Structural basis for the interaction of a hexameric replicative helicase with the regulatory subunit of human DNA polymerase α-primase

J Biol Chem. 2012 Aug 3;287(32):26854-66. doi: 10.1074/jbc.M112.363655. Epub 2012 Jun 14.

Abstract

DNA polymerase α-primase (Pol-prim) plays an essential role in eukaryotic DNA replication, initiating synthesis of the leading strand and of each Okazaki fragment on the lagging strand. Pol-prim is composed of a primase heterodimer that synthesizes an RNA primer, a DNA polymerase subunit that extends the primer, and a regulatory B-subunit (p68) without apparent enzymatic activity. Pol-prim is thought to interact with eukaryotic replicative helicases, forming a dynamic multiprotein assembly that displays primosome activity. At least three subunits of Pol-prim interact physically with the hexameric replicative helicase SV40 large T antigen, constituting a simple primosome that is active in vitro. However, structural understanding of these interactions and their role in viral chromatin replication in vivo remains incomplete. Here, we report the detailed large T antigen-p68 interface, as revealed in a co-crystal structure and validated by site-directed mutagenesis, and we demonstrate its functional importance in activating the SV40 primosome in cell-free reactions with purified Pol-prim, as well as in monkey cells in vivo.

Publication types

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

MeSH terms

  • Base Sequence
  • Blotting, Southern
  • DNA Polymerase I / chemistry
  • DNA Polymerase I / metabolism*
  • DNA Primase / chemistry
  • DNA Primase / metabolism*
  • DNA Primers
  • DNA Replication
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Simian virus 40 / genetics

Substances

  • DNA Primers
  • DNA Primase
  • DNA polymerase alpha-primase
  • DNA Polymerase I