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Interaction between PCNA and DNA ligase I is critical for joining of Okazaki fragments and long-patch base-excision

D S Levin1, A E McKenna, T A Motycka

  • 1Department of Molecular Medicine, Institute of Biotechnology, The University of Texas Health Science Center at San Antonio, 78245, USA.

Current Biology : CB
|August 26, 2000
PubMed

Insights

The interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA) is vital for DNA replication and repair. This binding is essential for joining Okazaki fragments during lagging-strand synthesis and for long-patch base-excision repair.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • DNA ligase I interacts with proliferating cell nuclear antigen (PCNA) through a conserved motif.
  • The biological significance of this PCNA-binding interaction for DNA ligase I function is not fully understood.

Purpose of the Study:

  • To investigate the functional importance of the PCNA-binding site on DNA ligase I.
  • To determine the role of this interaction in DNA replication and repair pathways.

Main Methods:

  • Site-directed mutagenesis to inactivate the PCNA-binding site of DNA ligase I.
  • Assays for catalytic activity, interaction with DNA polymerase beta, Okazaki fragment ligation, and complementation of a DNA ligase I mutant cell line (46BR.1G1).
  • Analysis of base-excision repair (BER) pathways in cell extracts.

Main Results:

  • Inactivating the PCNA-binding site did not affect DNA ligase I's catalytic activity or interaction with DNA polymerase beta.
  • Loss of PCNA binding significantly impaired Okazaki fragment joining during lagging-strand synthesis.
  • A functional PCNA-binding site was necessary for DNA ligase I to complement the hypersensitivity of 46BR.1G1 cells to alkylating agents, indicating a PCNA-dependent repair pathway.
  • 46BR.1G1 cell extracts showed defects in long-patch BER but not short-patch BER.

Conclusions:

  • The interaction between PCNA and DNA ligase I is crucial for subnuclear targeting and coordinating lagging-strand synthesis.
  • This interaction plays a key role in long-patch base-excision repair.
  • The study provides the first evidence for the biological significance of PCNA in long-patch BER.

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