The human homologue of fission yeast cdc27, p66, is a component of active human DNA polymerase δ

Koh Shikata, Satoshi Ohta, Kouichi Yamada, Chikashi Obuse, Hiroshi Yoshikawa, Toshiki Tsurimoto

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

An essential eukaryotic DNA polymerase, DNA polymerase δ (pol δ), synthesizes DNA processively in the presence of proliferating cell nuclear antigen (PCNA). Recently, a 66 kDa polypeptide (p66) that displays significant homology within its PCNA binding domain to that of fission yeast cdc27 was identified as a component of mouse and calf thymus poi δ. Our studies show that p66 interacts tightly with other subunits of pol δ during size fractionation of human cell extracts, and co-immunoprecipitates with these subunits along with PCNA-dependent polymerase activity. Active human poi δ could be reconstituted by co-expressing p125, p50, and p66 recombinant baculoviruses, but not by co-expressing p125 and p50 alone. Interaction studies demonstrated that p66 stabilizes the association between p125 and p50. Pull-down assays with PCNA-linked beads demonstrated that p66 increases the overall affinity of poi δ for PCNA. These results indicate that p66 is a functionally important subunit of human poi δ that stabilizes the poi δ complex and increases the affinity of poi δ for PCNA.

Original languageEnglish
Pages (from-to)699-708
Number of pages10
JournalJournal of biochemistry
Volume129
Issue number5
DOIs
Publication statusPublished - May 2001
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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