Replication of UV-irradiated single-stranded DNA by DNA polymerase ...

Proc. Nati. Acad. Sci. USA Vol. 83, pp. 4599-4603, July 1986

Biochemistry

Replication of UV-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli: Evidence for bypass of pyrimidine photodimers (SOS response/UV mutagenesis)

Zvi LIVNEH* Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305; and Department of Biochemistry, The Weizmann Institute of Science, Rehovot 76100, Israel

Communicated by I. Robert Lehman, January 6, 1986

ABSTRACT Replication of UV-irradiated circular singlestranded phage M13 DNA by Escherichia coli RNA polymerase (EC 2.7.7.6) and DNA polymerase HI holoenzyme (EC 2.7.7.7) in the presence of single-stranded DNA binding protein yielded full-length as well as partially replicated products. A similar result was obtained with phage G4 DNA primed with E. coli DNA primase, and phage 4X174 DNA primed with a synthetic oligonucleotide. The fraction of full-length DNA was several orders of magnitude higher than predicted if pyrimidine photodimers were to constitute absolute blocks to DNA replication. Recent models have suggested that pyrimidine photodimers are absolute blocks to DNA replication and that SOS-induced proteins are required to allow their bypass. Our results demonstrate that, under in vitro replication conditions, E. coli DNA polymerase m holoenzyme can insert nucleotides opposite pyrimidine dimers to a significant extent, even in the absence of SOS-induced proteins.

MATERIALS AND METHODS DNA Preparations. Ml3mp8 single-stranded (ss) DNA (11) and OX174 ss DNA were prepared as described (12, 13). M13Goril ss DNA was a gift from D. Soltis and G4 ss DNA was a gift from M. Stayton. Enzymes and Proteins. Escherichia coli DNA polymerase III holoenzyme and ss DNA-binding protein (SSB) were purified as described (14-16). Other E. coli replication proteins were gifts from the groups of A. Kornberg and I. R. Lehman as follows: RNA polymerase (EC 2.7.7.6), J. Kaguni; DNA primase, J. Flynn; DNA polymerase I, J. Kelly; DNA ligase (EC 6.5.1.2), I. R. Lehman. Phage T4 endonuclease V (EC 3.1.25.1) was a gift from P. Seawell and A. Ganesan. Bovine serum albumin (Pentex) was purchased from Miles. Biochemicals. Deoxyribonucleoside triphosphates and ribonucleoside triphosphates were obtained from P-L Biochemicals. [a-32P]dTTP (800 Ci/mmol; 1 Ci = 37 GBq) was obtained from Amersham and New England Nuclear.' UV-Irradiated ss DNA. DNA (72 ,ug/ml) in 10 mM Tris*HCl, pH 7.5/1 mM EDTA was spread on Parafilm as droplets (3 ,ul each) and UV-irradiated (254 nm) using a low-pressure mercury germicidal lamp. The dose rate was 1 J-m-sec-' as determined by a Latarjet UV meter. The average number of pyrimidine dimers per DNA molecule was determined by acid hydrolysis of [3H]thymidine-labeled ss DNA as described (17) and was found to be 1.2 x l0o5 pyrimidine dimers per nucleotide per J-m-2 at doses up to 300 J.m-2. Replication of ss DNA (SS -- RF Reaction). The standard reaction mixture included (in 25 ,l): 20 mM Tris HCl at pH 7.5, bovine serum albumin at 80 ,ug/ml, 8 mM dithiothreitol, 4% (vol/vol) glycerol, 8 mM MgCl2, 2 mM ATP, GTP, CTP, and UTP at 0.1 mM each, dATP, dGTP, dCTP, and dTTP at 50 ,uM each, 2-10 ,Ci of [a-32P]dTTP (800 Ci/mmol), 220 pmol (as nucleotide) of unirradiated or UV-irradiated ss DNA, 0.6 ,g of SSB, and 0.3 ,ug of DNA polymerase III holoenzyme (fraction V, 7 x 105 units/mg) (14). For priming at the M13 origin, 0.03 ,ug of RNA polymerase was added; for priming at the G4 origin, 0.1 ,g of DNA primase was added. 4X174 ss DNA was primed with the synthetic oligodeoxynucleotide 5'-ATTGCCCGGCGTACG3' (a gift from M. O'Donnell), complementary to the 4X174 sequence at positions 2794-2808 (18). Replication of the primed 4X174 ss DNA was performed in the absence of ribonucleoside triphosphates. To obtain closed circular duplex DNA, reaction mixtures were supplemented with 0.2 ,g of DNA polymerase I, 0.5 ,ug of E. coli DNA ligase, and 40

