The peroxidation-derived DNA adduct, 6-oxo-M₁dG, is a strong block to replication by human DNA polymerase η

Journal Article · 18 July 2023 · Journal of Biological Chemistry

DOI: https://doi.org/10.1016/j.jbc.2023.105067 · OSTI ID:1992172

Richie-Jannetta, Robyn ^[1]; Pallan, Pradeep ^[1]; Kingsley, Philip J. ^[1]; Kamdar, Nikhil ^[1]; Egli, Martin ^[1]; Marnett, Lawrence J. ^[1]

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1. Vanderbilt Univ., Nashville, TN (United States). School of Medicine

The DNA adduct 6-oxo-M₁dG, (3-(2'-deoxy-β-D-erythro-pentofuranosyl)-6-oxo-pyrimido(1,2alpha)purin-10(3H)-one) is formed in the genome via oxidation of the peroxidation-derived adduct M₁dG. However, the effect of 6-oxo-M₁dG adducts on subsequent DNA replication is unclear. Here we investigated the ability of the human Y-family polymerase hPol η to bypass 6-oxo-M₁dG. Using steady-state kinetics and analysis of DNA extension products by liquid chromatography–tandem mass spectrometry, we found hPol η preferentially inserts a dAMP or dGMP nucleotide into primer–templates across from the 6-oxo-M₁dG adduct, with dGMP being slightly preferred. We also show primer–templates with a 3'-terminal dGMP or dAMP across from 6-oxo-M₁dG were extended to a greater degree than primers with a dCMP or dTMP across from the adduct. In addition, we explored the structural basis for bypass of 6-oxo-M₁dG by hPol η using X-ray crystallography of both an insertion-stage and an extension-stage complex. In the insertion-stage complex, we observed that the incoming dCTP opposite 6-oxo-M₁dG, although present during crystallization, was not present in the active site. We found the adduct does not interact with residues in the hPol η active site but rather forms stacking interactions with the base pair immediately 3' to the adduct. In the extension-stage complex, we observed the 3' hydroxyl group of the primer strand dGMP across from 6-oxo-M₁dG is not positioned correctly to form a phosphodiester bond with the incoming dCTP. Taken together, these results indicate 6-oxo-M₁dG forms a strong block to DNA replication by hPol η and provide a structural basis for its blocking ability.

• Research Organization: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
• Sponsoring Organization: USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH)
• Grant/Contract Number: AC02-06CH11357; P30 CA-068485; W-31109-Eng-38
• OSTI ID: 1992172
• Alternate ID(s): OSTI ID: 2470090
• Journal Information: Journal of Biological Chemistry, Vol. 299, Issue 8; ISSN 0021-9258
• Publisher: American Society for Biochemistry and Molecular BiologyCopyright Statement
• Country of Publication: United States
• Language: English