Distinct pools of proliferating cell nuclear antigen associated to DNA replication sites interact with the p125 subunit of DNA polymerase delta or DNA ligase I

Exp Cell Res. 2004 Feb 15;293(2):357-67. doi: 10.1016/j.yexcr.2003.10.025.

Abstract

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and cell cycle control. PCNA is a homotrimeric ring that, when encircling DNA, is not easily extractable. Consequently, the dynamics of protein-protein interactions established by PCNA at DNA replication sites is not well understood. We have used DNase I to release DNA-bound PCNA together with replication proteins including the p125-catalytic subunit of DNA polymerase delta (p125-pol delta), DNA ligase I, cyclin A, and cyclin-dependent kinase 2 (CDK2). Interaction with these proteins was investigated by immunoprecipitation with antibodies binding near the interdomain connector loop or to the C-terminal domain of PCNA, respectively, or with antibodies to p125-pol delta or DNA ligase I. PCNA interaction with p125-pol delta or DNA ligase I was detected only by the latter antibodies, and found to be mutually exclusive. In contrast, antibodies to PCNA co-immunoprecipitated only CDK2. A GST-p21(waf1/cip1) C-terminal peptide displaced p125-pol delta and DNA ligase I, but not CDK2, from PCNA. These results suggest that PCNA trimers bound to DNA during the S phase are organized as distinct pools able to bind selectively different partners. Among them, p125-pol delta and DNA ligase I interact with PCNA in a mutually exclusive manner.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies / metabolism
  • Antibodies / pharmacology
  • Binding Sites / physiology
  • CDC2-CDC28 Kinases / genetics
  • CDC2-CDC28 Kinases / metabolism
  • Catalytic Domain / genetics
  • Cell Cycle / genetics*
  • Cell Line
  • Cyclin A / genetics
  • Cyclin A / metabolism
  • Cyclin-Dependent Kinase 2
  • DNA / metabolism*
  • DNA Ligase ATP
  • DNA Ligases / genetics
  • DNA Ligases / metabolism*
  • DNA Polymerase III / genetics
  • DNA Polymerase III / metabolism*
  • DNA Replication / genetics*
  • Deoxyribonucleases / metabolism
  • Fetus
  • Humans
  • Peptide Fragments / metabolism
  • Peptide Fragments / pharmacology
  • Proliferating Cell Nuclear Antigen / genetics
  • Proliferating Cell Nuclear Antigen / metabolism*
  • Protein Binding / physiology
  • Protein Structure, Tertiary / genetics

Substances

  • Antibodies
  • Cyclin A
  • LIG1 protein, human
  • Peptide Fragments
  • Proliferating Cell Nuclear Antigen
  • DNA
  • CDC2-CDC28 Kinases
  • CDK2 protein, human
  • Cyclin-Dependent Kinase 2
  • DNA Polymerase III
  • Deoxyribonucleases
  • DNA Ligases
  • DNA Ligase ATP