Capturing a glycosylase reaction intermediate in DNA repair by freeze-trapping of a pH-responsive hOGG1 mutant

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388963%3A_____%2F25%3A00637864" target="_blank" >RIV/61388963:_____/25:00637864 - isvavai.cz</a>

Výsledek na webu

<a href="https://doi.org/10.1093/nar/gkaf718" target="_blank" >https://doi.org/10.1093/nar/gkaf718</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1093/nar/gkaf718" target="_blank" >10.1093/nar/gkaf718</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Capturing a glycosylase reaction intermediate in DNA repair by freeze-trapping of a pH-responsive hOGG1 mutant

Popis výsledku v původním jazyce

The human 8-oxoguanine DNA glycosylase 1 (hOGG1) is a bifunctional DNA repair enzyme that possesses both glycosylase and AP-lyase activity. Its AP-lyase reaction mechanism had been revealed by crystallographic capturing of the intermediate adduct. However, no intermediate within the glycosylase reaction was reported to date and the relevant reaction mechanism thus remained unresolved. In this work, we studied the glycosylase reaction of hOGG1 by time-resolved crystallography and spectroscopic/enzymological analyses. To trigger the glycosylase reaction within a crystal, we created a pH-responsive mutant of hOGG1 in which lysine 249 (K249) has been replaced by histidine (H), and designated hOGG1(K249H). Using hOGG1(K249H), a reactive intermediate state of the hOGG1(K249H)–DNA complex was captured in crystal upon pH activation. An unprecedented, ribose-ring-opened hemiaminal structure at the 8-oxoguanine (oxoG) site was found. Based on the structure of the reaction intermediate and QM/MM (quantum mechanics/molecular mechanics) calculations, a glycosylase reaction pathway of hOGG1(K249H) was identified where the aspartic acid 268 (D268) acts as a proton donor to O4′ of oxoG. Moreover, enzymologically derived pKa (4.5) of a catalytic residue indicated that the observed pKa can be attributed to the carboxy group of D268. Thus, a reaction mechanism of the glycosylase reaction by hOGG1(K249H) has been proposed.

Název v anglickém jazyce

Capturing a glycosylase reaction intermediate in DNA repair by freeze-trapping of a pH-responsive hOGG1 mutant

Popis výsledku anglicky

The human 8-oxoguanine DNA glycosylase 1 (hOGG1) is a bifunctional DNA repair enzyme that possesses both glycosylase and AP-lyase activity. Its AP-lyase reaction mechanism had been revealed by crystallographic capturing of the intermediate adduct. However, no intermediate within the glycosylase reaction was reported to date and the relevant reaction mechanism thus remained unresolved. In this work, we studied the glycosylase reaction of hOGG1 by time-resolved crystallography and spectroscopic/enzymological analyses. To trigger the glycosylase reaction within a crystal, we created a pH-responsive mutant of hOGG1 in which lysine 249 (K249) has been replaced by histidine (H), and designated hOGG1(K249H). Using hOGG1(K249H), a reactive intermediate state of the hOGG1(K249H)–DNA complex was captured in crystal upon pH activation. An unprecedented, ribose-ring-opened hemiaminal structure at the 8-oxoguanine (oxoG) site was found. Based on the structure of the reaction intermediate and QM/MM (quantum mechanics/molecular mechanics) calculations, a glycosylase reaction pathway of hOGG1(K249H) was identified where the aspartic acid 268 (D268) acts as a proton donor to O4′ of oxoG. Moreover, enzymologically derived pKa (4.5) of a catalytic residue indicated that the observed pKa can be attributed to the carboxy group of D268. Thus, a reaction mechanism of the glycosylase reaction by hOGG1(K249H) has been proposed.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10610 - Biophysics

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2025

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Nucleic Acids Research

ISSN

0305-1048

e-ISSN

1362-4962

Svazek periodika

53

Číslo periodika v rámci svazku

14

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

11

Strana od-do

gkaf718

Kód UT WoS článku

001542476000001

EID výsledku v databázi Scopus

2-s2.0-105012498873