Human-Saccharomyces cerevisiae proliferating cell nuclear antigen hybrids - Oligomeric structure and functional characterization using in vitro DNA replication

The proliferating cell nuclear antigen (PCNA) is a highly conserved protein required for the assembly of the DNA polymerase delta (pol delta) holoenzy me, Because PCNAs from Saccharomyces cerevisiae and human do not complement each other using in vitro or in vivo assays, hybrids of the two proteins w ould help identify region(s) involved in the assembly of the pol S holoenzy me, Two mutants of human PCNA, HU1 (D21E) and HU3 (D120E), and six hybrids of human and S, cerevisiae PCNA, HC1, HC5, CH2, CH3, CH4, and CH5, were pre pared by swapping corresponding regions between the two proteins, In soluti on, all PCNA assembled into trimers, albeit to different extents, These PCN A variants were tested for stimulation of pol delta and in vitro replicatio n of M13 and SV40 DNA as well as to stimulate the ATPase activity of replic ation factor C (RF-C), Our data suggest that in addition to the interdomain connecting loop and C terminus, an additional site in the N terminus is re quired for pol delta interaction. PCNA mutants and hybrids that stimulated pol delta and RF-C were deficient in M13 and SV40 DNA replication assays, i ndicating that PCNA-induced pol delta stimulation and RF-C-mediated loading are not sufficient for coordinated DNA synthesis at a replication fork.