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. 2021 Jan-Jun:296:100642.
doi: 10.1016/j.jbc.2021.100642. Epub 2021 Apr 8.

Enzymatic bypass and the structural basis of miscoding opposite the DNA adduct 1,N2-ethenodeoxyguanosine by human DNA translesion polymerase η

Pratibha P Ghodke  1 , Jyotirling R Mali  2 , Amritraj Patra  1 , Carmelo J Rizzo  3 , F Peter Guengerich  4 , Martin Egli  5
Affiliations

Enzymatic bypass and the structural basis of miscoding opposite the DNA adduct 1,N2-ethenodeoxyguanosine by human DNA translesion polymerase η

Pratibha P Ghodke et al. J Biol Chem. 2021 Jan-Jun.

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.

Keywords: DNA damage; DNA polymerase; DNA replication; DNA–protein interaction; X-ray crystallography; etheno DNA adducts; mass spectrometry; translesion synthesis.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Major etheno (ε) adducts. The numbering pattern is shown for 1,N2-ε-G.
Figure 2
Figure 2
Mechanisms of formation of 1,N2-ε-G in DNA and RNA.A, reaction with 2-chlorooxirane (vinyl chloride epoxide), based on 13C labeling (5); B, reaction of the lipid peroxidation product 4-hydroxynonenal (11).
Figure 3
Figure 3
hpol η-mediated bypass across from 1,N2-ε-G in template 5′-T(εG)A-3′ (1). PAGE: 20%, 7 M urea. A, full-length extension assay: hpol η (120 nM) elongated Primer_1 opposite G, and 1,N2-ε-G-containing DNA templates in the presence of a mixture of dNTPs (500 μM). All reactions were done at 37 °C for 5-, 30-, 60-, and 120-min (time gradients indicated with wedges). Lanes: 1 to 5 for unmodified template; 6 to 10 for 1,N2-ε-G-modified template. Single-nucleotide insertion assays: hpol η (10 nM) was incubated with B, Primer_2/control template 1, and C, Primer_2/1,N2-ε-G modified template 1 (5′-T(εG)A-3′) as well as individual dNTPs (100 μM). Lanes: 1 to 3 for dATP, 4 to 6 for dCTP, 7 to 9 for dGTP, 10 to 12 for dTTP. All reactions were done at 37 °C for 5-, 10-, and 30-min. P indicates the FAM-labeled Primer_1. See Experimental procedures and Table S1 for the oligonucleotide sequences used.
Figure 4
Figure 4
hpol η-mediated postlesion full-length and single-nucleotide insertion assays using 1,N2-ε-G in template 5′-T(εG)A-3′ (1) and Primer_2. PAGE (20%, 7 M urea): A, full-length extension assay: hpol η (120 nM) elongated Primer_2 opposite G, and 1,N2-ε-G-containing DNA templates in the presence of a mixture of dNTPs (500 μM). All reactions were done at 37 °C for 5-, 30-, 60-, and 120-min (time gradients indicated with wedges). Lanes: 1 to 5 for unmodified template; 6 to 10 for 1,N2-ε-G-modified template. Single-nucleotide insertion assays: hpol η (10 nM) was incubated with B, Primer_2/control template 1, and C, Primer_2/1,N2-ε-G modified template 1 (5′-T(εG)A-3′), as well as individual dNTPs (100 μM). Lanes: 1 to 3 for dATP, 4 to 6 for dCTP, 7 to 9 for dGTP, 10 to 12 for dTTP. All reactions were done at 37 °C for 5-, 10-, and 30-min. P indicates the FAM-labeled Primer_2. See Experimental procedures and Table S1 for the oligonucleotide sequences used.
Figure 5
Figure 5
hpol η-mediated postlesion full-length and single-nucleotide insertion assays using 1,N2-ε-G in template 5′-T(εG)A-3′ (1) and Primer_3. PAGE (20%, 7 M urea). A, full-length extension assay: hpol η (120 nM) elongated Primer_3 opposite G, and 1,N2-ε-G-containing DNA templates in the presence of a mixture of dNTPs (500 μM). All reactions were done at 37 °C for 5-, 30-, 60-, and 120-min (time gradients indicated with wedges). Lanes: 1 to 5, unmodified template; 6 to 10, 1,N2-ε-G-modified template. Single-nucleotide insertion assays: hpol η (10 nM) was incubated with B, Primer_2/control template 1, and C, Primer_3/1,N2-ε-G modified template 1 (5′-T(εG)A-3′), as well as individual dNTPs (100 μM). Lanes: 1 to 3 for dATP, 4 to 6 for dCTP, 7 to 9 for dGTP, 10 to 12 for dTTP. All reactions were done at 37 °C for 5-, 10-, and 30-min. P indicates the FAM-labeled Primer_3. See Experimental procedures and Table S1 for the oligonucleotide sequences used.
Figure 6
Figure 6
Extracted ion chromatogram and CID spectrum of m/z 836.64 ion.A, chromatogram; B, mass spectrum. The m/z 836.64 ion (−3, tR 4.34) is associated with the extended product sequence 5′-pCATAGTGA-3′ for 1,N2-ε-G template 5′-T(εG)A-3′ (1)–Primer_4 complex. a-B fragments represented in red, W fragments in blue, and base losses in green.
Figure 7
Figure 7
Active site conformation in the ternary hpol η insertion step complex with dAMPnPP opposite 1,N2-ε−G in the 5′-T(εG)A-3′ template sequence context (oligonucleotide 1).A, view into the DNA major groove. B, rotated by 90° and viewed perpendicular to the adenine and adduct base planes. Selected active site residues are colored by atom with carbon atoms shown in purple (1,N2-ε−G), orange (incoming dAMPnPP), or pink (Arg-61 and Gln-38 from the finger domain and Asp/Glu coordinating Mg2+ ions that are shown as light green spheres). The remaining template and primer residues are colored in yellow and H-bonds involving the incoming nucleotide are drawn with dashed lines.
Figure 8
Figure 8
Active site conformation in the ternary hpol η insertion step complex with dCMPnPP opposite 1,N2-ε−G in the 5′-T(εG)A-3′ template sequence context (oligonucleotide 1).A, view into the DNA major groove. B, rotated by 90° and viewed perpendicular to the cytosine and adduct base planes. Selected active site residues are colored by atom with carbon atoms shown in purple (1,N2-ε−G), orange (incoming dCMPnPP), or pink (Arg-61 and Gln-38 from the finger domain and Asp/Glu coordinating Mg2+ ions that are shown as light green spheres). The remaining template and primer residues are colored in yellow and H-bonds involving the incoming nucleotide are drawn with dashed lines. It is unlikely that N1 of 1,N2-ε−G and N3 of dCMPnPP are H-bonded because the pH of the crystallization solution is too high for cytosine to be protonated at N3 and a tautomeric form of the adduct with the hydrogen on N1 cannot be invoked.
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