Proc. Natl. Acad. Sci. USA Vol. 93, pp. 1376-1380, February 1996 Biochemistry Reconstitution of repair-gap 1V mutagenesis with purified proteins from Escherichia coli: A role for DNA polymerases III and II (DNA repair/excision repair/error-prone repair/carcinogenesis) GuY TOMER*, ORNA COHEN-FIX*, MICHAEL O'DONNELLt, MYRON GOODMANt, AND Zvi LIVNEH*§ *Department of Biochemistry, Weizmann Institute of Science, Rehovot 76100, Israel; tDepartment of Microbiology, Cornell University Medical College, New York, NY 10021; and tDepartment of Molecular Biology, University of Southern California, Los Angeles, CA 90089 Communicated by I. Robert Lehman, Stanford University Medical Center, Stanford, CA, October 26, 1995 ABSTRACT Using a cell-free system for UV mutagenesis, identified two pathways: type I, or replicative UV mutagenesis, we have previously demonstrated the existence of a mutagenic that depended on DNA replication, and type II, or repair-gap pathway associated with nucleotide-excision repair gaps. UV mutagenesis, that depended on nucleotide excision repair Here, we report that this pathway can be reconstituted by (15, 16). Here, we report the reconstitution and characteriza- using six purified proteins: UvrA, UvrB, UvrC, DNA helicase tion of repair-gap UV mutagenesis using purified components. II, DNA polymerase III core, and DNA ligase. This establishes the minimal requirements for repair-gap UV mutagenesis. DNA polymerase II could replace DNA polymerase III, al- MATERIALS AND METHODS though less effectively, whereas DNA polymerase I, the major Materials. The sources of materials were as follows: unla- repair polymerase, could not. DNA sequence analysis of beled dNTPs and creatine phosphate, Boehringer Mannheim; mutations generated in the in vitro reaction revealed a spec- [a-32P]dNTPs (400 Ci/mmol; 1 Ci = 37 GBq), Amersham; trum typical of mutations targeted to UV lesions. These eosin yellow and methylene blue, Riedel de Haen; and bac- observations suggest that repair-gap UV mutagenesis is per- teriological media, Difco. Plasmid pOC2 is a 5.0-kb pBR322 formed by DNA polymerase III, and to a lesser extent by DNA derivative carrying the cro, kan, and bla genes (15). It was UV polymerase II, by filling-in ofa rare class of excision gaps that irradiated (254 nm) as described (17) at doses of 100, 200, or contain UV lesions. 400 Jm-2 to produce 5, 10, or 20 photodimers per molecule, respectively. UvrA (fraction IV, 0.33 mg/ml), UvrB (fraction DNA repair is the major defense mechanism of cells against V, 0.21 mg/ml), and UvrC (fraction IV, 0.02 mg/ml) were DNA damage and its deleterious effects, primarily cancer (1, purified as described (18, 19). UvrD (1.5 mg/ml) was kindly 2). A major repair mechanism both in prokaryotes and in given to us by R. Bryant (Johns Hopkins University, Balti- eukaryotes is nucleotide excision repair (3). In the bacterium more). DNA polymerase II (Pol II; fraction IV; 0.5 mg/ml) Escherichia coli, this process is initiated by the UvrABC repair was purified as described (20). DNA polymerase III (Pol III) nuclease that incises the DNA at both sides of the lesion, at a core (1 mg/ml) was prepared by reconstitution from the distance of 12 or 13 nucleotides, followed by removal of the individual purified subunits (21). E. coli DNA photolyase damaged oligonucleotide by DNA helicase 11 (4, 5). The (fraction IV, 1.0 mg/ml) was a gift from A. Sancar (University excision gap is filled-in by DNA polymerase I (Pol l), followed of North Carolina, Chapel Hill). Creatine kinase, RNase I, E. by ligation by DNA ligase. This process is error free and leads coli DNA ligase, T4 DNA ligase, and DNA polymerase I (Pol to restoration of the integrity of the genetic information. I) were from Boehringer Mannheim. Unrepaired DNA lesions are frequently processed into Excision Repair. The reaction mixture (25 ,ul) contained 40 mutations by genetically regulated pathways. The classical mM Tris HCl (pH 7.6); 85 mM KCl; 15 mM MgCl2; 1 mM example of this type of active mutagenesis is UV mutagenesis dithiothreitol; 1 mM EDTA; 50 ,ug of bovine serum albumin in E. coli (also termed error-prone repair, or SOS mutagen- per ml; 2 mM ATP; 40 mM creatine phosphate; 0.4 mg of esis), which depends on the UV-inducible genes recA, umuD, creatine kinase per ml; 100 ,uM each of dATP, dCTP, and and umuC that belong to the SOS stress regulon (reviewed in dTTP; 10 AM [a-32P]dGTP; 0.3 ,ug (90 fmol circles) of refs. 6 and 7). Similar mechanisms are found in eukaryotes (1, UV-irradiated plasmid pOC2; 12 nM each of UvrA, UvrB, and 8, 9). UV mutagenesis is believed to occur opposite a UV