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.
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.
García-Escudero
Ramón
R
Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049, Madrid, Spain.
García-Díaz
Miguel
M
Salas
María L
ML
Blanco
Luis
L
Salas
José
J
D000357:African Swine Fever / Q000473:pathology; D000358:African Swine Fever Virus / Q000201:enzymology*; D000818:Animals; D020029:Base Pairing; D019757:Carbon-Oxygen Lyases / Q000378:metabolism*; D002384:Catalysis; D018360:Crystallography, X-Ray; D019951:DNA Polymerase beta / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair; D004279:DNA, Viral / Q000502:physiology*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009841:Oligonucleotides / Q000737:chemistry*; D019761:Phosphorus-Oxygen Lyases / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D017931:DNA Primers; D004279:DNA, Viral; D009841:Oligonucleotides; D011994:Recombinant Proteins; C423906:5'-deoxyribose phosphate lyase; C109534:DNA polymerase X; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D019761:Phosphorus-Oxygen Lyases