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

• Published in: Proceedings of the National Academy of Sciences - PNAS 1999-08, Vol.96 (17), p.9515-9520
• Main Authors: Kelman, Z, Zuo, S, Arroyo, M.P, Wang, T.S.F, Hurwitz, J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Amino acids; Biochemistry; biological resistance; Biological Sciences; Cell growth; Cells; City states; damage; Deoxyribonucleic acid; DNA; dna modification; DNA Replication; DNA, Fungal - biosynthesis; DNA-binding proteins; DNA-directed DNA polymerase; DNA-Directed DNA Polymerase - metabolism; enzyme activity; Genetic mutation; Genetic vectors; Models, Molecular; Molecular biology; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutant proteins; mutants; phenotype; proliferating cell nuclear antigen; Proliferating Cell Nuclear Antigen - chemistry; Proliferating Cell Nuclear Antigen - genetics; Proliferating Cell Nuclear Antigen - radiation effects; Proteins; Schizosaccharomyces - chemistry; Schizosaccharomyces pombe; Structure-Activity Relationship; ultraviolet radiation; Ultraviolet Rays; Yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.96.17.9515

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 of Schizosaccharomyces 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 proteins, A251V and K253E, exhibited cold-sensitive phenotypes. Several yeast strains harboring mutations, including those at the acidic C-terminal region, showed elevated sensitivity to DNA damage. The ability of the mutant PCNA proteins to stimulate DNA synthesis by Pol(delta) and Pol(epsilon) also was studied 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) activities 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(epsilon) and for cell survival after DNA damage. The C terminus of sliding clamps from other organisms has been shown to be important for clamp loading as well as polymerase interactions. The relationship between the conserved sequence in this region in different organisms is discussed.