Tài liệu Báo cáo khoa học: Ionic strength and magnesium affect the specificity of Escherichia coli

Ionic strength and magnesium affect the specificity of

Escherichia coli and human 8-oxoguanine-DNA

glycosylases

Viktoriya S. Sidorenko1

, Grigory V. Mechetin1

, Georgy A. Nevinsky1,2 and Dmitry O. Zharkov1,2

1 SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia

2 Department of Natural Sciences, Novosibirsk State University, Russia

In all living organisms DNA is subject to ongoing

damage by various environmental and endogenous

factors [1]. One of the most frequently encountered

base lesions is 8-oxo-7,8-dihydroguanine (8-oxoG),

produced by oxidative stress to the steady-state level

of 1 · 106 guanines in human DNA [2]. 8-oxoG is

mutagenic due to its ability to form a stable Hoog￾sten pair with A [3] and its propensity to direct the

incorporation of dAMP by DNA polymerases [4]. If

left uncorrected, the resulting 8-oxoG:A mispair is

converted to a T:A pair in the next round of replica￾tion, producing a G:C fi T:A transversion mutation,

the type frequently encountered in human cancers

[5,6].

The consequences of 8-oxoG’s appearance in DNA

are counteracted by a three-tier enzymatic ‘GO system’

[7–9], part of general base-excision repair system [10].

In bacteria, once it has emerged in DNA in the context

of a G:C pair, the 8-oxoG base is excised from the

8-oxoG:C pair by formamidopyrimidine-DNA glycosy￾lase (Fpg, EC 3.2.2.23); in eukaryotes it is excised by

8-oxoguanine-DNA glycosylase OGG1, followed by

Keywords

8-oxoguanine; DNA damage; DNA

glycosylase; DNA repair; substrate

specificity

Correspondence

D. O. Zharkov, SB RAS Institute of

Chemical Biology and Fundamental

Medicine, Novosibirsk 630090, Russia

Fax: +7 383 333 3677

Tel: +7 383 335 6226

E-mail: [email protected]

(Received 20 February 2008, revised 18

April 2008, accepted 23 May 2008)

doi:10.1111/j.1742-4658.2008.06521.x

An abundant oxidative lesion, 8-oxo-7,8-dihydroguanine (8-oxoG), often

directs the misincorporation of dAMP during replication. To prevent muta￾tions, cells possess an enzymatic system for the removal of 8-oxoG. A key

element of this system is 8-oxoguanine-DNA glycosylase (Fpg in bacteria,

OGG1 in eukaryotes), which must excise 8-oxoG from 8-oxoG:C pairs but

not from 8-oxoG:A. We investigated the influence of various factors,

including ionic strength, the presence of Mg2+ and organic anions, poly￾amides, crowding agents and two small heterocyclic compounds (biotin

and caffeine) on the activity and opposite-base specificity of Escherichia coli

Fpg and human OGG1. The activity of both enzymes towards 8-oxoG:A

decreased sharply with increasing salt and Mg2+ concentration, whereas

the activity on 8-oxoG:C was much more stable, resulting in higher oppo￾site-base specificity when salt and Mg2+ were at near-physiological concen￾trations. This tendency was observed with both Cl) and glutamate as the

major anions in the reaction mixture. Kinetic and binding parameters for

the processing of 8-oxoG:C and 8-oxoG:A by Fpg and OGG1 were deter￾mined under several different conditions. Polyamines, crowding agents,

biotin and caffeine affected the activity and specificity of Fpg or OGG1

only marginally. We conclude that, in the intracellular environment, the

specificity of Fpg and OGG1 for 8-oxoG:C versus 8-oxoG:A is mostly due

to high ionic strength and Mg2+.

Abbreviations

8-oxoG, 8-oxo-7,8-dihydroguanine; AP, apurinic ⁄ apyrimidinic; KGlu, potassium glutamate; THF, tetrahydrofuran.

FEBS Journal 275 (2008) 3747–3760 ª 2008 The Authors Journal compilation ª 2008 FEBS 3747