Kinetics, structure, and mechanism of 8-Oxo-7,8-dihydro-2'-deoxyguanosine bypass by human DNA polymerase η.
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.
From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and.
Amritraj
A
Patra
From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and.
Leslie D
LD
Nagy
From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and.
Qianqian
Q
Zhang
From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and.
Yan
Y
Su
the Laboratory of Food and Nutrition Toxicology, Eidgenössische Technische Hochschule-Zentrum, CH-8092 Zürich, Switzerland.
Livia
L
Müller
From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and.
F Peter
FP
Guengerich
From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and martin.egli@vanderbilt.edu.
Martin
M
Egli
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D000099270:Y-Family DNA Polymerases
D004259:DNA-Directed DNA Polymerase; D006147:Guanine; D009711:Nucleotides; C024829:8-hydroxyguanine; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases
Crystal Structure; Kinetics; Mass Spectrometry (MS); Oxidative Stress; Polymerase η; Transversion Mutation; X-ray Crystallography