DNA polymerase X of African swine fever virus: insertion fidelity on gapped DNA substrates and AP lyase activity support a role in base excision repair of viral DNA

J Mol Biol. 2003 Mar 7;326(5):1403-12. doi: 10.1016/s0022-2836(03)00019-6.

Abstract

DNA polymerase X (pol X) from African swine fever virus (ASFV) is the smallest naturally ocurring DNA-directed DNA polymerase (174 amino acid residues) described so far. Previous biochemical analysis has shown that ASFV pol X is a highly distributive, monomeric enzyme, lacking a proofreading 3'-5' exonuclease. Also, ASFV pol X binds intermediates of the single-nucleotide base excision repair (BER) process, and is able to efficiently repair single-nucleotide gapped DNA. In this work, we perform an extensive kinetic analysis of single correct and incorrect nucleotide insertions by ASFV pol X using different DNA substrates: (i) a primer/template DNA; (ii) a 1nt gapped DNA; (iii) a 5'-phosphorylated 1nt gapped DNA. The results obtained indicate that ASFV pol X exhibits a general preference for insertion of purine deoxynucleotides, especially dGTP opposite template C. Moreover, ASFV pol X shows higher catalytic efficiencies when filling in gapped substrates, which are increased when a phosphate group is present at the 5'-margin of the gap. Interestingly, ASFV pol X misinserts nucleotides with frequencies from 10(-4) to 10(-5), and the insertion fidelity varies depending on the substrate, being more faithful on a phosphorylated 1nt gapped substrate. We have analyzed the capacity of ASFV pol X to act on intermediates of BER repair. Although no lyase activity could be detected on preincised 5'-deoxyribose phosphate termini, ASFV pol X has lyase activity on unincised abasic sites. Altogether, the results support a role for ASFV pol X in reparative BER of damaged viral DNA during ASFV infection.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • African Swine Fever / pathology
  • African Swine Fever Virus / enzymology*
  • Animals
  • Base Pairing
  • Carbon-Oxygen Lyases / metabolism*
  • Catalysis
  • Crystallography, X-Ray
  • DNA Polymerase beta / chemistry
  • DNA Polymerase beta / metabolism
  • DNA Primers / chemistry
  • DNA Repair*
  • DNA, Viral / physiology*
  • DNA-(Apurinic or Apyrimidinic Site) Lyase
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / metabolism
  • Kinetics
  • Models, Molecular
  • Oligonucleotides / chemistry*
  • Phosphorus-Oxygen Lyases / metabolism*
  • Protein Conformation
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Substrate Specificity

Substances

  • DNA Primers
  • DNA, Viral
  • Oligonucleotides
  • Recombinant Proteins
  • 5'-deoxyribose phosphate lyase
  • DNA polymerase X
  • DNA Polymerase beta
  • DNA-Directed DNA Polymerase
  • Carbon-Oxygen Lyases
  • DNA-(Apurinic or Apyrimidinic Site) Lyase
  • Phosphorus-Oxygen Lyases