Pyrimidine photodimers, the major UV-induced lesions in DNA, are believed to constitute an absolute block to DNA replication, which then resumes at a site distal to the dimer, generating a single-stranded gap within the DNA duplex (refs. 1-3; but see ref. 4). Current models suggest that recA protein binds to the single-stranded region, resulting in its activation as a specific protease, which can then lead to the cleavage of lexA repressor. As a consequence, at least 16 cellular genes are induced, generating a diversity of phenomena including inhibition of cell division, enhanced postreplication repair, and mutagenesis. The process of mutagenesis, which requires the recA (1), umuC, and umuD (5, 6) gene products, is of particular interest. It has been suggested that mutagenesis occurs by error-prone replication through the pyrimidine dimers, possibly by an altered, error-prone, DNA polymerase (1). In vitro studies have suggested that DNA polymerase III (EC 2.7.7.7) is involved in UV mutagenesis (7, 8).'Lackey et al. (9) have isolated an altered DNA polymerase I from SOS-induced cells that shows a high frequency of miscoding in vitro. However, the significance of this activity in vivo is unclear in view of the normal UV-mutability observed with polA mutants (1). In undertaking a biochemical analysis of UV-induced mutagenesis, we began by examining the replication of UV-irradiated circular single-stranded DNAs from phages M13, G4, and 4X174 with purified enzymes isolated from wild-type cells ["SS -* RF reaction" (10)]. We have found that, contrary to expectations, pyrimidine photodimers do not constitute an absolute block to DNA replication and can be bypassed to a significant extent, possibly with formation of mutations, even in the absence of SOS-induced proteins.

Abbreviations: ss DNA, single-stranded DNA; SSB, ss DNAbinding protein; kb, kilobase(s); RF, replicative form. *Present address: Department of Biochemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.

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Biochemistry: Livneh

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Proc. Natl. Acad. Sci. USA 83 (1986)

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FIG. 1. Incorporation of [a-32P]dTTP into acid-insoluble material replication of UV-irradiated M13Goril ss DNA. The reaction mixtures included (in 25 ,1l): 20 mM Tris'HCl at pH 7.5, bovine serum albumin at 80 ,ug/ml, 8 mM dithiothreitol, 4% glycerol, 8 mM MgCl2, 2 mM ATP, GTP, CTP, and UTP at 0.1 mM each, dATP, dGTP, dCTP, and dTTP at 50 ,uM each, 2 ,uCi of [a-32P]dTTP (800 Ci/mmol), 220 pmol (as nucleotide) of unirradiated or UV-irradiated ss DNA, 0.6 ,ug of SSB, 0.1 Mg of DNA primase, and 0.3 ,ug of DNA polymerase III holoenzyme (fraction V, 7 x 101 units/mg). The mixtures were incubated at 32°C for 15 min, after which the reaction was stopped by the addition of 0.1 ml of 0.1 M sodium pyrophosphate, and the DNA was precipitated with 1 ml of 10% trichloroacetic acid containing 0.1 M sodium pyrophosphate for 20 min on ice. Subsequently the mixtures were filtered through Whatman GF/C filters, and the amount of 32P-labeled dTMP incorporated into the DNA was determined by a liquid scintillation counter, using a toluene-based scintillation fluid. o-o, Replication of UV-irradiated M13Goril ss DNA; --, theoretical curve based on a Poisson distribution of an average of X pyrimidine photodimers per DNA molecule. The calculated incorporation, assuming each photodimer is a complete block to replication is 1/(X + 1) (see text for further discussion).

upon

,uM NAD. DNA polymerase I excises the RNA primer and synthesizes a DNA segment instead, thus enabling ligation by DNA ligase. Incubations were for 5-30 min at 32°C. The reactions were stopped by adding EDTA to 20 mM and NaDodSO4 to 0.5%, and the mixtures were kept on ice until assayed. DNA Synthesis. Incorporation of [a-32P]dTTP into trichloroacetic acid-insoluble material was determined as described (19).

