DNA polymerase lambda directly binds to proliferating cell nuclear antigen through its confined C-terminal region

Genes Cells. 2005 Jul;10(7):705-15. doi: 10.1111/j.1365-2443.2005.00868.x.

Abstract

DNA polymerase lambda (Pol lambda) was recently identified as a new member of the family X of DNA polymerases. Here, we show that Pol lambda directly binds to proliferating cell nuclear antigen (PCNA), an auxiliary protein for DNA replication and repair enzymes, both in vitro and in vivo. A pull-down assay using deletion mutants of Pol lambda showed that the confined C-terminal region of Pol lambda directly binds to PCNA. Furthermore, a synthetic peptide of 20-mers derived from the C-terminal region of Pol lambda competes with full-length Pol lambda for binding to PCNA. The residues between amino acids 518 and 537 of Pol lambda are required for binding to PCNA, and are different from the consensus PCNA interacting motif (PIM). Pol lambda associates with PCNA in vivo by immunoprecipitation analysis and EGFP-tagged Pol lambda co-localizes with PCNA as spots within a nucleus using fluorescent microscopy. Through direct binding, PCNA suppressed the distributive nucleotidyltransferase activity of Pol lambda. Pol micro, which also belongs to the family X of DNA polymerases, binds to PCNA by a pivotal amino acid residue.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Cell Nucleus / metabolism*
  • DNA Nucleotidylexotransferase / metabolism
  • DNA Polymerase beta / genetics
  • DNA Polymerase beta / metabolism*
  • DNA Replication
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Immunoprecipitation
  • In Vitro Techniques
  • Microscopy, Fluorescence
  • Mutagenesis, Site-Directed
  • Mutation
  • Proliferating Cell Nuclear Antigen / genetics
  • Proliferating Cell Nuclear Antigen / metabolism*
  • Protein Binding
  • Protein Structure, Tertiary

Substances

  • Proliferating Cell Nuclear Antigen
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • DNA Polymerase beta
  • DNA polymerase beta2
  • DNA Nucleotidylexotransferase