UvrC; 20 nM DNA helicase II (UvrD); 40 nM DNA polymer- lesion located in a segment of single-stranded DNA (ssDNA). ase; and 1 unit of E. coli or phage T4 DNA ligase. When the Such premutagenic sites can be generated by an interruption bacterial ligase was used, 50 ,uM NAD was added. The reaction of DNA replication at the lesion (7, 10) or by nucleotide components were mixed on ice and then incubated at 37°C for excision repair of closely opposed UV lesions (11, 12), where up to 1 h. The DNA was then deproteinized with proteinase the removal of a UV lesion from one strand results in a ssDNA K and fractionated on a neutral 0.8% agarose gel. The gel was excision gap containing the second UV lesion. The key step in then dried and scanned by using a Fuji BAS 1000 Bioimaging UV mutagenesis is thought to be a translesion DNA synthesis Analyzer. reaction (also termed bypass synthesis), in which an incorrect In Vitro UV Mutagenesis Reaction. The reaction mixture (75 nucleotide is incorporated opposite the lesion (6, 7, 13, 14). ,ul) contained 40 mM Tris HCl (pH 7.6); 85 mM KCl; 15 mM With the goal of elucidating the molecular mechanism of MgCl2; 1 mM dithiothreitol; 1 mM EDTA; 50 ,ug of bovine UV mutagenesis, we have recently established a cell-free assay serum albumin per ml; 2 mM ATP; 40 mM creatine phosphate; system for UV mutagenesis using protein extracts prepared 0.4 mg of creatine kinase per ml; 100 ,tM each of dATP, dCTP, from E. coli cells (15). Using this assay system, we have dGTP, and dTTP; 0.9 ,ug (270 fmol circles) of UV-irradiated plasmid pOC2; 12 nM each of UvrA, UvrB, and UvrC; 20 nM The publication costs of this article were defrayed in part by page charge DNA helicase II (UvrD); 40 nM DNA polymerase; and 1 unit payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 1376 Biochemistry: Tomer et al. Proc. Natl. Acad. Sci. USA 93 (1996) 1377 of E. coli or phage T4 DNA ligase. When the bacterial ligase was used, 50 ,uM NAD was added. The reaction components were mixed on ice and then incubated at 37°C for up to 1 h. The reaction was stopped by adding EDTA and SDS to final concentrations of 20 mM and 1%, respectively. Under these x X | ~~~~~~POlHII conditions deamination of cytosine-containing cyclobutyl 80 core dimers occurs with a half-life of nearly 10 h (22) and thus does not affect the mutagenesis results. Cro- mutations generated 60 during the in vitro reaction were detected as previously de- scribed (15, 16) by transforming an indicator strain which gives rise to dark red colonies when transformed with a Cro- plasmid and white colonies when transformed with a wild-type cro plasmid. A new indicator strain, E. coli WBY11T (22), was 20 used that carried a AumuDCS95::cat mutation (23) in addition to the ArecA mutation. Each experiment was performed three 0 times, and a typical experiment contained 1.5-3 x 105 trans- 0 20 40 60 80 formants. Transformation frequency was calculated by divid- Time, min ing the number of mutants by the total number of transfor- mants. Standard deviations of the mutation frequencies were FIG. 1. Kinetics of in vitro reconstituted repair-gap UV mutagen- esis. The in vitro reactions contained UV-irradiated plasmid pOC2 ± 15-20%. DNA Sequence Analysis of Mutants. In vitro UV mutagen- (400 J.m-2), UvrA, UvrB, UvrC, DNA helicase II, DNA ligase, and Pol III core (closed circles), Pol II (closed triangles), or Pol I (open esis reactions were carried out with UV-irradiated plasmid squares). Cro- mutations were assayed by transforming the indicator pOC2 (400 J m-2), and the reaction products were bioassayed strain with the reaction products, as described in Materials and for Cro- mutations as described above. Plasmid DNA was Methods. extracted from mutant (dark red) colonies obtained from independent transformation reactions by using the Promega ase (Fig. 1). The mutagenic reaction was dependent on the Wizard Miniprep DNA purification system. The sequence of UvrA, UvrB, and UvrC proteins, on DNA polymerase, on the cro gene carried by these plasmids was determined in the UV-irradiation, on ATP, and on Mg2+ (Table 1). A modest Biological Services Unit in our institute by automated DNA mutagenic effect was observed in the absence of DNA helicase sequence analysis using Taq DyeDeoxy Terminator Cycle II (UvrD), suggesting that the DNA polymerases can displace Sequencing in an Applied Biosystems model 373 DNA se- the incised oligonucleotide. DNA ligase was not required (data quencer. not shown); presumably ligation can occur in the cell. The same requirements were observed for reactions with either Pol II or RESULTS Pol III core (Table 1).