7M87

Human DNA Pol eta S113A with dA-ended primer and dATP: in crystallo reaction for 230 s

Summary for 7M87

• Entry DOI: 10.2210/pdb7m87/pdb
• Descriptor: DNA polymerase eta, DNA (5'-D(*CP*AP*TP*TP*TP*TP*GP*AP*CP*GP*CP*T)-3'), DNA (5'-D(*AP*GP*CP*GP*TP*CP*AP*AP*A)-3'), ... (9 entities in total)
• Functional Keywords: dna polymerase, time resolved crystallography, deprotonation, dna binding protein, transferase-dna complex, transferase/dna
• Biological source: Homo sapiens (Human); More
• Total number of polymer chains: 3
• Total formula weight: 56033.32

Authors

Gregory, M.T.,Gao, Y.,Yang, W. (deposition date: 2021-03-29, release date: 2021-06-02, Last modification date: 2023-10-18)

Primary citation

Gregory, M.T.,Gao, Y.,Cui, Q.,Yang, W.
Multiple deprotonation paths of the nucleophile 3'-OH in the DNA synthesis reaction.
Proc.Natl.Acad.Sci.USA, 118:-, 2021

PubMed Abstract: DNA synthesis by polymerases is essential for life. Deprotonation of the nucleophile 3'-OH is thought to be the obligatory first step in the DNA synthesis reaction. We have examined each entity surrounding the nucleophile 3'-OH in the reaction catalyzed by human DNA polymerase (Pol) η and delineated the deprotonation process by combining mutagenesis with steady-state kinetics, high-resolution structures of in crystallo reactions, and molecular dynamics simulations. The conserved S113 residue, which forms a hydrogen bond with the primer 3'-OH in the ground state, stabilizes the primer end in the active site. Mutation of S113 to alanine destabilizes primer binding and reduces the catalytic efficiency. Displacement of a water molecule that is hydrogen bonded to the 3'-OH using the 2'-OH of a ribonucleotide or 2'-F has little effect on catalysis. Moreover, combining the S113A mutation with 2'-F replacement, which removes two potential hydrogen acceptors of the 3'-OH, does not reduce the catalytic efficiency. We conclude that the proton can leave the O3' via alternative paths, supporting the hypothesis that binding of the third Mg initiates the reaction by breaking the α-β phosphodiester bond of an incoming deoxyribonucleoside triphosphate (dNTP).

PubMed: 34088846
DOI: 10.1073/pnas.2103990118

Experimental method

X-RAY DIFFRACTION (1.85 Å)