Highly mutagenic replication by DNA polymerase V (UmuC) provides a mechanistic basis for SOS untargeted mutagenesis Ayelet Maor-Shoshani, Nina Bacher Reuven, Guy Tomer, and Zvi Livneh* Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel Communicated by Evelyn M. Witkin, Rutgers, The State University of New Jersey, New Brunswick, NJ, November 16, 1999 (received for review September 20, 1999) When challenged by DNA-damaging agents, Escherichia coli cells DNA polymerase termed pol IV, which tends to produce respond by inducing the SOS stress response, which leads to an frameshifts (24). The role of pol IV in E. coli cells is not clear, increase in mutation frequency by two mechanisms: translesion because dinB mutants are proficient both in untargeted and replication, a process that causes mutations because of misinser- targeted SOS mutagenesis (20, 25). tion opposite the lesions, and an inducible mutator activity, which Here we report that pol V (UmuC), in the presence of UmuDЈ, acts at undamaged sites. Here we report that DNA polymerase V RecA, and ssDNA-binding protein (SSB), is highly mutagenic (pol V; UmuC), which previously has been shown to be a lesion- and exhibits a specificity for transversion mutations. These bypass DNA polymerase, was highly mutagenic during in vitro protein requirements and mutagenic specificity suggest that gap-filling replication of a gapped plasmid carrying the cro re- replication of ssDNA regions by pol V, in the presence of porter gene. This reaction required, in addition to pol V, UmuD؅, UmuDЈ, RecA, and SSB, is the mechanistic basis of SOS RecA, and single-stranded DNA (ssDNA)-binding protein. pol V untargeted mutagenesis. produced point mutations at a frequency of 2.1 ؋ 10؊4 per nucleotide (2.1% per cro gene), 41-fold higher than DNA polymer- Materials and Methods ase III holoenzyme. The mutational spectrum of pol V was domi- Materials. The sources of materials used were as follows: nucle- BIOCHEMISTRY nated by transversions (53%), which were formed at a frequency otides and DTT, Boehringer Mannheim; ethidium bromide, .of 1.3 ؋ 10؊4 per nucleotide (1.1% per cro gene), 74-fold higher Sigma; and [␣-32P]dTTP, Amersham than with pol III holoenzyme. The prevalence of transversions and the protein requirements of this system are similar to those of in Proteins. The fusion maltose-binding protein (MBP)-UmuC pro- vivo untargeted mutagenesis (SOS mutator activity). This finding tein and UmuDЈ were purified as described previously (9, 26). suggests that replication by pol V, in the presence of UmuD؅, RecA, pol III holoenzyme, SSB, and RecA were purified according to and ssDNA-binding protein, is the basis of chromosomal SOS published procedures (refs. 27–29, respectively), except that a untargeted mutagenesis. phosphocellulose purification step was added for RecA. DNA polymerase II was a gift from M. Goodman (University of he SOS stress response is induced in Escherichia coli by Southern California, Los Angeles), and the E. coli MutM (Fpg) Tsingle-stranded DNA (ssDNA) gaps formed when DNA protein was a gift from J. Laval (Institute Gustave Roussy, lesions that have escaped repair block replication (1–3). Unable Villejuif, France) and S. Boiteux (Commissariat Energie Atom- to remove the lesion from such gap͞lesion structures, the cells ique, Fontenay Aux Roses, France). Uracil DNA N-glycosylase activate a tolerance response, which involves filling in the DNA was purchased from United States Biochemical; pol I, exonu- gaps without removal of the lesion, thereby restoring genome clease III, BSA, and proteinase K were from Boehringer Mann- continuity. Thereafter, a second attempt to eliminate the lesion heim; S1 nuclease was from Promega, and restriction nuclease by DNA repair can be made. Filling in of the gap is done by AatII, dam methylase, and T4 DNA ligase were from New patching of a homologous DNA segment from the fully repli- England Biolabs. cated sister chromatid via recombinational repair (4, 5) or by translesion replication, which requires the SOS-inducible pro- Gapped Plasmid. Plasmid pOC2 is a pBR322 derivative carrying teins UmuC, UmuDЈ, and RecA (6, 7). The latter process is the cro gene, which was used previously in our laboratory for mutagenic, because of the miscoding promoted by most DNA mutagenesis studies (30–34). Treatment of pOC2 with the lesions. Recently, it was found that UmuC is a DNA polymerase, restriction nuclease AatII in the presence of ethidium bromide termed DNA polymerase V (pol V), with a remarkable capa- (35) produced two populations of plasmid, each nicked in one of bility to replicate through DNA lesions that severely block other the two complementary strands (Fig. 1). Subsequently, exonu- DNA polymerases (8, 9). clease III was added to extend the nicks into gaps. Notice that In addition to this mutagenesis process, which is targeted to the cro region was single-stranded and, therefore, could be DNA