Site-specific mutagenesis of Drosophila proliferating cell nuclear antigen enhances its effects on calf thymus DNA polymerase delta

BMC Biochem. 2004 Aug 13;5:13. doi: 10.1186/1471-2091-5-13.

Abstract

Background: We and others have shown four distinct and presumably related effects of mammalian proliferating cell nuclear antigen (PCNA) on DNA synthesis catalyzed by mammalian DNA polymerase delta(pol delta). In the presence of homologous PCNA, pol delta exhibits 1) increased absolute activity; 2) increased processivity of DNA synthesis; 3) stable binding of synthetic oligonucleotide template-primers (t1/2 of the pol deltaPCNAtemplate-primer complex >/=2.5 h); and 4) enhanced synthesis of DNA opposite and beyond template base lesions. This last effect is potentially mutagenic in vivo. Biochemical studies performed in parallel with in vivo genetic analyses, would represent an extremely powerful approach to investigate further, both DNA replication and repair in eukaryotes.

Results: Drosophila PCNA, although highly similar in structure to mammalian PCNA (e.g., it is >70% identical to human PCNA in amino acid sequence), can only substitute poorly for either calf thymus or human PCNA (approximately 10% as well) in affecting calf thymus pol delta. However, by mutating one or only a few amino acids in the region of Drosophila PCNA thought to interact with pol delta, all four effects can be enhanced dramatically.

Conclusions: Our results therefore suggest that all four above effects depend at least in part on the PCNA-pol delta interaction. Moreover unlike mammals, Drosophila offers the potential for immediate in vivo genetic analyses. Although it has proven difficult to obtain sufficient amounts of homologous pol delta for parallel in vitro biochemical studies, by altering Drosophila PCNA using site-directed mutagenesis as suggested by our results, in vitro biochemical studies may now be performed using human and/or calf thymus pol delta preparations.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Cattle
  • DNA / metabolism
  • DNA Polymerase III / chemistry
  • DNA Polymerase III / metabolism*
  • DNA Replication
  • Drosophila Proteins / chemistry
  • Drosophila Proteins / genetics
  • Drosophila Proteins / physiology*
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Proliferating Cell Nuclear Antigen / chemistry
  • Proliferating Cell Nuclear Antigen / genetics
  • Proliferating Cell Nuclear Antigen / physiology*
  • Protein Binding
  • Protein Conformation
  • Protein Interaction Mapping
  • Protein Structure, Tertiary
  • Species Specificity
  • Thymus Gland / enzymology

Substances

  • Drosophila Proteins
  • Proliferating Cell Nuclear Antigen
  • DNA
  • DNA Polymerase III