Analysis of Products by Agarose Gel Electrophoresis. The incorporation of [a-32P]dTTP into acid-insoluble materials was determined by assaying 5-/l samples from each reaction mixture prior to analysis on agarose gels. All samples that were loaded onto the gels contained similar amounts of incorporated [a-32P]dTTP, 20 mM EDTA, 0.1 Ag of carrier plasmid DNA (plasmid pMOB45), 5% glycerol, 0.02% bromocresol green dye, and 0.2 M NaOH in 1% NaDodSO4. Horizontal 1% agarose gels made with 30 mM NaOH/1 mM EDTA (20) were used for electrophoresis in 30 mM NaOH/1 mM EDTA at 45 V for 16-20 hr. Care was taken to ensure that the running solution formed a contact with the gel edges but did not overflow the gel. This eliminates contamination ofthe gel by unreacted [a-32P]dTTP. For analysis on neutral agarose gels, the NaOH was omitted from the loading mixture. Electrophoresis was performed in 0.8% agarose gels in 90 mM Tris/borate, pH 8.1/2.5 mM EDTA containing ethidium bromide at 0.5 ,g/ml, at 35 V for 14-16 hr. After electrophoresis, the gels were dried and autoradiographed with Kodak XAR-5 x-ray film. The autoradiograms were scanned with a Helena Laboratories (Beaumont, TX) Quick Scan, Jr. densitometer. The scans were quantitated by cutting out the peaks on the tracing paper and weighing them on an analytical balance. T4 Endonuclease V Digestion. The DNA was incubated in a buffer containing 10 mM Tris HCl at pH 8, 10 mM EDTA, 0.1 M NaCl, bovine serum albumin at 0.1 mg/ml, and 2 x 105 units of T4 endonuclease V, at 37°C for 60 min.

RESULTS When UV-irradiated circular ss M13 DNA was replicated with RNA polymerase, DNA polymerase III holoenzyme, and SSB, there was a decrease in nucleotide incorporation as a function of UV dose (Fig. 1). The incorporation was inversely proportional to UV dose, hence, directly proportional to the distance between pyrimidine dimers. It was, however, 2- to 3-fold higher than that predicted from the Poisson distribution, assuming that each dimer represents a complete block to replication (Fig. 1; Table 1). When analyzed by electrophoresis in alkaline agarose gels (Fig. 2) the length distribution of products revealed a large proportion of polynucleotides longer than the average interdimer distances, including a high proportion of fulllength molecules. Similar results were obtained with G4 or M13Goril ss DNA (21) primed with DNA primase, with M13mp8 ss DNA (11) primed with RNA polymerase, and with 4X174 ss DNA primed with a synthetic 15-residue oligodeoxynucleotide. The synthesis of full-length molecules is therefore independent of the source of ss DNA and the mode of priming and is primarily a function of the DNA

Table 1. Product analysis of replicated UV-irradiated M13Goril ss DNA % full-length DNA in products % incorporation Average no. of Ratio Ratio Foundt Found* Expectedt dimers per circle Expectedt 1 100 1 100 100 0 100(100) 7.4 25 (5.0) 3.4 2.4 17 40 5 380 17 (1.7) 0.045 2.7 24 9 10 1i0 11 (0.7) 10-4 6 3.2 19 16.4 with DNA to primase, as in the priming was described 1, using Fig. ss DNA legend replicated UV-irradiated M13Goril and the products were analyzed by gel electrophoresis. *Radiolabeled dTTP incorporated into acid-insoluble material. tCalculated from the Poisson distribution. Expected incorporation = 1/(X + 1); expected full-length = Xe-X, in which X is the average number of dimers per molecule. See text. *The length distribution was obtained by densitometric tracing of the autoradiogram in Fig. 2. The numbers in parentheses give the percentage of molecules that were fully replicated. These values are obtained by dividing the observed fraction of full-length DNA by the average number of photodimers per molecule. UV dose, J-m -2 0 45 90 150


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Proc. Natl. Acad. Sci. USA 83 (1986) RNA polymerase

