The 3'-5' proofreading exonuclease of archaeal family-B DNA polymerase hinders the copying of template strand deaminated bases

Nucleic Acids Res. 2009 Dec;37(22):7603-11. doi: 10.1093/nar/gkp800.

Abstract

Archaeal family B polymerases bind tightly to the deaminated bases uracil and hypoxanthine in single-stranded DNA, stalling replication on encountering these pro-mutagenic deoxynucleosides four steps ahead of the primer-template junction. When uracil is specifically bound, the polymerase-DNA complex exists in the editing rather than the polymerization conformation, despite the duplex region of the primer-template being perfectly base-paired. In this article, the interplay between the 3'-5' proofreading exonuclease activity and binding of uracil/hypoxanthine is addressed, using the family-B DNA polymerase from Pyrococcus furiosus. When uracil/hypoxanthine is bound four bases ahead of the primer-template junction (+4 position), both the polymerase and the exonuclease are inhibited, profoundly for the polymerase activity. However, if the polymerase approaches closer to the deaminated bases, locating it at +3, +2, +1 or even 0 (paired with the extreme 3' base in the primer), the exonuclease activity is strongly stimulated. In these situations, the exonuclease activity is actually stronger than that seen with mismatched primer-templates, even though the deaminated base-containing primer-templates are correctly base-paired. The resulting exonucleolytic degradation of the primer serves to move the uracil/hypoxanthine away from the primer-template junction, restoring the stalling position to +4. Thus the 3'-5' proofreading exonuclease contributes to the inability of the polymerase to replicate beyond deaminated bases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Archaeal Proteins / chemistry
  • Archaeal Proteins / metabolism*
  • DNA Primers
  • DNA-Directed DNA Polymerase / chemistry
  • DNA-Directed DNA Polymerase / metabolism*
  • Deamination
  • Exodeoxyribonucleases / chemistry
  • Exodeoxyribonucleases / metabolism*
  • Hypoxanthine / chemistry
  • Hypoxanthine / metabolism
  • Pyrococcus furiosus / enzymology*
  • Templates, Genetic
  • Uracil / chemistry
  • Uracil / metabolism*

Substances

  • Archaeal Proteins
  • DNA Primers
  • Hypoxanthine
  • Uracil
  • Pfu DNA polymerase
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases