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.

Insights into the effect of minor groove interactions and metal cofactors on mutagenic replication by human DNA polymerase β

“…Third, the nascent base-pair binding pocket of polβ is highly sensitive to structural and electronic perturbations caused by nucleobase lesions, particularly alkylated lesions. For example, polβ incorporates dCTP opposite N7-methylguanine 300-fold less efficiently than opposite guanine [47]. In addition, although N7-methylguanine:T forms a Watson-Crick-like conformation in the absence of protein contact [48], polβ does not permit the formation of a Watson-Crick-like N7-methylguaine:T base pair at the insertion site and induces a staggered base-pair conformation [47].…”

“…For example, polβ incorporates dCTP opposite N7-methylguanine 300-fold less efficiently than opposite guanine [47]. In addition, although N7-methylguanine:T forms a Watson-Crick-like conformation in the absence of protein contact [48], polβ does not permit the formation of a Watson-Crick-like N7-methylguaine:T base pair at the insertion site and induces a staggered base-pair conformation [47]. The enzyme’s active site architecture appears to sense the steric and/or electronic perturbation of the N7-methylguanine moiety.…”

Structural Basis for Error-Free Bypass of the 5-N-Methylformamidopyrimidine-dG Lesion by Human DNA Polymerase η and Sulfolobus solfataricus P2 Polymerase IV

“…24,25 Previous crystallographic studies using the chemically stable 2′-fluoro-m 7 dG analog in a DNA sequence, either in complex with E. coli DNA glycosylase AlkA 26 or human DNA polymerase β 27 revealed that this polymerase bypasses m 7 dG accurately and that the lesion forms a canonical Watson-Crick base pair with incoming dCTP.…”

Structural and Kinetic Studies of the Effect of Guanine N7 Alkylation and Metal Cofactors on DNA Replication

“…49 When N7mG adduct is at the templating position, polβ adopts a closed conformation, showing that small alkyl group at the N7 position of G minimally affect polβ conformation. The polβ’s conformational difference between the N7bnG:dCTP* and N7mG:dCTP* structures suggests that the steric bulkiness of N7-alkylG adducts may significantly affect the conformational reorganization of some DNA polymerases.…”

“…49 Diffraction data were collected at 100 K at the beamline 5.0.3 at the Advanced Light Source, Lawrence Berkeley National Laboratory. All diffraction data were processed using HKL2000.…”