Effects of Xenopus laevis mitochondrial single-stranded DNA-binding protein on primer-template binding and 3'-->5' exonuclease activity of DNA polymerase gamma

J Biol Chem. 1996 Aug 2;271(31):18939-46. doi: 10.1074/jbc.271.31.18939.

Abstract

Mitochondrial DNA (mtDNA) is replicated by DNA polymerase gamma by a strand displacement mechanism involving mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB stimulates the overall rate of DNA synthesis on singly-primed M13 DNA mainly by stimulating the processivity of DNA synthesis rather than by stimulating primer recognition. We used electrophoretic mobility shift methods to study the effects of mtSSB on primer-template recognition by DNA pol gamma. Preliminary experiments showed that single mtSSB tetramers bind tightly to oligo(dT) single strands containing 32 to 48 residues. An oligonucleotide primer-template was designed with an 18-mer primer annealed to the 3'-portion of a 71-mer template containing 40 dT residues at its 5'-end as a binding site for mtSSB. DNA pol gamma bound to this primer-template either in the absence or presence of mtSSB in complexes that remained intact and enzymatically active following native gel electrophoresis. Association of mtSSB with the 5'-dT40-tail in the 18:71-mer primer-template reduced the binding of DNA polymerase gamma and the efficiency of primer extension. Binding of mtSSB to single-stranded DNA was also observed to block the action of the 3'-->5' exonuclease of DNA polymerase gamma. The size of fragments protected from 3'-->5' exonuclease trimming increases with increasing ionic strength in a manner consistent with the known salt dependence of the binding site size of Escherichia coli SSB.

Publication types

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

MeSH terms

  • Animals
  • Bacterial Proteins / metabolism
  • Base Sequence
  • Binding Sites
  • DNA Polymerase III / genetics
  • DNA Polymerase III / metabolism*
  • DNA Primers / genetics
  • DNA Primers / metabolism
  • DNA, Mitochondrial / genetics
  • DNA, Mitochondrial / metabolism*
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism*
  • DNA-Binding Proteins / metabolism*
  • Escherichia coli / metabolism
  • Exodeoxyribonuclease V
  • Exodeoxyribonucleases / genetics
  • Exodeoxyribonucleases / metabolism
  • Female
  • In Vitro Techniques
  • Molecular Sequence Data
  • Viral Proteins / metabolism
  • Xenopus laevis

Substances

  • Bacterial Proteins
  • DNA Primers
  • DNA, Mitochondrial
  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Viral Proteins
  • gp32 protein, Enterobacteria phage T4
  • DNA Polymerase III
  • Exodeoxyribonucleases
  • Exodeoxyribonuclease V