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replication, the G4 origin on a 2.2-kilobase-pair DNA fragment inserted into the intergenic region (21). Priming at either origin yielded a similar fraction of full-length DNA products after replication by DNA polymerase III holoenzyme, although the distributions of the shorter DNA products were not identical. The differences in distributions are probably due to differences in termination (Fig. 2). When replication mixtures were supplemented with DNA polymerase I (to excise the RNA primer) and DNA ligase (to seal the remaining nick) and the products were analyzed by electrophoresis in neutral agarose gels containing ethidium bromide, a high proportion of supercoiled products was observed. Denaturation of the products with alkali followed by renaturation did not alter the migration of the replicative form (RF) I product, verifying that it is covalently closed circular duplex DNA (Fig. 3). The assumption that a pyrimidine dimer is a complete block to DNA replication implies that any full-length product is the result of replication of that fraction ofthe UV-irradiated DNA molecules lacking dimers. Assuming a random distribution of pyrimidine dimers among DNA molecules, this fraction (no) can be predicted from the Poisson equation, NM = Xe-X, in which X is the average number of dimers per molecule (22). When a number rather than a length distribution is used the equation becomes No = ek The distribution we measure is the length distribution of products. To translate the measured fraction of full-length DNA to the fraction of molecules that were fully replicated, one has to divide the measured values by the average number of photodimers per molecule. For M13Goril ss DNA carrying averages of 5, 10, and 16.4 photodimers per molecule, the measured fractions of full-length products were 25%, 17%, and 11%, respectively (Table 1). These represent 5%, 1.7%, and 0.7% of the molecules, respectively, being fully replicated. Thus the assay amplifies the signal generated by fully replicated molecules and enables their accurate determination. Our results show that the observed fraction of fulllength products was up to several orders of magnitude higher than expected (Table 1). Furthermore, the ratio of found to expected increased with UV dose from a value of 7 at a dose of 45 J m-2, through 400 at 90 J m-2, up to 105 at 150 J m-2. These results suggest strongly that DNA molecules that

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FIG. 2. (A) Autoradiogram of replicated UV-irradiated M13Goril DNA analyzed by alkaline gel electrophoresis. Replication mixtures were as described in the legend to Fig. 1. Mixtures were incubated at 320C for 20 min, after which reactions were stopped by the addition of EDTA to a final concentration of 20 mM and NaDodSO4 to a final concentration of 1%. Samples containing similar amounts of incorporated [a-32P]dTMP, 20 mM EDTA, 0.1 Jug of carrier DNA (plasmid pMOB45), 5% glycerol, 0.02% bromocresol green dye, 0.2 M NaOH, and 1% NaDodSO4 were loaded onto horizontal 1% agarose gels and electrophoresed in 30 mM NaOH/1 mM EDTA at 45 V for 16-20 hr. After electrophoretic separation the gels were dried and autoradiographed. kb, Kilobases. (B) Densitometry trace of the autoradiogram (replication primed with DNA primase). Densitometer sensitivity was adjusted for each lane to yield the largest signal within the linear range of the instrument to allow an accurate determination of the fraction of fully replicated molecules. Thus the peaks of full-length DNAs from different lanes cannot be compared directly. The comparison is made by determining the fraction of full-length DNA among replication products in each lane (see Table 1). Usually different exposures were required to allow scanning of different lanes.

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FIG. 3. Autoradiogram of replicated UV-irradiated M13mp8 ss DNA analyzed by neutral agarose gel electrophoresis. Replication mixtures were as described in the legend to Fig. 1 with 0.03 Ag of RNA polymerase and with the addition of 0.2 ,ug of DNA polymerase I, 0.5 A&g of DNA ligase, and 40 ,uM NAD. The products were analyzed by neutral agarose gel electrophoresis in the presence of ethidium bromide. Each reaction mixture was analyzed also after a denaturation-renaturation treatment, which consisted of mild denaturation by NaOH (pH 12.5) followed by neutralization with 1 M HCL. Under those conditions only covalently closed circular DNA remains in its native form, whereas nicked circular and linear duplex DNAs denature to the single-stranded forms. RF I, supercoiled DNA; RF II, circular nicked duplex DNA; RF III, linear duplex DNA.

