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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. ColloquiumManaging DNA polymerases: Coordinating DNA replication, DNA repair, and DNA recombinationMark D.Sutton and Graham C.Walker* Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139 Two important and timely questions with respect to DNA replication, DNA recombination, and DNA repair are: (i) what controls which DNA polymerase gains access to a particular primer-terminus, and (ii) what determines whether a DNA polymerase hands off its DNA substrate to either a different DNA polymerase or to a different protein(s) for the completion of the specific biological process? These questions have taken on added importance in light of the fact that the number of known template-dependent DNA polymerases in both eukaryotes and in prokaryotes has grown tremendously in the past two years. Most notably, the current list now includes a completely new family of enzymes that are capable of replicating imperfect DNA templates. This UmuC-DinB-Rad30-Rev1 superfamily of DNA polymerases has members in all three kingdoms of life. Members of this family have recently received a great deal of attention due to the roles they play in translesion DNA synthesis (TLS), the potentially mutagenic replication over DNA lesions that act as potent blocks to continued replication catalyzed by replicative DNA polymerases. Here, we have attempted to summarize our current understanding of the regulation of action of DNA polymerases with respect to their roles in DNA replication, TLS, DNA repair, DNA recombination, and cell cycle progression. In particular, we discuss these issues in the context of the Gram-negative bacterium, Escherichia coli, that contains a DNA polymerase (Pol V) known to participate in most, if not all, of these processes. The boundaries that once separated the fields of DNA replication, recombination, and repair have become increasingly blurred in the last few years. Recent advances in each of these three fields have not only illuminated the molecular mechanisms of the individual processes, but have also provided significant insights into their interrelatedness and codependence. For example, recent studies indicate that the Escherichia coli RecA protein is not only required for homologous recombination, but is also required for efficient chromosomal DNA replication even under normal growth conditions (1, 2), as well as for the regulation of cellular responses to DNA damage and the replication of damaged DNA (3–5). Furthermore, DNA replication by specialized DNA polymerases, such as the umuDC-encoded DNA polymerase V in E. coli (6, 7), underlies the molecular mechanism of translesion DNA synthesis, a major source of mutagenesis in living cells (3, 4, 8). In this report, we have attempted to summarize not only our current understanding of how cells regulate the action of their various DNA polymerases, but also how this regulation may be coordinated with DNA replication, recombination, and repair. Although we discuss these issues as they are currently understood in both eukaryotes and prokaryotes, we pay special attention to how E. coli regulates the actions of its five different DNA polymerases, particularly Pol III and Pol V, because it represents the paradigm for the study of DNA replication, recombination, and repair at both the genetic and biochemical levels. A Superfamily of DNA Polymerases Involved in Replication of Imperfect DNA Templates. Recently, the field of translesion DNA synthesis and induced mutagenesis has generated a great deal of excitement because of the discovery that key gene products required for these processes, in both prokaryotes (9, 10) and in eukaryotes (11, 12), possess an intrinsic DNA polymerase activity (refs. 6, 7, and 13–20 and reviewed in refs. 21–24). A common, defining feature of these DNA polymerases is a remarkable ability to replicate imperfect DNA templates. Depending on the DNA polymerase, these include templates such as those containing a misaligned primer-template junction (13), an abasic site (6, 7), a cyclobutane dimer (15, 16, 25), or a pyrimidine-pyrimidone (6–4) photoproduct (25). These newly discovered DNA polymerases contain highly conserved blocks of amino acid sequences (26) and constitute a new superfamily of novel DNA polymerases termed the UmuC-DinB-Rad30-Rev1 superfamily because the two E. coli members, UmuC and DinB, and the two Saccharomyces cerevisiae members, Rad30p and Rev1p, define its four subfamilies. For brevity, we will refer to it as the UmuC superfamily because UmuC was its founding member (9, 10, 27, 28). Humans have at least four members of this superfamily. These include two members of the RAD30 subfamily, Pol η encoded by the hRAD30A/XP-V gene (16, 29) and Pol ι encoded by the hRAD30B gene (30); Pol κ encoded by the hDINB1 gene (18, 26); and hREV1 gene product (31). Pol η is mutated in individuals having the xeroderma pigmentosum-variant (XP-V) defect (32, 33). The xeroderma pigmentosum (XP) genetic disorder is characterized by an unusually high sensitivity to UV light (UV) that results from an inability to cope properly with U V-induced DNA lesions (reviewed in ref. 34). XP-V is unique in that it is the only one of the eight XP genetic complementation groups that is not deficient in nucleotide excision repair of DNA lesions (3). Biochemical characterization of human Pol η indicates that it is able to bypass cis-syn cyclobutane dimers in a relatively accurate fashion by inserting two adenines opposite the lesion (16, 29, 35). The current model to describe the molecular events underlying the response of XP-V individuals to UV suggests that, in the absence of a functional Pol η, cyclobutane dimers are bypassed by a different polymerase such as the error-prone polymerase Pol ζ (16, 29, 35). The reduced accuracy of translesion DNA synthesis (TLS) over cyclobutane dimers leads to an increased mutation frequency that contributes to the XP-V disorder. What Controls Which DNA Polymerase Acts at a Given Primer Terminus? The discovery of the UmuC superfamily of DNA polymerases, taken together with the recent discovery of additional 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: ssDNA, single-stranded DNA; PCNA, proliferating cell nuclear antigen; XP, xeroderma pigmentosum; XP-V, XP-variant.
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