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J Biol Chem. 2014 Jun 13;289(24):16867-82. doi: 10.1074/jbc.M114.551820. Epub 2014 Apr 23.

Kinetics, structure, and mechanism of 8-Oxo-7,8-dihydro-2'-deoxyguanosine bypass by human DNA polymerase η.

Author information

  • 1From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and.
  • 2the Laboratory of Food and Nutrition Toxicology, Eidgenössische Technische Hochschule-Zentrum, CH-8092 Zürich, Switzerland.
  • 3From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and martin.egli@vanderbilt.edu.

Abstract

DNA damage incurred by a multitude of endogenous and exogenous factors constitutes an inevitable challenge for the replication machinery. Cells rely on various mechanisms to either remove lesions or bypass them in a more or less error-prone fashion. The latter pathway involves the Y-family polymerases that catalyze trans-lesion synthesis across sites of damaged DNA. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) is a major lesion that is a consequence of oxidative stress and is associated with cancer, aging, hepatitis, and infertility. We have used steady-state and transient-state kinetics in conjunction with mass spectrometry to analyze in vitro bypass of 8-oxoG by human DNA polymerase η (hpol η). Unlike the high fidelity polymerases that show preferential insertion of A opposite 8-oxoG, hpol η is capable of bypassing 8-oxoG in a mostly error-free fashion, thus preventing GC→AT transversion mutations. Crystal structures of ternary hpol η-DNA complexes and incoming dCTP, dATP, or dGTP opposite 8-oxoG reveal that an arginine from the finger domain assumes a key role in avoiding formation of the nascent 8-oxoG:A pair. That hpol η discriminates against dATP exclusively at the insertion stage is confirmed by structures of ternary complexes that allow visualization of the extension step. These structures with G:dCTP following either 8-oxoG:C or 8-oxoG:A pairs exhibit virtually identical active site conformations. Our combined data provide a detailed understanding of hpol η bypass of the most common oxidative DNA lesion.

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

KEYWORDS:

Crystal Structure; Kinetics; Mass Spectrometry (MS); Oxidative Stress; Polymerase η; Transversion Mutation; X-ray Crystallography

PMID:
24759104
[PubMed - indexed for MEDLINE]
PMCID:
PMC4059130
[Available on 2015/6/13]

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