Distinct Structural Alterations in Proliferating Cell Nuclear Antigen Block DNA Mismatch Repair

Abstract

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.

PCNA and Msh2-Msh6 Activate an Mlh1-Pms1 Endonuclease Pathway Required for Exo1-Independent Mismatch Repair

“…How these trimerization defects affect Msh2-Msh6 binding is unclear but may result from the fact that the IDCL, which contains part of the PIP-box binding site, starts at the end of βI 1 , which is part of the trimer interface (Figure 1). Consistent with the requirement that these mutant PCNAs must be functional, the PCNA-C81R structure revealed only small changes at the interface (Dieckman et al, 2013), potentially reflecting the ability of PCNA-C81R to trimerize at high concentrations. Furthermore, PCNA-C81R did not show alternative assemblies like another mutant PCNA resulting from a trimer interface mutation, PCNA-E113G (Freudenthal et al, 2009), which has not been tested for its effect on MMR.…”

“…The pol30 mutations identified here could function by directly altering an Mlh1-Pms1 interaction surface on PCNA. In this regard, the structure of PCNA-C22Y revealed only modest and localized changes (Dieckman et al, 2013), arguing against large-scale structural changes. Alternatively, the pol30 mutations could function by a more indirect effect involving PCNA loading or proper association with DNA.…”

The Many Roles of PCNA in Eukaryotic DNA Replication

“…The crystal structures of these mutant proteins showed distinct structural alterations caused by these two substitutions [116]. Cysteine-22 is in a loop adjacent to one of the α-helices lining the central hole of the PCNA ring, and the C22Y mutation causes a significant shifting of several of these α-helices.…”

Dead-End Elimination with a Polarizable Force Field Repacks PCNA Structures

“…Several separation-of-function mutations in PCNA have been identified that inhibit various cellular processes including DNA mismatch repair as well as TLS (38,39). X-ray structures of wild-type PCNA, ubiquitin-modified PCNA, SUMO-modified PCNA, two separation-of-function mutant PCNA proteins that block mismatch repair, and two separation-of-function mutant proteins that block TLS have been determined (40)(41)(42).…”