Proliferating cell nuclear antigen (PCNA), the eukaryotic DNA sliding clamp, forms a ring-shaped homo-trimer that encircles double-stranded DNA. This protein is best known for its ability to confer high processivity to replicative DNA polymerases. However, it does far more than this, because it forms a mobile platform on the DNA that recruits many of the proteins involved in DNA replication, repair, and recombination to replication forks. X-ray crystal structures of PCNA bound to PCNA-binding proteins have provided insights into how PCNA recognizes its binding partners and recruits them to replication forks. More recently, X-ray crystal structures of ubiquitin-modified and SUMO-modified PCNA have provided insights into how these post-translational modifications alter the specificity of PCNA for some of its binding partners. This article focuses on the insights gained from structural studies of PCNA complexes and post-translationally modified PCNA.

Eukaryotic DNA polymerase delta (pol δ) is a member of the B family of polymerases and synthesizes most of the lagging strand during DNA replication. Yeast pol δ is a heterotrimer comprised of three subunits: the catalytic subunit (Pol3) and two accessory subunits (Pol31 and Pol32). Although it is one of the major eukaryotic replicative polymerase, the mechanism by which it incorporates nucleotides is unknown. Here we report both steady state and pre-steady state kinetic studies of the fidelity of pol δ. We found that pol δ incorporates nucleotides with an error frequency of 10 −4 to 10 −5 . Furthermore, we showed that for correct versus incorrect nucleotide incorporation, there are significant differences between both pre-steady state kinetic parameters (apparent K d dNTP and k pol ). Somewhat surprisingly, we found that pol δ synthesizes DNA at a slow rate with a k pol of ~1 s −1 . We suggest that, unlike its prokaryotic counterparts, pol δ requires replication accessory factors like proliferating cell nuclear antigen to achieve rapid rates of nucleotide incorporation.Eukaryotic DNA polymerase δ (pol δ), a member of the B family of DNA polymerases, is responsible for synthesizing the bulk of the lagging strand of genomic DNA during normal replication [1][2][3]. In addition, pol δ participates in nucleotide excision repair, base excision repair, and double strand break repair [4]. It also plays an important role in maintaining genome stability, as mutations in pol δ cause an increased frequency of cancer in mice [5][6][7] and have been found in cell lines derived from human cancers [8,9].Yeast pol δ is a heterotrimer, comprised of three subunits: Pol3 (125 kDa), Pol31 (55 kDa), and Pol32 (40 kDa) [10]. Pol3 is the catalytic subunit and possesses both DNA polymerase and 3′-5′ exonuclease catalytic activities [11,12]. Pol31 is an accessory subunit that is essential for viability [13]. By contrast, Pol32 is an accessory subunit that is not essential for viability; however, cells lacking this subunit show defects in DNA replication and repair [13]. Pol32 also contains the consensus proliferating cell nuclear antigen (PCNA) binding motif, and interactions with PCNA significantly increase the processivity of pol δ [14,15].The structure of the Pol3 catalytic subunit of pol δ bound to both DNA and incoming dNTP substrates has recently been determined [16]. Overall, the structure of the Pol3 subunit resembles that of the bacteriophage RB69 DNA polymerase, another member of the B † The project described was supported by Award Number GM081433 from the National Institute of General Medical Sciences to M.T.W and Award Number CA138546 from the National Cancer Institute to S.P. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences, the National Cancer Institute, or the National Institutes of Health. * To whom correspondence should be addressed: M. Todd Washington, Department of Biochemistry, 4-403 Bowen Scienc...

Translesion synthesis (TLS), the process by which DNA polymerases replicate through DNA lesions, is the source of most DNA damage-induced mutations. Sometimes TLS is carried out by replicative polymerases that have evolved to synthesize DNA on non-damaged templates. Most of the time, however, TLS is carried out by specialized translesion polymerases that have evolved to synthesize DNA on damaged templates. TLS requires the mono-ubiquitylation of the replication accessory factor proliferating cell nuclear antigen (PCNA). PCNA and ubiquitin-modified PCNA (UbPCNA) stimulate TLS by replicative and translesion polymerases. Two mutant forms of PCNA, one with an E113G substitution and one with a G178S substitution, support normal cell growth but inhibit TLS thereby reducing mutagenesis in yeast. A re-examination of the structures of both mutant PCNA proteins revealed substantial disruptions of the subunit interface that forms the PCNA trimer. Both mutant proteins have reduced trimer stability with the G178S substitution causing a more severe defect. The mutant forms of PCNA and UbPCNA do not stimulate TLS of an abasic site by either replicative Pol δ or translesion Pol η. Normal replication by Pol η was also impacted, but normal replication by Pol δ was much less affected. These findings support a model in which reduced trimer stability causes these mutant PCNA proteins to occasionally undergo conformational changes that compromise their ability to stimulate TLS by both replicative and translesion polymerases.

During DNA replication, mismatches and small loops in the DNA resulting from insertions or deletions are repaired by the mismatch repair (MMR) machinery. Proliferating cell nuclear antigen (PCNA) plays an important role in both mismatch-recognition and resynthesis stages of MMR. Previously, two mutant forms of PCNA were identified that cause defects in MMR with little, if any, other defects. The C22Y mutant PCNA protein completely blocks MutSα-dependent MMR, and the C81R mutant PCNA protein partially blocks both MutSα-dependent and MutSβ-dependent MMR. In order to understand the structural and mechanistic basis by which these two amino acid substitutions in PCNA proteins block MMR, we solved the X-ray crystal structures of both mutant proteins and carried out further biochemical studies. We found that these amino acid substitutions lead to subtle, distinct structural changes in PCNA. The C22Y substitution alters the positions of the α-helices lining the central hole of the PCNA ring, whereas the C81R substitution creates a distortion in an extended loop near the PCNA subunit interface. We conclude that the structural integrity of the α-helices lining the central hole and this loop are both necessary to form productive complexes with MutS α and mismatch-containing DNA.

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