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contain pyrimidine dimers have been fully replicated-i.e., pyrimidine dimers have been bypassed. This conclusion relies on the applicability of the Poisson distribution to our experimental situation. Since it assumes a random distribution of pyrimidine dimers, any deviation from randomness-e.g., clustering of pyrimidine dimers-would alter the predicted fraction of dimer-free molecules; however, no clustering of pyrimidine dimers has been observed in UV-irradiated ss M13 DNA (23). Moreover, if pyrimidine dimers were formed preferentially at a limited number of hot spots, rather than distributed equally among all pyrimidinepyrimidine sequences, then the value of no should be calculated from the binomial distribution, which gives values that are even lower than the Poisson distribution (22). To obtain a direct measure of the fraction of pyrimidine dimer-free ss DNA molecules within a population of UVirradiated ss DNA, the following experiment was performed. 32P-labeled M13mp8 and 4X174 ss DNAs were irradiated and treated with T4 endonuclease V, an enzyme that acts specifically on UV-irradiated double-stranded DNA, cleaving the DNA at pyrimidine dimers (24-27). The products were then analyzed in alkaline agarose gels. Since UV-irradiated ss DNA is a poor substrate for T4 endonuclease V (24-27), a large excess of enzyme was required, resulting in some cleavage of the control unirradiated ss DNA. As shown in Fig. 4, extensive degradation of the UV-irradiated ss DNA did occur. The fraction of dimer-free ss DNA as determined by this method was much less than the fraction of full-length products obtained after replication of the UV-irradiated ss DNA (compare Fig. 4A and Fig. 2B). The percentage of full-length molecules at each UV dose is shown in Fig. 4B. At a dose of 45 J-m-2 the results obtained after T4 endonuclease V cleavage were as predicted by the Poisson distribution. At higher doses the experimental values were considerably higher than predicted possibly as a result of incomplete cleavage by the T4 endonuclease V. Nevertheless, it is clear that at all doses tested, the fraction of full-length molecules obtained after replication is much higher than that expected from T4 endonuclease V cleavage.

DISCUSSION There is at present no direct evidence in vivo that pyrimidine dimers constitute absolute blocks to DNA replication in E. coli (4). Studies in vitro (3, 28) have, however, demonstrated that pyrimidine dimers reduce the extent of DNA synthesis and that replication frequently terminates at the site of a dimer (29, 30). Our results confirm these observations; however, our experiments, which were designed to examine all of the products of replication rather than interruptions in synthesis, have shown that DNA polymerase III holoenzyme in the presence of SSB can replicate through pyrimidine dimers. It is noteworthy that T4 DNA polymerase, in the presence of T4 gene 32 protein, is deficient in its ability to replicate through a dimer (unpublished results). The duplex DNA product obtained by replication of UV-irradiated ss DNA was resistant to T4 endonuclease V and to Micrococcus luteus "UV" endonuclease unless a high excess of enzyme was used or the product DNA was denatured prior to digestion (unpublished results). This would indicate that there is some alteration in the structure of the duplex opposite the replicated pyrimidine dimer. A related observation was reported by two groups (31, 32), who found that after replication of UV-irradiated DNA in vivo, in E. coli (31) or in mouse cells infected with the minute virus of mice (32), pyrimidine dimers, while still present in the DNA, become resistant to M. luteus UV endonuclease or T4 endonuclease V, respectively. The in vitro replication products were also insensitive to nuclease S1 (unpublished re-

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FIG. 4. (A) Densitometry trace of UV-irradiated 32P-labeled 4X174 ss DNA digested with T4 endonuclease V and analyzed by alkaline agarose gel electrophoresis. Digestion mixture contained 0.1 jig of unirradiated or UV-irradiated 32P-labeled 4X174 ss DNA, 10 mM Tris HCl at pH 8, 10 mM EDTA, 0.1 M NaCl, bovine serum albumin at 0.1 mg/ml, and 2 x iOs units of T4 endonuclease V. Mixtures were incubated at 37°C for 60 min, after which they were analyzed by alkaline agarose gel electrophoresis as described in Materials and Methods and in the legend to Fig. 2A. (B) Comparison of the fraction of full-length DNA obtained upon replication of UV-irradiated 4X174 ss DNA to the dimer-free portion in UVirradiated ss DNA. o, Theoretical dimer-free portion Nl in UVirradiated 4X174 ss DNA, calculated from the Poisson length distribution No = Xe-X in which X is the average number of pyrimidine dimers per DNA molecule. See text and Table 1 for more details. A, Pyrimidine dimer-free fraction in a population of UVirradiated 4X174 ss DNA, as determined by T4 endonuclease V cleavage. Data obtained from A. e, Fraction of full length obtained after replication of UV-irradiated 4X174 ss DNA.

