PDBsum entry 4o5c

Transferase, lyase/DNA

PDB id: 4o5c

Contents

Protein chain

327 a.a.

DNA/RNA

Metals

_NA ×2

Waters ×107

References listed in PDB file

Key reference

• Title: Transition-State destabilization reveals how human DNA polymerase β proceeds across the chemically unstable lesion n7-Methylguanine.
• Authors: M.C.Koag, Y.Kou, H.Ouzon-Shubeita, S.Lee.
• Ref.: Nucleic Acids Res, 2014, 42, 8755-8766. [DOI no: 10.1093/nar/gku554]
• PubMed id: 24966350

Note: In the PDB file this reference is annotated as "TO BE PUBLISHED". The citation details given above have been manually determined.

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.