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p21Cip1/Waf1 disrupts the recruitment of human Fen1 by proliferating-cell nuclear antigen into the DNA replication

U Chen1, S Chen, P Saha

  • 1Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

Proceedings of the National Academy of Sciences of the United States of America
|October 15, 1996
PubMed

Insights

Human proliferating-cell nuclear antigen (PCNA) binds to Fen1, a key DNA replication enzyme. This interaction is mutually exclusive with p21 binding, suggesting PCNA

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Fen1 (maturation factor 1) is a crucial 5'-3' exonuclease involved in DNA replication and repair.
  • Okazaki fragment maturation requires removal of RNA primers by Fen1.
  • Proliferating-cell nuclear antigen (PCNA) is a sliding clamp protein essential for DNA replication and repair.

Purpose of the Study:

  • To investigate the interaction between human Fen1 and human PCNA.
  • To elucidate the functional implications of the Fen1-PCNA complex in DNA metabolism.
  • To determine the relationship between PCNA interactions with Fen1 and p21.

Main Methods:

  • Biochemical assays to determine binding affinity (Kd) and stoichiometry of Fen1-PCNA interaction.
  • Analysis of protein-protein interactions in cell extracts.
  • Investigation of the effect of p21 on the Fen1-PCNA complex formation in vivo.

Main Results:

  • Human PCNA directly associates with human Fen1 with a Kd of 60 nM and a stoichiometry of three Fen1 molecules per PCNA trimer.
  • The interaction is mediated by a basic region at the C terminus of Fen1.
  • PCNA-bound Fen1, alongside polymerase delta, possesses key DNA replication activities (polymerase, 3'-5' exonuclease, 5'-3' exonuclease).
  • p21 also binds PCNA but forms a mutually exclusive complex, suggesting a conformational switch in PCNA.
  • Overexpression of p21 disrupts Fen1-PCNA interaction in vivo.

Conclusions:

  • The formation of a polymerase delta-PCNA-Fen1 complex provides essential activities for DNA replication.
  • PCNA exhibits mutually exclusive binding to either Fen1 or p21, regulating DNA replication and repair pathways.
  • p21's interaction with PCNA not only affects polymerase processivity but also uncouples Fen1 from the PCNA scaffold, impacting DNA metabolism.

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