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The following HTML text is provided to enhance online readability. Many aspects of typography translate only awkwardly to HTML. Please use the page image as the authoritative form to ensure accuracy. ColloquiumRoles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coliPhuong Pham*, Savithri Rangarajan*, Roger Woodgate†, and Myron F.Goodman*‡ *Departments of Biological Sciences and Chemistry, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, CA 90089–1340; and †Section on DNA Replication, Repair and Mutagenesis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892–2725 DNA polymerase V, composed of a heterotrimer of the DNA damage-inducible UmuC and UmuD2 proteins, working in conjunction with RecA, single-stranded DNA (ssDNA)-binding protein (SSB), β sliding clamp, and γ clamp loading complex, are responsible for most SOS lesion-targeted mutations in Escherichia coli, by catalyzing translesion synthesis (TLS). DNA polymerase II, the product of the damage-inducible polB (dinA) gene plays a pivotal role in replication-restart, a process that bypasses DNA damage in an error-free manner. Replication-restart takes place almost immediately after the DNA is damaged (≈2 min post-UV irradiation), whereas TLS occurs after pol V is induced ≈50 min later. We discuss recent data for pol V-catalyzed TLS and pol II-catalyzed replication-restart. Specific roles during TLS for pol V and each of its accessory factors have been recently determined. Although the precise molecular mechanism of pol II-dependent replication-restart remains to be elucidated, it has recently been shown to operate in conjunction with RecFOR and PriA proteins. Two seemingly unconnected questions arising during the early and mid 1970s were to decipher the biochemical basis of SOS mutagenesis in Escherichia coli, often referred to as SOS error-prone repair (1), and to determine a cellular role for E. coli DNA polymerase II. A tentative link between the two was established when it was determined that pol II was induced as part of the LexA-regulon (2). pol II was subsequently shown to be encoded by the DNA damage-inducible polB (dinA) gene (3–5). A ∆polB strain shows no measurable UV sensitivity, and SOS-induced mutagenesis occurs at normal levels (6, 7). However, a ∆polB ∆umuDC double mutant strain is more sensitive to killing by U V light than either of the single mutant strains, implying that the two SOS-induced polymerases might play compensatory roles in vivo (8). The ability of pols II and V to complement each other does not mean that these activities are functionally redundant, and indeed they are not. pol V is able to copy UV-damaged DNA in a process referred to as error-prone translesion synthesis (TLS). TLS generates mutations targeted specifically to DNA template damage sites (9–12). In contrast, pol II copies chromosomal DNA during error-free replication-restart (8). Although both polymerases are induced by DNA damage, they appear to function on widely disparate time frames—pol II-catalyzed replication-restart occurs 2 min post-UV irradiation whereas pol V-catalyzed TLS begins roughly 50 min later (8). In this paper, we discuss current models for the roles of pol V in TLS and pol II in replication-restart. Coping with DNA Damage in E. coliThere are over 40 genes induced on DNA damage in E. coli that have been identified recently by using microarray chip technology (13), of which at least 31 are known to be negatively regulated at the transcriptional level by the LexA protein (14). Many of these genes encode proteins required to repair DNA damage (15). The overriding importance of DNA repair is apparent from the observation that a single pyrimidine dimer is lethal in E. coli strains defective for excision and recombinational repair (16). These experiments were among the first to demonstrate the essential contribution of DNA repair to cell survival. Excision and recombination repair pathways are referred to as “error-free” because they do not result in an increase in mutation rate above spontaneous background levels (1). In contrast to error-free repair, damage-inducible TLS generates a significant mutational load (17). Most TLS depends on the damage inducible UmuD2 and UmuC proteins, which heterotrimerize to form E. coli pol V (UmuD2C; refs. 18–20). By copying lesions that block normal replication fork progression, pol V-induced mutations are primarily targeted directly opposite DNA template damage sites; however, pol V is also responsible for causing untargeted mutations at undamaged template sites (21). An in Vitro Model System for SOS Mutagenesis. Three commonly occurring DNA lesions that have been used as models to study SOS mutagenesis in vivo and in vitro are TT cis-syn cyclobutane dimers, TT (6–4) photoproducts, and abasic (apurinic/ apyrimidinic) moieties. TT dimers and (6–4) photoproducts arise from UV irradiation (1), and abasic sites occur either spontaneously or from the action of DNA glycosylases (22). TT (6–4) photoproducts and abasic moieties are strongly mutagenic (11, 23), but TT dimers are much less so (9). However, each of the lesions presents a strong block to DNA replication in vivo (9, 11, 23) and in in vitro model systems using purified DNA polymerases and polymerase accessory proteins (12). A model biochemical system devised by H. Echols and coworkers (24) has facilitated reconstitution of SOS mutagenesis in vitro (Fig. 1a). The proteins involved in copying blocking template lesions are pol V, RecA, SSB, and β sliding clamp and γ clamp loading complex (18). In accordance with Echols’ original suggestion, we have continued to refer to the group of proteins including pol V, RecA, SSB, and β clamp/γ clamp-loading complex by the term “pol V Mut,” where the designation “Mut” refers to a mutasomal complex (25). Although there is strong biochemical evidence that these proteins mutually interact proximal to a DNA template permitting bypass of the lesion This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA. Abbreviations: TLS, translesion synthesis; p/t, primer-template; ssDNA, single-stranded DNA; SSB, ssDNA-binding protein.
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