Lynne M. Dieckman

PCNA structure and function: insights from structures of PCNA complexes and post-translationally modified PCNA.

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.

Pre-Steady State Kinetic Studies of the Fidelity of Nucleotide Incorporation by Yeast DNA Polymerase δ

Eukaryotic DNA polymerase delta (pol delta) is a member of the B family of polymerases and synthesizes most of the lagging strand during DNA replication. Yeast pol delta is a heterotrimer comprised of three subunits: the catalytic subunit (Pol3) and two accessory subunits (Pol31 and Pol32). Although pol delta 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 delta. We found that pol delta 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 delta synthesizes DNA at a slow rate with a k(pol) of approximately 1 s(-1). We suggest that, unlike its prokaryotic counterparts, pol delta requires replication accessory factors like proliferating cell nuclear antigen to achieve rapid rates of nucleotide incorporation.

PCNA trimer instability inhibits translesion synthesis by DNA polymerase η and by DNA polymerase δ.

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.

Eukaryotic Y-Family Polymerases: A Biochemical and Structural Perspective

Classical DNA polymerases, which replicate DNA rapidly and with high fidelity, stall upon encountering DNA damage. Thus nonclassical polymerases, which have evolved to accommodate DNA damage, are necessary to overcome these replication blocks. These nonclassical polymerases mainly belong to the Y-family and replicate DNA slower and with lower fidelity than their classical counterparts. Y-family polymerases employ surprising strategies to incorporate nucleotides opposite DNA damage. These include the use of larger and less constrained active sites, the use of Hoogsteen base pairing, and the use of amino acid side chains as templates. Y-family polymerases also engage in protein–protein interactions that are important for their recruitment to stalled replication forks and the coordination of their activities on the DNA. These polymerases function within a dynamic network of protein–protein interactions that are mediated by intrinsically disordered regions of these enzymes. This review focuses on the biochemical and structural studies of the Y-family polymerases, which have provided clear insights into their function.

Distinct structural alterations in proliferating cell nuclear antigen block DNA mismatch repair.

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 MutSα and mismatch-containing DNA.

Identification of New Mutations at the PCNA Subunit Interface that Block Translesion Synthesis.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication and repair by interacting with a large number of proteins involved in these processes. Two amino acid substitutions in PCNA, both located at the subunit interface, have previously been shown to block translesion synthesis (TLS), a pathway for bypassing DNA damage during replication. To better understand the role of the subunit interface in TLS, we used random mutagenesis to generate a set of 33 PCNA mutants with substitutions at the subunit interface. We assayed the full set of mutants for viability and sensitivity to ultraviolet (UV) radiation. We then selected a subset of 17 mutants and measured their rates of cell growth, spontaneous mutagenesis, and UV-induced mutagenesis. All except three of these 17 mutants were partially or completely defective in induced mutagenesis, which indicates a partial or complete loss of TLS. These results demonstrate that the integrity of the subunit interface of PCNA is essential for efficient TLS and that even conservative substitutions have the potential to disrupt this process.

Salmonella enterica serovar Typhimurium has three transketolase enzymes contributing to the pentose phosphate pathway

The genus Salmonella is responsible for many illnesses in humans and other vertebrate animals. We report here that Salmonella enterica serovar Typhimurium harbors three transketolases that support the non-oxidative branch of the pentose phosphate pathway. BLAST analysis identified two genes, STM14_2885 and STM14_2886, that together encode a putative transketolase (TktC) with 46–47% similarity to the known TktA and TktB isoforms. Assessing the mRNA and protein expression for each of the three transketolases, we determined that all are expressed in WT cells and regulated to varying extents by the alternative sigma factor RpoS. Enzyme assays with lysates from WT and transketolase-knockout strains established that TktA is responsible for >88% of the transketolase activity in WT cells. We purified recombinant forms of each isoenzyme to assess the kinetics for canonical transketolase reactions. TktA and TktB had comparable values for Vmax (539–1362 μm NADH consumed/s), Km (80–739 μm), and catalytic efficiency (1.02 × 108-1.06 × 109 m−1/s) for each substrate tested. The recombinant form of TktC had lower Km values (23–120 μm), whereas the Vmax (7.8–16 μm NADH consumed/s) and catalytic efficiency (5.58 × 106 to 6.07 × 108 m−1/s) were 10–100-fold lower. Using a murine model of Salmonella infection, we showed that a strain lacking all three transketolases is avirulent in C57BL/6 mice. These data provide evidence that S. Typhimurium possesses three transketolases that contribute to pathogenesis.