lesions, a mutator activity is induced under SOS condi- replicated in only half of the molecules (Fig. 1). This limitation, tions, which produces mutations in the apparent absence of DNA however, did not interfere with the assay, because the unrepli- damage (untargeted mutagenesis) (10–12). Chromosomal un- cated DNA did not add a significant mutagenesis background targeted mutagenesis requires the SOS-inducible proteins RecA, (see below). The nicks were introduced upstream to the cro gene, UmuDЈ, and UmuC (1, 13, 14), the same proteins that are using 0.025 unit͞␮l of the restriction nuclease AatII, in the required for translesion replication. In addition, it exhibits a particular mutational specificity, namely, the selective genera- Abbreviations: ssDNA, single-stranded DNA; SSB, ssDNA-binding protein; pol III and V, DNA tion of transversions (14–17). Another pathway of untargeted polymerases III and V; MBP, maltose-binding protein; EMB, eosin͞methylene blue; MMR, mutagenesis is observed by transfecting UV-irradiated E. coli mismatch repair. ␭ cells with unirradiated phage (18). This phage untargeted *To whom reprint requests should be addressed. E-mail: [email protected]. mutagenesis requires the dinB, uvrA, and polA gene products The publication costs of this article were defrayed in part by page charge payment. This (19–22) and produces frameshift mutations (23). Recently, dinB article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. (a homologue of umuC) was shown to encode an error-prone §1734 solely to indicate this fact. PNAS ͉ January 18, 2000 ͉ vol. 97 ͉ no. 2 ͉ 565–570 Downloaded by guest on October 8, 2021 Fig. 1. Preparation of the gapped plasmid. Plasmid pOC2 (A) was nicked upstream to the cro gene with restriction enzyme AatII in the presence of ethidium bromide. This generated two subpopulations of nicked plasmid (B and C). Addition of exonuclease III extended the nicks into gaps in the 3Ј 3 5Ј direction. Half of the molecules contain the cro gene in the single-stranded region. presence of 0.11 ␮g͞␮l ethidium bromide, and 77 nM plasmid pOC2, at 37°C for 30 min. The DNA was precipitated with ethanol to remove the ethidium bromide, then extracted with Fig. 2. Outline of the cro replication fidelity assay. phenol, and precipitated again. The gap was generated in a reaction mixture containing 30 nM nicked pOC2, 1 unit͞␮l exonuclease III, 66 nM Tris⅐HCl (pH 7.5), 0.66 mM MgCl ,1mM Calculation of Mutation Frequency. The observed mutation fre- 2 quency per gene was calculated by dividing the number of DTT, and 90 mM NaCl. The reaction was carried out at 37°C for Ϫ 20 min to obtain a ssDNA region of approximately 350 nt. The dark-red Cro mutants by the total number of colonies on the size of the gap was deduced from the electrophoretic migration plate. To obtain the actual mutation frequency, two corrections i of the DNA after treatment with nuclease S1, which digested the were made. ( ) The subpopulation of the substrate that contained double-stranded cro transformed the indicator strain and led to single-stranded region in the plasmid. the formation of kanR colonies, but did not contribute a signif- icant number of CroϪ mutants (see Table 1). To compensate for In Vitro Replication Fidelity Assay. The standard gap-filling repli- this, the mutation frequency obtained with each of the DNA cation reaction mixture (50 ␮l) contained 20 mM Tris⅐HCl (pH polymerases was multiplied by 2. To check the accuracy of this 7.5), 8 ␮g͞ml BSA, 5 mM DTT, 0.1 mM EDTA, 4% glycerol, 1 correction factor, we pretreated the gapped plasmid with re- mM ATP, 10 mM MgCl , 0.5 mM each of dATP, dGTP, dTTP, 2 striction nuclease EcoRI, which cuts within cro. Because ssDNA and dCTP, and 1 ␮g (6.2 nM) gapped pOC2. The replication was is resistant to EcoRI, all molecules in which cro is not in the gap carried out with 0.5 ␮M UmuC fusion protein in the presence of ␮ Ј ␮ ␮ are linearized, leaving only gapped circles with cro in the gap. 4.8 M UmuD , 0.6 M SSB, 4.2 M RecA, and 200 units of T4 When this DNA was used as a substrate to determine the DNA ligase. Control reactions were performed with 1.5 nM pol frequencies of pol III holoenzyme and of pol V, we obtained III holoenzyme, 11 nM DNA pol I, or 11 nM DNA pol II. mutation frequencies that were 2-fold higher than with substrate Reactions were carried out at 37°C for 20 min, after which they that was not pretreated. This validates the multiplication of were terminated by heat inactivation at 65°C for 10 min. The mutations frequencies by 2. (ii) Whereas DNA synthesis by pol DNA then was methylated by adding 32 units of dam methylase, ␮ ⅐ III holoenzyme led to essentially quantitative filling in of the 80 M S-adenosylmethionine, 50 mM Tris HCl (pH 7.5), 10 mM single-stranded gap in the plasmid, the amount of DNA synthesis EDTA, and 5 mM 2-mercaptoethanol, in a total volume of 100 ␮ by pol V was 29.4% that of pol III holoenzyme (see Fig.