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 of 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 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 Poldelta and Polepsilon 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 Poldelta and Polepsilon 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 Poldelta and Polepsilon 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.
Kelman
Z
Z
Department of Molecular Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue/Box 97, New York, NY 10021, USA.
Zuo
S
S
Arroyo
M P
MP
Wang
T S
TS
Hurwitz
J
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D012568:Schizosaccharomyces / Q000737:chemistry*; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays
D004271:DNA, Fungal; D018809:Proliferating Cell Nuclear Antigen; D004259:DNA-Directed DNA Polymerase