The C-terminal region of Schizosaccaromyces pombe proliferating cell nuclear antigen is essential for DNA polymerase activity

Proliferating cell nuclear antigen (PCNA), the processivity factor (sliding clamp) of DNA polymerases (Pols), plays essential roles in DNA metabolism, In this report, we examined the functional role of the C-terminal region o f Schizosaccaromyces pombe PCNA both in vitro and in vivo. The deletion or Ala substitution of the last 9 aa (252-260A), as well as Ala replacement of only 4 aa (252-255A) at the C terminus, failed to substitute for the wild- type PCNA protein for cell growth in S, pombe, Two other PCNA mutant protei ns, A251V and K253E, exhibited cold-sensitive phenotypes. Several yeast str ains harboring mutations, including those at the acidic C-terminal region, showed elevated sensitivity to DNA damage. The ability of the mutant PCNA p roteins to stimulate DNA synthesis by Pol delta and Pol epsilon also was st udied in vitro, The mutant proteins that did not support cell growth and a mutant protein containing a single amino acid substitution at position 252, where Pro is replaced by Ala, stimulated Pol delta and Pol epsilon activit ies poorly, All mutant PCNA proteins, however, were assembled around DNA by the clamp loader, replication factor C, efficiently. Thus, the C-terminal region of PCNA is important for interactions with both Pol delta and Pol ep silon and for cell survival after DNA damage. The C terminus of sliding cla mps from other organisms has been shown to be important for clamp loading a s well as polymerase interactions. The relationship between the conserved s equence in this region in different organisms is discussed.