Effects of substitutions of arginine residues on the basic surface of herpes simplex virus UL42 support a role for DNA binding in processive DNA synthesis

J Virol. 2005 Sep;79(18):12025-34. doi: 10.1128/JVI.79.18.12025-12034.2005.

Abstract

The way that UL42, the processivity subunit of the herpes simplex virus DNA polymerase, interacts with DNA and promotes processivity remains unclear. A positively charged face of UL42 has been proposed to participate in electrostatic interactions with DNA that would tether the polymerase to a template without preventing its translocation via DNA sliding. An alternative model proposes that DNA binding by UL42 is not important for processivity. To investigate these issues, we substituted alanine for each of four conserved arginine residues on the positively charged surface. Each single substitution decreased the DNA binding affinity of UL42, with 14- to 30-fold increases in apparent dissociation constants. The mutant proteins exhibited no meaningful change in affinity for binding to the C terminus of the catalytic subunit of the polymerase, indicating that the substitutions exert a specific effect on DNA binding. The substitutions decreased UL42-mediated long-chain DNA synthesis by the polymerase in the same rank order in which they affected DNA binding, consistent with a role for DNA binding in polymerase processivity. Combining these substitutions decreased DNA binding further and impaired the complementation of a UL42 null virus in transfected cells. Additionally, using a revised mathematical model to analyze rates of dissociation of UL42 from DNAs of various lengths, we found that dissociation from internal sites, which would be the most important for tethering the polymerase, was relatively slow, even at ionic strengths that permit processive DNA synthesis by the holoenzyme. These data provide evidence that the basic surface of UL42 interacts with DNA and support a model in which DNA binding by UL42 is important for processive DNA synthesis.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Base Sequence
  • Conserved Sequence
  • DNA Replication
  • DNA, Viral / biosynthesis
  • DNA, Viral / genetics
  • DNA, Viral / metabolism*
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Exodeoxyribonucleases / chemistry*
  • Exodeoxyribonucleases / genetics
  • Exodeoxyribonucleases / metabolism*
  • Genetic Complementation Test
  • Herpesvirus 1, Human / genetics
  • Herpesvirus 1, Human / metabolism*
  • In Vitro Techniques
  • Kinetics
  • Models, Biological
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Viral Proteins / chemistry*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism*

Substances

  • DNA, Viral
  • Recombinant Fusion Proteins
  • Viral Proteins
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases
  • DNA polymerase, Simplexvirus