A highly conserved Tyrosine residue of family B DNA polymerases contributes to dictate translesion synthesis past 8-oxo-7,8-dihydro-2'-deoxyguanosine.
The harmfulness of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodG) damage resides on its dual coding potential, as it can pair with the correct dCMP (dC) or the incorrect dAMP (dA). Here, we investigate the translesional synthesis ability of family B 29 DNA polymerase on 8oxodG-containing templates. We show that this polymerase preferentially inserts dC opposite 8oxodG, its 3'-5' exonuclease activity acting indistinctly on both dA or dC primer terminus. In addition, 29 DNA polymerase shows a favoured extension of the 8oxodG/dA pair, but with an efficiency much lower than that of the canonical dG/dC pair. Additionally, we have analysed the role of the invariant tyrosine from motif B of family B DNA polymerases in translesional synthesis past 8oxodG, replacing the corresponding 29 DNA polymerase Tyr390 by Phe or Ser. The lack of the aromatic portion in mutant Y390S led to a lost of discrimination against dA insertion opposite 8oxodG. On the contrary, the absence of the hydroxyl group in the Y390F mutant precluded the favoured extension of 8oxodG:dA base pair with respect to 8oxodG:dC. Based on the results obtained, we propose that this Tyr residue contributes to dictate nucleotide insertion and extension preferences during translesion synthesis past 8oxodG by family B replicases.
de Vega
Miguel
M
Instituto de Biología Molecular Eladio Viñuela (CSIC), Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain. mdevega@cbm.uam.es
Salas
Margarita
M
D000080242:8-Hydroxy-2'-Deoxyguanosine; D020816:Amino Acid Motifs; D017103:Bacillus Phages / Q000201:enzymology; D020029:Base Pairing; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry*
D003848:Deoxyguanine Nucleotides; D009711:Nucleotides; C078206:8-oxodeoxyguanosine triphosphate; D014443:Tyrosine; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine