Monomeric Yeast PCNA Mutants Are Defective in Interacting with and Stimulating the ATPase Activity of RFC

Abstract

Yeast PCNA is a homo-trimeric, ring-shaped DNA polymerase accessory protein that can encircle duplex DNA. The integrity of this multimeric sliding DNA clamp is maintained through the protein-protein interactions at the interfaces of adjacent subunits. To investigate the importance of trimer stability for PCNA function, we introduced single amino acid substitutions at residues (A112T, S135F) that map to opposite ends of the monomeric protein. Recombinant wild-type and mutant PCNAs were purified from E. coli, and they were tested for their properties in vitro. Unlike the stable wild-type PCNA trimers, the mutant PCNA proteins behaved as monomers when diluted to low nanomolar concentrations. In contrast to what has been reported for a monomeric form of the beta clamp in E. coli, the monomeric PCNAs were compromised in their ability to interact with their associated clamp loader, replication factor C (RFC). Similarly, monomeric PCNAs were not effective in stimulating the ATPase activity of RFC. The mutant PCNAs were able to form mixed trimers with wild-type subunits, although these mixed trimers were unstable when loaded onto DNA. They were able to function as weak DNA polymerase delta processivity factors in vitro, and when the monomeric PCNA-41 (A112T, S135F double mutant) allele was introduced as the sole source of PCNA in vivo, the cells were viable and healthy. These pol30-41 mutants were, however, sensitive to UV irradiation and to the DNA damaging agent methylmethane sulfonate, implying that DNA repair pathways have a distinct requirement for stable DNA clamps.

Conformational Analysis of Processivity Clamps in Solution Demonstrates that Tertiary Structure Does Not Correlate with Protein Dynamics

“…Each gene was cloned into plasmid pET11T using the NdeI and BamHI sites (Nguyen et al, 1993), followed by transformation of E. coli strain BL21(DE3). For the overexpression of yeast PCNA and the T. kodakaraensis PCNAs, E. coli Rosetta(DE3) pLysS cells were transformed using plasmids pMM119 (Ionescu et al, 2002) (a generous gift from Dr. Michael McAlear, Wesleyan University), and pET-TK0535 or pETTK0582 (Ladner et al, 2011) (a generous gift from Dr. Zvi Kelmam, Center for Advanced Research in Biotechnology, UMBC), respectively.…”

Arabidopsis thaliana Y‐family DNA polymerase η catalyses translesion synthesis and interacts functionally with PCNA2

“…The inability of AtPolg plus PCNA1 to complement the rad30 phenotype was surprising as PCNA1 and PCNA2 differ at only eight sites and by one residue in length (Figure 6a). None of the eight sites is in the interdomain connecting loop or hydrophobic patch that are likely to interact with Polg (Bruning and Shamoo, 2004;Gulbis et al, 1996;Vidal et al, 2004), or coincides with any of 22 other sites where PCNA mutations confer UV-sensitivity in yeast (Amin and Holm, 1996;Ayyagari et al, 1995;Hoege et al, 2002;Ionescu et al, 2002;Zhang et al, 2006). Furthermore, seven of the residues that differ in AtPCNA1 may not be critical.…”

Proliferating cell nuclear antigen in neutrophil fate

“…In another study, substitution of alanine 112 into threonine and serine 135 into phenylalanine in PCNA also prevents in vitro assembly of the trimer and their interactions with the RFC. Nonetheless, these substitutions are not lethal in yeast but result in an increased sensitivity to UV radiation and other DNA damaging agents …”

“…Nonetheless, these substitutions are not lethal in yeast but result in an increased sensitivity to UV radiation and other DNA damaging agents. 84 Taking advantage of the fact that the PCNAY114A mutant is monomeric, we examined the effect of its overexpression in PLB985 cells compared with wildtype trimeric PCNA. We first observe that the It should be noted that within the nucleus, the PCNA trimer is organized around the DNA molecule (Fig.…”