The C-terminal zinc finger of the catalytic subunit of DNA polymerase delta is responsible for direct interaction with the B-subunit

Nucleic Acids Res. 2004 Jun 1;32(10):3005-16. doi: 10.1093/nar/gkh623. Print 2004.

Abstract

DNA polymerase delta (Pol delta) plays a central role in eukaryotic chromosomal DNA replication, repair and recombination. In fission yeast, Pol delta is a tetrameric enzyme, comprising the catalytic subunit Pol3 and three smaller subunits, Cdc1, Cdc27 and Cdm1. Previous studies have demonstrated a direct interaction between Pol3 and Cdc1, the B-subunit of the complex. Here it is shown that removal of the tandem zinc finger modules located at the C-terminus of Pol3 by targeted proteolysis renders the Pol3 protein non-functional in vivo, and that the C-terminal zinc finger module ZnF2 is both necessary and sufficient for binding to the B-subunit in vivo and in vitro. Extensive mutagenesis of the ZnF2 module identifies important residues for B-subunit binding. In particular, disruption of the ZnF2 module by substitution of the putative metal-coordinating cysteines with alanine abolishes B-subunit binding and in vivo function. Finally, evidence is presented suggesting that the ZnF region is post-translationally modified in fission yeast cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Catalytic Domain
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • DNA Polymerase III / chemistry*
  • DNA Polymerase III / genetics
  • DNA Polymerase III / metabolism*
  • Fungal Proteins / chemistry*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • Molecular Sequence Data
  • Mutation / genetics
  • Protein Binding
  • Protein Subunits / chemistry
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Two-Hybrid System Techniques
  • Zinc Fingers* / genetics

Substances

  • CDC1 protein, S cerevisiae
  • Cell Cycle Proteins
  • Fungal Proteins
  • POL3 protein, S cerevisiae
  • Protein Subunits
  • Saccharomyces cerevisiae Proteins
  • DNA Polymerase III