sults): thus if looped-out structures are formed during the replication they are unlikely to be very long, probably three nucleotides or less (33). The simplest interpretation of the structural alteration

Biochemistry: Livneh opposite the pyrimidine dimer is that a mutation has been generated-e.g., only one nucleotide has been inserted opposite the photodimers. If applicable in vivo, this implies the existence of a branch of UV mutagenesis that is constitutive and independent of SOS-induced functions. Indeed, Kato et al. (34), investigating forward mutation in the cI gene of phage X, found that 10-15% of the mixed-burst mutations scored were recA independent. Recently, Tessman (35) has demonstrated umuC- and recA-independent mutagenesis in UV-irradiated phage S13. It has recently been suggested that pyrimidine-pyrimidine (4-6) photoproducts may be involved in UV mutagenesis in addition to pyrimidine dimers (36). In UV-irradiated doublestranded DNA, pyrimidine adducts amount to approximately 10% ofthe number ofpyrimidine dimers; however, in ss DNA this value is reduced to 2% (37). Thus the substrates used in our experiments contained between 0.1 and 0.3 adduct per molecule, a number too small to allow measurement of bypass. Interestingly, the large fragment of DNA polymerase I has recently been shown to catalyze polymerization opposite pyrimidine dimers (38). However, the bypass required Mn2+ and a high concentration of deoxynucleoside triphosphates. Recent studies reveal that DNA polymerase I can bypass DNA lesions such as psoralen monoadducts (39), apurinic sites (40, 41), and guanine-8-aminofluorene adducts (42). Thus it would appear that even bulky modifications of bases in DNA are not complete blocks to the major repair polymerase in E. coli, DNA polymerase I. The same may be true for the major replicative polymerase in E. coli, DNA polymerase III holoenzyme, as shown in this paper for the case of pyrimidine photodimers. Our findings suggest that when DNA polymerase III holoenzyme encounters a pyrimidine photodimer, it pauses, and may then take one of two possible pathways: The major route is termination, which produces a persistent singlestranded region downstream from the pyrimidine dimer. This, as the current model suggests, would activate recA protein and generate the SOS response (43). The minor pathway is bypass, in which replication proceeds through the pyrimidine photodimer. We estimate that the probability of bypass of a pyrimidine dimers is at least 30% (unpublished results). The connection between targeted SOS-induced UV mutagenesis and the constitutive bypass of pyrimidine dimers is presently unclear; however, our findings do raise the possibility that proteins induced by the SOS response may alter the efficiency and/or the specificity of the replication through pyrimidine dimers by DNA polymerase III holoenzyme, thereby leading to increased levels of mutagenesis. Alternatively, a completely different mechanism for SOS-induced UV mutagenesis may exist. I am grateful to I. R. Lehman for his interest and support and for his constructive criticism of the manuscript. I am grateful to A. Kornberg and his coworkers, and in particular to M. O'Donnell, J. Kaguni, and N. Dixon, for gifts of enzymes and many stimulating discussions. Z.L. is a special fellow of the Leukemia Society of America. This study was supported in part by Grant 1743 from the Council for Tobacco Research, USA, Inc. 1. Witkin, E. M. (1976) Bacteriol. Rev. 40, 869-907. 2. Caillet-Fauquet, P., Defais, M. & Radman, M. (1977) J. Mol. Biol. 117, 95-112. 3. Villani, G., Boiteux, S. & Radman, M. (1978) Proc. Natl. Acad. Sci. USA 75, 3037-3041. 4. Lawrence, C. W. (1981) Mol. Gen. Genet. 182, 511-513.


