Crystal structures of PCNA mutant proteins defective in gene silencing suggest a novel interaction site on the front face of the PCNA ring

Abstract

Proliferating cell nuclear antigen (PCNA), a homotrimeric protein, is the eukaryotic sliding clamp that functions as a processivity factor for polymerases during DNA replication. Chromatin association factor 1 (CAF-1) is a heterotrimeric histone chaperone protein that is required for coupling chromatin assembly with DNA replication in eukaryotes. CAF-1 association with replicating DNA, and the targeting of newly synthesized histones to sites of DNA replication and repair requires its interaction with PCNA. Genetic studies have identified three mutant forms of PCNA in yeast that cause defects in gene silencing and exhibit altered association of CAF-1 to chromatin in vivo, as well as inhibit binding to CAF-1 in vitro. Three of these mutant forms of PCNA, encoded by the pol30-6, pol30-8, and the pol30-79 alleles, direct the synthesis of PCNA proteins with the amino acid substitutions D41A/D42A, R61A/D63A, and L126A/I128A, respectively. Interestingly, these double alanine substitutions are located far away from each other within the PCNA protein. To understand the structural basis of the interaction between PCNA and CAF-1 and how disruption of this interaction leads to reduced gene silencing, we determined the X-ray crystal structures of each of these mutant PCNA proteins. All three of the substitutions caused disruptions of a surface cavity on the front face of the PCNA ring, which is formed in part by three loops comprised of residues 21–24, 41–44, and 118–134. We suggest that this cavity is a novel binding pocket required for the interaction between PCNA and CAF-1, and that this region in PCNA also represents a potential binding site for other PCNA-binding proteins.

PCNA accelerates the nucleotide incorporation rate by DNA polymerase δ

“…Pcna-6 shows a minor defect in cell growth, but a high sensitivity to hydroxyurea (24). The crystal structures of PCNA (44) and the pcna-6 mutant (45) are shown in Figure 4B. The main distinguishing characteristics are decreased hydrophilicity on the surface side that binds Pol δ (23,46–48).…”

The replisome guides nucleosome assembly during DNA replication

“…It has been proposed that this weak binding to PCNA by CAF-1 may promote nucleosome assembly without disrupting normal DNA replication events [106]. The results from a structural analysis of PCNA mutant proteins with defective gene silencing showed that the mutation sites were all located in a cavity at a distance from canonical binding sites, indicating a second binding mode for the CAF-1-PCNA interaction [107]. PCNA can recruit CAF-1, while CAF-1 itself shows DNA-binding activity.…”

Mutations in the PCNA DNA Polymerase Clamp ofSaccharomyces cerevisiaeReveal Complexities of the Cell Cycle and Ploidy on Heterochromatin Assembly

“…Additionally, many DNA repair factors contain PCNA-Interacting Protein (PIP) or PIP-like motifs (reviewed in Boehm and Washington (2016)). The pol30-6 and pol30-79 mutations disrupt the cleft of PCNA that bind PIP motifs (Kondratick et al 2018), which could prevent the recruitment of DNA repair factors to sites of DNA damage. These results could explain the high levels of DNA damage and mitotic recombination in pol30-6 and pol30-79.…”