Direct interaction of proliferating cell nuclear antigen with the p125 catalytic subunit of mammalian DNA polymerase δ

Peng Zhang, Jin Yao Mo, Aymee Perez, Argentina Leon, Li Liu, Nayef Mazloum, Heng Xu, Marietta Y.W.T. Lee

Research output: Contribution to journalArticle

71 Citations (Scopus)

Abstract

The formation of a complex between DNA polymerase δ(pol δ) and its sliding clamp, proliferating cell nuclear antigen (PCNA), is responsible for the maintenance of processive DNA synthesis at the leading strand of the replication fork. In this study, the ability of the p125 catalytic subunit of DNA polymerase δ to engage in protein-protein interactions with PCNA was established by biochemical and genetic methods, p125 and PCNA were shown to co-immunoprecipitate from either calf thymus or HeLa extracts, or when they were ectopically co-expressed in Cos 7 cells. Because pol δ is a multimeric protein, this interaction could be indirect. Thus, rigorous evidence was sought for a direct interaction of the p125 catalytic subunit and PCNA. To do this, the ability of recombinant p125 to interact with PCNA was established by biochemical means, p125 co-expressed with PCNA in Sf9 cells was shown to form a physical complex that can be detected on gel filtration and that can be cross-linked with the bifunctional cross-linking agent Sulfo-EGS (ethylene glycol bis (sulfosuccinimidylsuccinate)). An interaction between p125 and PCNA could also be demonstrated in the yeast two hybrid system. Overlay experiments using biotinylated PCNA showed that the free p125 subunit interacts with PCNA. The PCNA overlay blotting method was also used to demonstrate the binding of synthetic peptides corresponding to the N2 region of pol δ and provides evidence for a site on pol δ that is involved in the protein-protein interactions between PCNA and pol δ. This region contains a sequence that is a potential member of the PCNA binding motif found in other PCNA-binding proteins. These studies provide an unequivocal demonstration that the p125 subunit of pol δ interacts with PCNA.

Original languageEnglish
Pages (from-to)26647-26653
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Issue number38
DOIs
Publication statusPublished - 17 Sep 1999
Externally publishedYes

Fingerprint

Proliferating Cell Nuclear Antigen
DNA-Directed DNA Polymerase
Catalytic Domain
Proteins
Catalytic DNA
Sf9 Cells
Thymus
Two-Hybrid System Techniques
Ethylene Glycol
Clamping devices
Hybrid systems
Yeast
Thymus Gland
Gel Chromatography
Molecular Biology
Carrier Proteins
Demonstrations

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Direct interaction of proliferating cell nuclear antigen with the p125 catalytic subunit of mammalian DNA polymerase δ. / Zhang, Peng; Mo, Jin Yao; Perez, Aymee; Leon, Argentina; Liu, Li; Mazloum, Nayef; Xu, Heng; Lee, Marietta Y.W.T.

In: Journal of Biological Chemistry, Vol. 274, No. 38, 17.09.1999, p. 26647-26653.

Research output: Contribution to journalArticle

Zhang, Peng ; Mo, Jin Yao ; Perez, Aymee ; Leon, Argentina ; Liu, Li ; Mazloum, Nayef ; Xu, Heng ; Lee, Marietta Y.W.T. / Direct interaction of proliferating cell nuclear antigen with the p125 catalytic subunit of mammalian DNA polymerase δ. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 38. pp. 26647-26653.
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