9Y62

Human DNA polymerase beta crosslinked ternary complex with Sp-dCTP-alpha-S

This is a non-PDB format compatible entry.

Summary for 9Y62

• Entry DOI: 10.2210/pdb9y62/pdb
• Descriptor: DNA polymerase beta, DNA (5'-D(*GP*TP*CP*GP*G)-3'), DNA (5'-D(*GP*CP*TP*GP*AP*TP*GP*CP*GP*C)-3'), ... (9 entities in total)
• Functional Keywords: transferase/dna, replication
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 4
• Total formula weight: 49402.13

Authors

Gaur, A.,Suo, Z. (deposition date: 2025-09-06, release date: 2026-06-17, Last modification date: 2026-06-24)

Primary citation

Betancourt, D.,Gaur, A.,Seay, T.W.,Zalenski, N.,Suo, Z.
Conformational gating governs nucleotide incorporation by a DNA-crosslinked polymerase.
Nucleic Acids Res., 54:-, 2026

PubMed Abstract: Base excision repair is a major pathway that repairs single-base DNA damage. We recently demonstrated that human DNA polymerase β (hPolβ) fills single-nucleotide gaps after Schiff base formation but before β-elimination, implying that its dRP lyase domain remains covalently crosslinked to DNA during gap-filling synthesis. Because uncrosslinked Polβ dissociates rapidly from DNA (∼3 s-1), mechanistic investigation has been challenging. To elucidate the kinetic mechanism of correct incorporation by DNA-crosslinked hPolβ, we generated a catalytically active crosslinked hPolβ‒DNA complex and performed pre-steady-state kinetic, thermodynamic, and structural analyses. Sulfur elemental effects of 3.7 ± 0.4 and 24 ± 4 for correct and incorrect nucleotide incorporation, respectively, suggest the chemical step is rate-limiting for incorrect, but not for correct, nucleotide incorporation. Pulse-chase and pulse-quench assays revealed a 33% difference in reaction amplitude, establishing the existence of a ternary intermediate preceding the chemical step. Eyring analysis identified a high activation free energy barrier, while the lack of viscosity dependence rules out large domain motions, indicating that the rate-limiting pre-chemical step involves local active-site rearrangements. Together with structurally characterized intermediates, these findings establish the first minimal kinetic mechanism for correct nucleotide incorporation by a DNA-crosslinked polymerase and identify local active-site rearrangements as the rate-limiting step.

PubMed: 42227337
DOI: 10.1093/nar/gkag539

Experimental method

X-RAY DIFFRACTION (2.1 Å)