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Activity of the Purified Mutagenesis Proteins Umuc, Umud', and Reca

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Activity of the Purified Mutagenesis Proteins Umuc, Umud', and Reca Proc. Natl. Acad. Sci. USA Vol. 89, pp. 10777-10781, November 1992 Biochemistry Activity of the purified mutagenesis proteins UmuC, UmuD', and RecA in replicative bypass of an abasic DNA lesion by DNA polymerase III (SOS respon/fdelity of DNA repfcatlon/umutatlo) MALINI RAJAGOPALANt, CHI Lut, ROGER WOODGATEtt, MIKE O'DONNELL§, MYRON F. GOODMAN$, AND HARRISON ECHOLSt tDivision of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720; 1Department of Microbiology, Cornell University Medical College, New York, NY 10021; and IDepartment of Biological Science, University of Southern California, Los Angeles, CA 90089 Contributed by Harrison Echols, August 17, 1992 ABSTRACT The introduction of a replication-inhibiting SOS mutagenesis (C. Bonner, S. Creighton and M.F.G., lesion Into the DNA ofEscherichia colU generates the Induced, unpublished work). multigene SOS response. One component of the SOS response Together, the studies noted above give clear indications is a marked increase in mutation rate, de t on RecA that SOS mutagenesis is a consequence ofreplicative bypass protein and the induced muta p us UmuC and of DNA lesions mediated by a damage-localized nucleopro- UmuD. A variety of previous indirect ap es have Indi- tein complex involving RecA, UmuC-UmuD', and pol III-a cated that SOS mutagenesi results from replicative bypass of "mutasome" (14, 17). However, direct evidence for such a the DNA lesion by DNA polymerase (pol ) me in pathway has been lacking in the absence of a defined bio- a reaction mediated by RecA, UmuC, and a prcsd form of chemical system. In the work reported here, we have used UmuD termed UmuD'. To study the bc is of SOS purified proteins to demonstrate replicative bypass of an mutagenesis, we have recostutd replicative bypass with a abasic lesion in a reaction requiring UmuC, UmuD', and defined in vitro system cotg purified proteIns and a DNA RecA. Thus we have concluded that the UmuC-UmuD' substrate with a singe abasc DNA lesion. The replicative complex and RecA act to rescue an otherwise stalled pol III bypass reaction requires po0 m1, UmuC, UmuD', and RecA. holoenzyme at a replication-blocking DNA lesion. The nonprocessed UmuD protein does not replace UmuD' but inhibits the bypass activity of UmuD', perhaps by sequestering MATERIALS AND METHODS UmuD' In a heterodimer. Our experiments demonstrate di- rectly that the UmuC-UmuD' complex and RecA act to rescue Materials. T4 polynucleotide kinase, Fsp I restriction en- an otherwise stalled po1 II holoenzyme at a replication- zyme, DNA ligase, and 4X174 single-stranded DNA (ssDNA) blocking DNA lesion. were obtained from New England Biolabs; ultrapure ATP and deoxynucleoside triphosphates and DNA polymerase I (Kle- A population of Escherichia coli bacteria reacts to a repli- now fragment) were from Pharmacia; [y32P]ATP (3000 Ci/ cation-blocking DNA lesion by inducing the multigene SOS mmol; 1 Ci = 37 GBq) was from Amersham; and polyethylene response (1-4). A remarkable and intensely studied aspect of glycol (PEG) 6000 was from Sigma. Sequenase was obtained the SOS response is an induced mutagenic pathway that from United States Biochemical. The oligonucleotide template depends on the RecA protein and the UmuC and UmuD with a specific abasic site was prepard as described (27). proteins (4-14). SOS mutagenesis is regulated by at least two Other oligonucleotides used were either from Appligene sequential reactions. First, RecA protein is activated by (Pleasanton, CA) or from the Microchemical Facility at the DNA damage to mediate the proteolytic cleavage of the University of California, Berkeley. Purified proteins used in LexA repressor for SOS-controlled genes, including umuC the assays were prepared as described: a (28), as (29), y and umuD (2, 13). In a second RecA-dependent proteolytic complex (30), pol III* (31), DNA polymerase 11 (32), RecA step, UmuD is processed to UmuD', the active agent in (19), UmuC (17), UmuD (16), and UmuD' (17). The , protein mutagenesis (9, 15, 16). Based on direct evidence for a was a gift from Arthur Kornberg (Stanford University). physical interaction, the UmuC-UmuD' complex is pre- DNA Template Construction. The DNA template used in sumed to be the functional unit for mutagenesis (17). Genetic the replicative bypass assays was a 5.4-kilobase (kb) linear experiments have indicated that RecA probably has a third, ssDNA with an abasic lesion located 30 bases from the 5' end. more direct role in SOS mutagenesis, in addition to the two Circular 4X174 ssDNA was linearized at a unique site by regulatory functions (9-12). annealing a 20-mer oligonucleotide to the ssDNA and cutting Based on the available evidence, the pathway for SOS with Fsp I. The 3' end ofthe linear ssDNA was ligated to the mutagenesis has been presumed to involve replication past 60-mer containing the abasic lesion. The ligation step was the lesion by DNA polymerase in an altered low-fidelity made possible by annealing a 35-mer oligonucleotide which mode mediated by UmuC, UmuD', and RecA (14, 17-19). bridged the linear 4X174 DNA and the 60-mer to create a Genetic and physiological experiments have implicated DNA stretch of double-stranded DNA for ligase action (+5X174/ polymerase III (pol III) in the mutagenic pathway (20, 21). 