Monomeric yeast PCNA mutants are defective in interacting with and stimulating the ATPase activity of RFC.
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.
Ionescu
Costin N
CN
Molecular Biology and Biochemistry Department, Wesleyan University, Middletown, Connecticut 06459-0175, USA.
Shea
Kathleen A
KA
Mehra
Rajendra
R
Prundeanu
Lucia
L
McAlear
Michael A
MA
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D008741:Methyl Methanesulfonate / Q000633:toxicity; D008969:Molecular Sequence Data; D017354:Point Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D021122:Protein Subunits; D051818:Replication Protein C; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000528:radiation effects; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; D008741:Methyl Methanesulfonate; D000251:Adenosine Triphosphatases; D051818:Replication Protein C