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

36 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 - Jan 1 2001

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Schizosaccharomyces
Proliferating Cell Nuclear Antigen
DNA-Directed DNA Polymerase
Yeast
Thymus
Baculoviridae
Fractionation
Cell Extracts
Thymus Gland
Assays
Cells
Association reactions
Peptides
DNA

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Cite this

The human homologue of fission yeast cdc27, p66, is a component of active human DNA polymerase δ. / Shikata, Koh; Ohta, Satoshi; Yamada, Kouichi; Obuse, Chikashi; Yoshikawa, Hiroshi; Tsurimoto, Toshiki.

In: Journal of biochemistry, Vol. 129, No. 5, 01.01.2001, p. 699-708.

Research output: Contribution to journalArticle

Shikata, Koh ; Ohta, Satoshi ; Yamada, Kouichi ; Obuse, Chikashi ; Yoshikawa, Hiroshi ; Tsurimoto, Toshiki. / The human homologue of fission yeast cdc27, p66, is a component of active human DNA polymerase δ. In: Journal of biochemistry. 2001 ; Vol. 129, No. 5. pp. 699-708.
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