Tài liệu Báo cáo khoa học: The stereochemistry of benzo[a]pyrene-2¢-deoxyguanosine adducts affects

The stereochemistry of benzo[a]pyrene-2¢-deoxyguanosine

adducts affects DNA methylation by SssI and HhaI DNA

methyltransferases

Oksana M. Subach1

, Diana V. Maltseva1

, Anant Shastry2

, Alexander Kolbanovskiy2

,

Saulius Klimasˇauskas3

, Nicholas E. Geacintov2 and Elizaveta S. Gromova1

1 Chemistry Department, Moscow State University, Russia

2 Department of Chemistry, New York University, NY, USA

3 Laboratory of Biological DNA Modification, Institute of Biotechnology, Vilnius, Lithuania

The polycyclic aromatic hydrocarbons are a well￾known class of ubiquitous environmental pollutants

which are generated by incomplete combustion of

organic matter. These compounds require metabolic

activation to highly reactive diol epoxides to elicit their

detrimental genotoxic effects [1]. Benzo[a]pyrene

(B[a]P), one of the most widely studied polycyclic

aromatic hydrocarbons, is metabolically activated

in vivo to the highly mutagenic [2] and tumorigenic [3]

(+)-7R,8S-diol 9S,10R-epoxide of benzo[a]pyrene

Keywords

benzo[a]pyrene-2¢-deoxyguanosine adducts;

DNA methyltansferases; environmental

pollutants; stereochemistry

Correspondence

E. S. Gromova, Chemistry Department,

Moscow State University, Moscow,

119992, Russia

Fax: +7495 939 31 81

Tel: +7495 939 31 44

E-mail: gromova@genebee.msu.ru

(Received 19 July 2006, revised 19 January

2007, accepted 21 February 2007)

doi:10.1111/j.1742-4658.2007.05754.x

The biologically most significant genotoxic metabolite of the environmental

pollutant benzo[a]pyrene (B[a]P), (+)-7R,8S-diol 9S,10R-epoxide, reacts

chemically with guanine in DNA, resulting in the predominant formation

of (+)-trans-B[a]P-N2

-dG and, to a lesser extent, (+)-cis-B[a]P-N2

-dG

adducts. Here, we compare the effects of the adduct stereochemistry and

conformation on the methylation of cytosine catalyzed by two purified

prokaryotic DNA methyltransferases (MTases), SssI and HhaI, with the

lesions positioned within or adjacent to their CG and GCGC recognition

sites, respectively. The fluorescence properties of the pyrenyl residues of the

(+)-cis-B[a]P-N2

-dG and (+)-trans-B[a]P-N2

-dG adducts in complexes with

MTases are enhanced, but to different extents, indicating that aromatic

B[a]P residues are positioned in different microenvironments in the DNA–

protein complexes. We have previously shown that the (+)-trans-isomeric

adduct inhibits both the binding and methylating efficiencies (kcat) of both

MTases [Subach OM, Baskunov VB, Darii MV, Maltseva DV, Alexandrov

DA, Kirsanova OV, Kolbanovskiy A, Kolbanovskiy M, Johnson F,

Bonala R, et al. (2006) Biochemistry 45, 6142–6159]. Here we show that the

stereoisomeric (+)-cis-B[a]P-N2

-dG lesion has only a minimal effect on the

binding of these MTases and on kcat. The minor-groove (+)-trans adduct

interferes with the formation of the normal DNA minor-groove contacts

with the catalytic loop of the MTases. However, the intercalated base￾displaced (+)-cis adduct does not interfere with the minor-groove DNA–

catalytic loop contacts, allowing near-normal binding of the MTases and

undiminished kcat values.

Abbreviations

AdoHcy, S-adenosyl-L-homocysteine; AdoMet, S-adenosyl-L-methionine; B[a]P, benzo[a]pyrene; B[a]PDE, r7,t8-dihydroxy-t9,10-epoxy￾7,8,9,10-tetrahydrobenzo[a]pyrene; B[a]P-DNA, DNA containing benzo[a]pyrene; C5 MTase, C5-cytosine DNA methyltransferase; EMSA,

electrophoretic mobility shift assay; kcat, multiple turnover rate constant; Kd, dissociation constant; M.SssI, SssI DNA methyltransferase;

M.HhaI, HhaI DNA methyltransferase; MTase, DNA methyltransferase; V0, initial rate of methylation; Vmax, maximal rate of methylation.

FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS 2121