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Sequential switching of binding partners on PCNA during in vitro Okazaki fragment maturation

Daniel Dovrat1, Joseph L Stodola2, Peter M J Burgers2

  • 1Department of Life Sciences and National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be'er Sheva 84105, Israel; and.

Proceedings of the National Academy of Sciences of the United States of America
|September 18, 2014
PubMed

Insights

Proliferating cell nuclear antigen (PCNA) coordinates DNA replication. Studies show PCNA heterotrimers with one functional site efficiently mature Okazaki fragments, suggesting sequential partner switching, not simultaneous binding, in eukaryotes.

Area of Science:

  • Molecular Biology
  • DNA Replication and Repair

Background:

  • The homotrimeric sliding clamp proliferating cell nuclear antigen (PCNA) is crucial for Okazaki fragment maturation.
  • PCNA coordinates DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1), and DNA ligase I (Lig1) activities.
  • The mechanism of partner switching on PCNA and the role of its binding sites remain unclear.

Purpose of the Study:

  • To investigate the mechanism of partner switching on PCNA during Okazaki fragment maturation.
  • To determine the necessity of multiple binding sites on PCNA for coordinating partner activities.
  • To elucidate the functional advantages, if any, of simultaneous versus sequential partner binding to PCNA.

Main Methods:

  • Generation of PCNA heterotrimers with one or two non-functional mutant monomers.
  • Assessment of PCNA heterotrimer stability and structure.
  • In vitro assays to evaluate Okazaki fragment maturation efficiency with PCNA heterotrimers.

Main Results:

  • PCNA heterotrimers with only one functional binding site efficiently coordinated Pol δ, FEN1, and Lig1 for Okazaki fragment maturation.
  • Limiting PCNA binding sites did not significantly impair partner switching efficiency.
  • Simultaneous binding of multiple partners to PCNA is unnecessary and offers no significant functional advantage for Okazaki fragment maturation in vitro.

Conclusions:

  • Eukaryotic PCNA functions via sequential partner switching, not simultaneous binding, during DNA replication and repair.
  • This contrasts with the 'toolbelt' model observed in bacterial and archaeal sliding clamps.
  • PCNA's coordination of DNA replication and repair partners is robust even with limited binding sites.

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