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Enzymatic bypass and the structural basis of miscoding opposite the DNA adduct 1,N
- Source :
- The Journal of Biological Chemistry
- Publication Year :
- 2021
-
Abstract
- Etheno (ε)-adducts, e.g., 1,N2-ε−guanine (1,N2-ε-G) and 1,N6-ε−adenine (1,N6-ε-A), are formed through the reaction of DNA with metabolites of vinyl compounds or with lipid peroxidation products. These lesions are known to be mutagenic, but it is unknown how they lead to errors in DNA replication that are bypassed by DNA polymerases. Here we report the structural basis of misincorporation frequencies across from 1,N2-ε-G by human DNA polymerase (hpol) η. In single-nucleotide insertions opposite the adduct 1,N2-ε-G, hpol η preferentially inserted dGTP, followed by dATP, dTTP, and dCTP. This preference for purines was also seen in the first extension step. Analysis of full-length extension products by LC-MS/MS revealed that G accounted for 85% of nucleotides inserted opposite 1,N2-ε-G in single base insertion, and 63% of bases inserted in the first extension step. Extension from the correct nucleotide pair (C) was not observed, but the primer with A paired opposite 1,N2-ε-G was readily extended. Crystal structures of ternary hpol η insertion-stage complexes with nonhydrolyzable nucleotides dAMPnPP or dCMPnPP showed a syn orientation of the adduct, with the incoming A staggered between adducted base and the 5’-adjacent T, while the incoming C and adducted base were roughly coplanar. The formation of a bifurcated H-bond between incoming dAMPnPP and 1,N2-ε-G and T, compared with the single H-bond formed between incoming dCMPnPP and 1,N2-ε-G, may account for the observed facilitated insertion of dGTP and dATP. Thus, preferential insertion of purines by hpol η across from etheno adducts contributes to distinct outcomes in error-prone DNA replication.
- Subjects :
- dNMPnPP, 2′-deoxynucleoside-5′-[(α,β)-imido]triphosphate
CID, collision-induced dissociation
1,N2-ε-G, 1,N2-ethenodeoxyguanosine or 1,N2-ethenoguanine
DNA-Directed DNA Polymerase
DNA polymerase
DNA replication
dCMPnPP, 2′-deoxycytosine-5′-[(α,β)-imido]triphosphate
Crystallography, X-Ray
UPLC, ultraperformance liquid chromatography
DNA Adducts
h, human
Tandem Mass Spectrometry
Humans
translesion synthesis
X-ray crystallography
mass spectrometry
ESI, electrospray ionization
Deoxyguanosine
EIC, extracted ion chromatogram
UDG, uracil DNA glycosylase
DNA damage
dAMPnPP, 2′-deoxyadenosine-5′-[(α,β)-imido]triphosphate
pol, DNA polymerase
DNA–protein interaction
FAM, 6-carboxyfluorescein
Chromatography, Liquid
Research Article
etheno DNA adducts
Subjects
Details
- ISSN :
- 1083351X
- Volume :
- 296
- Database :
- OpenAIRE
- Journal :
- The Journal of biological chemistry
- Accession number :
- edsair.pmid..........45ed173b81a8588859e7de392898885b