35-mer/60-mer in ratio 1:2:3). The success of the ligation Genetic studies indicate that DNA polymerase I is not step (>95%) was verified by DNA sequencing. required (22, 23). DNA polymerase II exhibits the interesting Replication Assay. Standard replication reaction mixtures property of SOS induction (24-26); however, deletion and (10 Al) contained 20 mM Tris-HCl (pH 7.5), 8 mM MgC12, 5 insertion mutations in the gene for polymerase II do not alter Abbreviations: pol III, DNA polymerase III; ssDNA, single- stranded DNA. The publication costs ofthis article were defrayed in part by page charge *Present address: Section on Viruses and Cellular Biology, National payment. This article must therefore be hereby marked "advertisement" Institutes ofChild Health and Human Development, National Insti- in accordance with 18 U.S.C. §1734 solely to indicate this fact. tutes of Health, Bethesda, MD 20892. 10777 10778 Biochemistry: Rajagopalan et al. Proc. Natl. Acad. Sci. USA 89 (1992) mM dithiothreitol, 0.1 mM EDTA, 25 mM sodium glutamate, AB 1 mM ATP, 4%6 (vol/vol) glycerol, 40 gg of bovine serum 31 5 ' 5.4kb albumin per ml, and 5% (wt/vol) PEG 6000. The PEG was P2- P1*_ required for an effective replicative bypass reaction. The Ssb, RecA, primed DNA substrate (2.5 nM) was preincubated for 5 min UmuC, UmuD' 5 min at 300C with 3.1 ,M Ssb, 100 nM 13 protein, 3.6 nM ycomplex, 50 nM UmuC, 1.3 ,uM UmuD', and 2.5 AM RecA. Reactions Polymerase, 10 mi were then brought back to ice, and respective polymerases d NTPs 10 min were added. The reconstituted pol III complexes were used 3' at 20 nM and made as described (29). When used, pol III* was _ * _~~~~~~~~I 5' at 12-20 nM. No y complex was added to pol III* reactions. Replication was initiated by the addition of deoxynucleoside PRODUCTS SEPARATED ON DENATURING GELS triphosphates (60 AuM each). Replication was carried out for 1. Replication Block * - 85 b 10 min at 370C, followed by quenching with 20 Al of 20 mM EDTA in 95% formamide. The DNA was then denatured by 2. Mlsincorporatlon *I 86 b heating, and the replicated primer products were separated 3. Bypass *- by electrophoresis in 10%6 polyacrylamide gels containing >86 b urea and were visualized by autoradiography of dried gels. FIG. 1. Assay for translesion replication. The assay used a doubly primed, linear ssDNA substrate (5.4 kb) with a single abasic lesion located 65 nucleotides from the 3' end ofthe radioactive primer RESULTS (P1). Replication products were separated on denaturing acrylamide Assay for the RepUcative Bypass of an Abasic Lesion. To gels. Only replication products from the end-labeled primer (P1) can develop a biochemical system to study replicative bypass of be visualized on autoradiograms of dried gels. DNA lesions, we used an assay system that is highly sensitive to limited bypass. In principle, an oligonucleotide with a with a similar DNA template carrying a cyclobutane pyrim- lesion at a specific site fulfills this need (24,27,33). However, idine dimer, we also obtained bypass of the lesion site by pol assembly of pol III holoenzyme on a primer-template DNA III dependent on UmuC, UmuD', and RecA (M.R., C. requires at least 36 nucleotides from the 3' and the 5' end of Lawrence, and H.E., unpublished work); thus our in vitro the primer (M.O., unpublished data). To circumvent this replicative bypass reaction was not limited to abasic sites. problem, we used a 5.4-kb ssDNA substrate with an abasic The experiments in Fig. 2 were all done with a reconstituted site located 30 nucleotides upstream from the 5' end (Fig. 1). pol III holoenzyme, which carried subunits essential for The 5.4-kb linear ssDNA was replicated from two 20-mer processivity but lacked the e exonuclease subunit ("a holo- oligonucleotide primers. Primer 1 (P1), which anneals to the enzyme") (29). We used this enzyme because the exonu- substrate 65 nucleotides upstream from the abasic site, was clease can limit the very low level of bypass in the absence labeled at the 5' end with ['t-32P]ATP. Replication from the of the mutagenesis proteins (our unpublished data). How- unlabeled primer 2 (P2), located 347 nucleotides downstream ever, as shown below, pol III holoenzyme with exonuclease from the 3' end of the template, was included to overcome also exhibited a bypass reaction in the presence of possible technical complications arising from long regions of UmuC, ssDNA.
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