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5. Kato, T. & Shinoura, Y. (1977) Mol. Gen. Genet. 156, 121-131. 6. Elledge, S. J. & Walker, G. C. (1983) J. Mol. Biol. 164, 175-192. 7. Bridges, B. A., Motershead, R. P. & Sedgewick, S. G. (1976) Mol. Gen. Genet. 144, 53-58. 8. Brotcorne-Lannoye, A., Maenhaut-Michel, G. & Radman, M. (1985) Mol. Gen. Genet. 199, 64-69. 9. Lackey, D., Krauss, S. W. & Linn, S. (1985) J. Biol. Chem. 260, 3178-3184. 10. Meyer, R. R., Shlomai, J., Kobori, J., Bates, D. L., Rowen, L., McMaken, T., Ueda, K. & Kornberg, A. (1979) Cold Spring Harbor Symp. Quant. Biol. 43, 289-293. 11. Messing, J. & Vieira, J. (1982) Gene 19, 269-276. 12. Van Den Hondel, C. A., Weijers, A., Konings, R. N. H. & Schoemakers, J. G. G. (1975) Eur. J. Biochem. 53, 559-567. 13. Razin, A., Sedat, J. W. & Sinsheimer, R. L. (1970) J. Mol. Biol. 53, 251-259. 14. McHenry, C. & Kornberg, A. (1977) J. Biol. Chem. 252, 6478-6485. 15. Chase, J. W., Whittier, R. F., Auerbach, J., Sancar, A. & Rupp, W. D. (1980) Nucleic Acids Res. 8, 3215-3227. 16. Weiner, J. H., Bertsch, L. L. & Kornberg, A. (1975) J. Biol. Chem. 250, 1972-1980. 17. Setlow, R. B. & Carrier, W. L. (1966) J. Mol. Biol. 17, 237-254. 18. Sanger, F., Coulson, A. R., Friedmann, T., Air, G. M. & Smith, M. (1978) J. Mol. Biol. 125, 225-246. 19. Livneh, Z. & Lehman, I. R. (1982) Proc. Natl. Acad. Sci. USA 79, 3171-3175. 20. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 171-172. 21. Kaguni, J. & Ray, D. S. (1979) J. Mol. Biol. 135, 863-878. 22. Selby, S. M., ed. (1970) CRC Handbook for Mathematics (Chemical Rubber, Cleveland, OH), 4th Ed., pp. 952-957. 23. Brunk, C. (1975) in Molecular Mechanisms for Repair of DNA, eds. Hanawalt, P. & Setlow, R. B. (Plenum, New York), pp. 61-65. 24. Minton, K., Durphy, M., Taylor, R. & Friedberg, E. C. (1975) J. Biol. Chem. 250, 2823-2829. 25. Nakabeppu, Y., Yamashita, K. & Sekiguchi, M. (1982) J. Biol. Chem. 257, 2556-2562. 26. Seawell, P. C., Simon, T. J. & Ganesan, A. K. (1980) Biochemistry 19, 1685-1691. 27. Gordon, L. K. & Haseltine, W. A. (1980) J. Biol. Chem. 255, 12047-12050. 28. Poddar, R. K. & Sinsheimer, R. L. (1971) Biophys. J. 11, 355-369. 29. Moore, P. & Strauss, B. (1979) Nature (London) 278, 664-666. 30. Moore, P. D., Bose, K. K., Rabkin, S. D. & Strauss, B. S. (1981) Proc. Natl. Acad. Sci. USA 78, 110-114. 31. Sedliakova, M., Brozmanova, J., Masek, F. & Kleibel, K. (1981) Biophys. J. 36, 429-441. 32. Vos, J. M. & Rommelaere, J. (1982) Biochimie 64, 839-844. 33. Dodgson, J. B. & Wells, R. D. (1977) Biochemistry 16, 2374-2379. 34. Kato, T., Rothman, R. H. & Clark, A. J. (1977) Genetics 87, 1-18. 35. Tessman, I. (1985) Proc. Natl. Acad. Sci. USA 82, 6614-6618. 36. Brash, D. E. & Haseltine, W. A. (1982) Nature (London) 298, 189-192. 37. Patrick, M. H. (1977) Photochem. Photobiol. 25, 357-372. 38. Rabkin, S. D., Moore, P. D. & Strauss, B. S. (1983) Proc. Natl. Acad. Sci. USA 80, 1541-1545. 39. Piette, J. G. & Hearst, J. E. (1983) Proc. Natl. Acad. Sci. USA 80, 5540-5544. 40. Kunkel, T. A., Shearman, C. W. & Loeb, L. A. (1981) Nature (London) 291, 349-351. 41. Sagher, D. & Strauss, B. (1983) Biochemistry 22, 4518-4526. 42. O'Connor, D. & Stohrer, G. (1985) Proc. Natl. Acad. Sci. USA 82, 2325-2329. 43. Little, J. W. & Mount, D. W. (1982) Cell 29, 11-22.