title: Transition-state destabilization reveals how human DNA polymerase β proceeds across the chemically unstable lesion N7-methylguanine.
abstract: N7-Methyl-2'-deoxyguanosine (m7dG) is the predominant lesion formed by methylating agents. A systematic investigation on the effect of m7dG on DNA replication has been difficult due to the chemical instability of m7dG. To gain insights into the m7dG effect, we employed a 2'-fluorine-mediated transition-state destabilzation strategy. Specifically, we determined kinetic parameters for dCTP insertion opposite a chemically stable m7dG analogue, 2'-fluoro-m7dG (Fm7dG), by human DNA polymerase β (polβ) and solved three X-ray structures of polβ in complex with the templating Fm7dG paired with incoming dCTP or dTTP analogues. The kinetic studies reveal that the templating Fm7dG slows polβ catalysis ∼ 300-fold, suggesting that m7dG in genomic DNA may impede replication by some DNA polymerases. The structural analysis reveals that Fm7dG forms a canonical Watson-Crick base pair with dCTP, but metal ion coordination is suboptimal for catalysis in the polβ-Fm7dG:dCTP complex, which partially explains the slow insertion of dCTP opposite Fm7dG by polβ. In addition, the polβ-Fm7dG:dTTP structure shows open protein conformations and staggered base pair conformations, indicating that N7-methylation of dG does not promote a promutagenic replication. Overall, the first systematic studies on the effect of m7dG on DNA replication reveal that polβ catalysis across m7dG is slow, yet highly accurate.
authors: Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA SeongminLee@austin.utexas.edu.
authors: Myong-Chul
authors: MC
authors: Koag
authors: Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA SeongminLee@austin.utexas.edu.
authors: Yi
authors: Y
authors: Kou
authors: Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA.
authors: Hala
authors: H
authors: Ouzon-Shubeita
authors: Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA SeongminLee@austin.utexas.edu.
authors: Seongmin
authors: S
authors: Lee
mesh_terms: D004247:DNA / Q000737:chemistry; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000737:chemistry; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000737:chemistry; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008345:Manganese / Q000737:chemistry; D008958:Models, Molecular; D013942:Thymine Nucleotides / Q000737:chemistry
chemical_list: D003845:Deoxycytosine Nucleotides; D013942:Thymine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D008345:Manganese; D006147:Guanine; C008450:7-methylguanine; D004247:DNA; D019951:DNA Polymerase beta; D003849:Deoxyguanosine; C024157:thymidine 5'-triphosphate