DESTABILIZED PCNA TRIMERS SUPPRESS DEFECTIVE RFC1 PROTEINS IN-VIVO AND IN-VITRO

Replication factor C (RFC) and the proliferating cell nuclear antigen (PCNA) are two essential DNA polymerase accessory proteins that are re quired for numerous aspects of DNA metabolism including DNA replicatio n, DNA repair, and telomere metabolism, PCNA is a homotrimeric ring-sh aped sliding DNA clamp that can facilitate DNA replication by tetherin g DNA polymerase delta or DNA polymerase epsilon to the DNA template. RFC is the 5-subunit multiprotein complex that loads PCNA onto DNA at primer-template junctions in an ATP-dependent reaction. All five of th e RFC subunits share a set of related sequences (RFC boxes) that inclu de nucleotide-binding consensus sequences. We report here that a mutat ion in the gene encoding the large subunit of yeast RFC gives rise to DNA metabolism defects that can be observed in vivo and in vitro. The rfc1-1 substitution (D513N) lies within the widely conserved RFC box V III consensus sequence and results in phenotypes including DNA replica tion defects, increased sensitivity to DNA damaging agents, and elonga ted telomeres. Mutant Rfc1-1 complexes exhibit in vitro DNA replicatio n defects that are sensitive to ATP concentrations, and these defects can be suppressed by mutant PCNA proteins which contain substitutions that destabilize the homotrimeric sliding DNA clamp.