Structure of a DNA polymerase alpha-primase domain that docks on the SV40 helicase and activates the viral primosome

J Biol Chem. 2010 May 28;285(22):17112-22. doi: 10.1074/jbc.M110.116830. Epub 2010 Mar 16.

Abstract

DNA polymerase alpha-primase (pol-prim) plays a central role in DNA replication in higher eukaryotes, initiating synthesis on both leading and lagging strand single-stranded DNA templates. Pol-prim consists of a primase heterodimer that synthesizes RNA primers, a DNA polymerase that extends them, and a fourth subunit, p68 (also termed B-subunit), that is thought to regulate the complex. Although significant knowledge about single-subunit primases of prokaryotes has accumulated, the functions and regulation of pol-prim remain poorly understood. In the SV40 replication model, the p68 subunit is required for primosome activity and binds directly to the hexameric viral helicase T antigen, suggesting a functional link between T antigen-p68 interaction and primosome activity. To explore this link, we first mapped the interacting regions of the two proteins and discovered a previously unrecognized N-terminal globular domain of p68 (p68N) that physically interacts with the T antigen helicase domain. NMR spectroscopy was used to determine the solution structure of p68N and map its interface with the T antigen helicase domain. Structure-guided mutagenesis of p68 residues in the interface diminished T antigen-p68 interaction, confirming the interaction site. SV40 primosome activity of corresponding pol-prim mutants decreased in proportion to the reduction in p68N-T antigen affinity, confirming that p68-T antigen interaction is vital for primosome function. A model is presented for how this interaction regulates SV40 primosome activity, and the implications of our findings are discussed in regard to the molecular mechanisms of eukaryotic DNA replication initiation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Viral, Tumor / chemistry
  • DNA Polymerase I / chemistry*
  • DNA Primase / chemistry*
  • DNA Primers / genetics
  • DNA Replication
  • Magnetic Resonance Spectroscopy
  • Molecular Conformation
  • Mutagenesis
  • Protein Binding
  • Protein Conformation
  • Protein Interaction Mapping
  • Protein Structure, Tertiary
  • Simian virus 40 / enzymology*
  • Two-Hybrid System Techniques

Substances

  • Antigens, Viral, Tumor
  • DNA Primers
  • DNA Polymerase I
  • DNA Primase
  • DNA polymerase alpha-primase