Visualizing sequence-governed nucleotide selectivities and mutagenic consequences through a replicative cycle: processing of a bulky carcinogen N2-dG lesion in a Y-family DNA polymerase

Biochemistry. 2009 Jun 9;48(22):4677-90. doi: 10.1021/bi802363f.

Abstract

Understanding how DNA polymerases process lesions remains fundamental to determining the molecular origins of mutagenic translesion bypass. We have investigated how a benzo[a]pyrene-derived N(2)-dG adduct, 10S-(+)-trans-anti-[BP]-N(2)-dG ([BP]G*), is processed in Dpo4, the well-characterized Y-family bypass DNA polymerase. This polymerase has a slippage-prone spacious active site region. Experimental results in a 5'-C[BP]G*G-3' sequence context reveal significant selectivity for dGTP insertion that predominantly yields -1 deletion extension products. A less pronounced error-prone nonslippage pathway that leads to full extension products with insertion of A > C > G opposite the lesion is also observed. Molecular modeling and dynamics simulations follow the bypass of [BP]G* through an entire replication cycle for the first time in Dpo4, providing structural interpretations for the experimental observations. The preference for dGTP insertion is explained by a 5'-slippage pattern in which the unmodified G rather than G* is skipped, the incoming dGTP pairs with the C on the 5'-side of G*, and the -1 deletion is produced upon further primer extension which is more facile than nucleotide insertion. In addition, the simulations suggest that the [BP]G* may undergo an anti/syn conformational rearrangement during the stages of the replication cycle. In the minor nonslippage pathway, the nucleotide insertion preferences opposite the lesion are explained by relative distortions to the active site region. These structural insights, provided by the modeling and dynamics studies, augment kinetic and limited available crystallographic investigations with bulky lesions, by providing molecular explanations for lesion bypass activities over an entire replication cycle.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Archaeal Proteins / chemistry
  • Archaeal Proteins / genetics
  • Base Sequence / genetics
  • Benzopyrenes / chemistry*
  • Carcinogens / chemistry*
  • Catalytic Domain / genetics
  • DNA Adducts / chemistry*
  • DNA Polymerase beta / chemistry*
  • DNA Polymerase beta / genetics
  • DNA Polymerase beta / metabolism
  • DNA Replication* / genetics
  • DNA, Archaeal / chemistry
  • DNA, Archaeal / genetics
  • Deoxyguanosine / analogs & derivatives*
  • Deoxyguanosine / chemistry
  • Deoxyguanosine / genetics
  • Mutagenesis, Insertional*
  • Protein Processing, Post-Translational* / genetics
  • Sequence Deletion
  • Sulfolobus solfataricus / enzymology*
  • Sulfolobus solfataricus / genetics

Substances

  • Archaeal Proteins
  • Benzopyrenes
  • Carcinogens
  • DNA Adducts
  • DNA, Archaeal
  • benzo(a)pyrene N2-dG adduct
  • DNA Polymerase beta
  • Deoxyguanosine