, through 1/4 in. of Lexan) sufficient for maximum reversal of UV-inactivated colony-forming ability, and the other half was kept dark. Both were then prepared for electroporation and used in assays of DNA samples containing reduced apurinic sites: open circles, photoreversed; filled circles, dark.

References

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