Kinetics, Structure, and Mechanism of 8-Oxo-7,8-dihydro-2′-deoxyguanosine Bypass by Human DNA Polymerase η

Patra, A.; Nagy, Leslie D.; Zhang, Q.; Su, Yan; Müller, Leonie; Guengerich, F. Peter; Egli, Martin

doi:10.1074/jbc.m114.551820

Cited by 81 publications

(155 citation statements)

References 67 publications

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“…With either conformation adopted by 8-oxodG, the 8-oxodG:rCTP pair was shifted toward the major groove compared with the structure of hpol ⅐(8-oxodG):dCTP (PDB code 4O3P) (29). The closest distance between 2Ј-OH of rCTP and Phe-18 was 3.3 Å, and the distance between 3Ј-OH and the steric gate residue was 3.4 Å.…”

Section: Resultsmentioning

confidence: 88%

“…4B. Noticeably, the electron density of 8-oxodG did not fit either a pure anti conformation as seen at the active site of hpol ⅐(8-oxodG): dCTP or a pure syn conformation as in hpol ⅐(8-oxodG):dATP (29). Instead, the electron density was indicative of two alternative conformations of 8-oxodG.…”

Section: Resultsmentioning

confidence: 91%

“…5). In comparison with the hpol ⅐dG:dCTP structure (PDB code 4O3N) (29), the position of the phenyl ring of Phe-18 was almost identical. In addition, the distance between the 3Ј-OH of the primer end (two conformations) to P␣ of the incoming rCTP was 4.5 or 4.3 Å, about 1 Å further than that in a Data shown in parentheses are from the highest resolution shell.…”

Section: Resultsmentioning

confidence: 99%

“…Diffraction data were collected on the 21-ID-F or 21-ID-G beamline at the Advanced Photon Source (Life Sciences Collaborative Access Team, Argonne National Laboratory, Argonne, IL). Data were integrated and scaled using HKL2000 (40), processed with Phaser MR (41) for molecular replacement phasing (PDB code 4O3N as the search model for hpol ⅐dG⅐rCTP, and PDB code 4O3P for hpol ⅐dG⅐rCTP and hpol ⅐dG⅐rATP) (29), and refined with PHE-NIX (42). Model building was performed with ARP/ ARP classic (43,44) and COOT (45).…”

Section: Methodsmentioning

confidence: 99%

“…hpol is also involved in translesion synthesis of other DNA lesions, e.g. 8-oxodG (29,30), abasic sites (31,32), and cisplatin or other therapeutic drug-induced DNA damage (33)(34)(35)(36)(37)(38).…”

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confidence: 99%

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Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η

Egli

Guengerich

2016

Journal of Biological Chemistry

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Ribonucleotides and 2-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2-OH group. Y-family human DNA polymerase (hpol ) is of interest because of its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2-deoxyguanosine and cyclobutane pyrimidine dimer but with rates that are 10 3 -fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that the hpol scaffolds the incoming rNTP to pair with the template base (dG) or 7,8-dihydro-8-oxo-2-deoxyguanosine with a significant propeller twist. As a result, the 2-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and P␣ of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18. This is the first crystal structure of a DNA polymerase with an incoming rNTP opposite a DNA lesion.RNA and DNA are fundamental to life in all forms. The two nucleic acid polymers are composed of ribonucleotides and 2Ј-deoxyribonucleotides as the basic units, respectively. However, ribonucleotides have been found in DNA; they constitute a large proportion of the "DNA lesions" in the genome. In mice, they have been shown to be collectively the most frequently occurring DNA lesions, even more than abasic sites and 7,8-dihydro-8-oxo-2Ј-deoxyguanosine (8-oxodG).2 The presence of ribonucleotides in DNA increases the possibility of spontaneous hydrolysis, causing DNA to break more frequently. These ribonucleotides are mainly removed from DNA by the RNase H2 pathway (1-5). DNA polymerases introduce ribonucleotides into DNA by misinserting them (6). Concentrations of cellular rNTPs are 1-6 orders of magnitude higher than those of dNTPs, depending on the cell type and the stage of the cell cycle (6 -9). To discriminate dNTP from rNTP, a steric gate residue (typically a tyrosine or phenylalanine) is generally conserved in DNA polymerases and thought to create a clash with the extra hydroxyl group of the incoming rNTP (10 -17). Although limited in extent, ribonucleotide incorporation has still been observed by a variety of DNA polymerases, from replicative ones with relatively high fidelity and small active sites (e.g. pol ⑀ and pol ␦) to error-prone X-family pol and pol ␤ and Y-family pol (6,8,(17)(18)(19)(20)(21).Compared with other DNA polymerases, those in the Yfamily are known for high ...

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Section: Resultsmentioning

confidence: 88%

Section: Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η

Egli

Guengerich

2016

Journal of Biological Chemistry

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Steady‐State Kinetic Analysis of DNA Polymerase Single‐Nucleotide Incorporation Products

O’Flaherty

Guengerich

2014

CP Nucleic Acid Chemistry

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This unit describes the experimental procedures for the steady-state kinetic analysis of DNA synthesis across DNA nucleotides (native or modified) by DNA polymerases. In vitro primer extension experiments with a single nucleoside triphosphate species followed by denaturing polyacrylamide gel electrophoresis of the extended products is described. Data analysis procedures and fitting to steady-state kinetic models is presented to highlight the kinetic differences involved in the bypass of damaged versus undamaged DNA. Moreover, explanations concerning problems encountered in these experiments are addressed. This approach provides useful quantitative parameters for the processing of damaged DNA by DNA polymerases.

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Mechanism of Error‐Free Bypass of the Environmental Carcinogen N‐(2′‐Deoxyguanosin‐8‐yl)‐3‐aminobenzanthrone Adduct by Human DNA Polymerase η

et al. 2016

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The environmental pollutant 3‐nitrobenzanthrone produces bulky aminobenzanthrone (ABA) DNA adducts with both guanine and adenine nucleobases. A major product occurs at the C8 position of guanine (C8‐dG‐ABA). These adducts present a strong block to replicative polymerases but, remarkably, can be bypassed in a largely error‐free manner by the human Y‐family polymerase η (hPol η). Here, we report the crystal structure of a ternary Pol⋅DNA⋅dCTP complex between a C8‐dG‐ABA‐containing template:primer duplex and hPol η. The complex was captured at the insertion stage and provides crucial insight into the mechanism of error‐free bypass of this bulky lesion. Specifically, bypass involves accommodation of the ABA moiety inside a hydrophobic cleft to the side of the enzyme active site and formation of an intra‐nucleotide hydrogen bond between the phosphate and ABA amino moiety, allowing the adducted guanine to form a standard Watson–Crick pair with the incoming dCTP.

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Kinetics, Structure, and Mechanism of 8-Oxo-7,8-dihydro-2′-deoxyguanosine Bypass by Human DNA Polymerase η

Cited by 81 publications

References 67 publications

Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η

Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η

Steady‐State Kinetic Analysis of DNA Polymerase Single‐Nucleotide Incorporation Products

Mechanism of Error‐Free Bypass of the Environmental Carcinogen N‐(2′‐Deoxyguanosin‐8‐yl)‐3‐aminobenzanthrone Adduct by Human DNA Polymerase η

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