Schizosaccharomyces pombe proliferating cell nuclear antigen mutations affect DNA polymerase delta processivity

We introduced nine site-directed mutations into seven conserved fission yeast proliferative cell nuclear antigen (PCNA) residues, Leu(2), Asp(63), Arg(64), Gly(69), Gln(201), Glu(259), and Glu(260), either as single or as double mutants, Both the recombinant wild type and mutant PCNAs were able to form homotrimers in solution and to sustain growth of a null pcna strain (Delta pcna). Wild type Schizosaccharomyces pombe PCNA and PCNA proteins with mutations in Asp(63), Gln(201), Glu(259), Or Glu(260) to Ala were able to stimulate DNA synthetic activity and to enhance the processivity of calf thymus DNA polymerase delta holoenzyme similar to calf thymus PCNA, Mutations of Leu(2) to Val or Arg(64) to Ala, either singly eras a double mutant, yielded PCNA mutant proteins that had reduced capacity in enhancing the processivity of DNA polymerase delta but showed no deficiency in stimulation of the ATPase activity of replication factor C, S, pombe Delta pcna strains sustained by these two mutant-pcna alleles had moderate defects in growth and displayed elongated phenotypes, These cells, however, were not sensitive to UV irradiation, Together, these in vitro and in vivo studies suggest that the side chains of Leu(2) and Arg(64) in one face of the PCNA trimer ring structure are two of the several sites involved in tethering DNA polymerase delta for processive DNA synthesis during DNA replication.