Investigation of the mechanisms of DNA binding of the human G/T glycosylase using designed inhibitors

Investigation of the mechanisms of DNA binding of the human G/T glycosylase using designed inhibitors

^*Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138; and ^‡Istituto di Richerce di Biologica Molecolare, P. Angeletti, Via Pontina km. 30.600-Pomezia, Rome, Italy

Abstract

Deamination of 5-methylcytosine residues in DNA gives rise to the G/T mismatched base pair. In humans this lesion is repaired by a mismatch-specific thymine DNA glycosylase (TDG or G/T glycosylase), which catalyzes specific excision of the thymine base through N-glycosidic bond hydrolysis. Unlike other DNA glycosylases, TDG recognizes an aberrant pairing of two normal bases rather than a damaged base per se. An important structural issue is thus to understand how the enzyme specifically targets the T (or U) residue of the mismatched base pair. Our approach toward the study of substrate recognition and processing by catalytic DNA binding proteins has been to modify the substrate so as to preserve recognition of the base but to prevent its excision. Here we report that replacement of 2′-hydrogen atoms with fluorine in the substrate 2′-deoxyguridine (dU) residue abrogates glycosidic bond cleavage, thereby leading to the formation of a tight, specific glycosylase–DNA complex. Biochemical characterization of these complexes reveals that the enzyme protects an ≈20-bp stretch of the substrate from DNase I cleavage, and directly contacts a G residue on the 3′ side of the mismatched U derivative. These studies provide a mechanistic rationale for the preferential repair of deaminated CpG sites and pave the way for future high-resolution studies of TDG bound to DNA.

Footnotes

↵ † Present address: Department of Cell Biology and Genetics, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.
↵ § Present address: Institute for Medical Radiobiology, August Forel-Strasse 7, 8029 Zurich, Switzerland.
↵ ¶ To whom reprint requests should be addressed.
JoAnne Stubbe, Massachusetts Institute of Technology, Cambridge, MA
ABBREVIATIONS:

m5C,

5-methylcytosine;

AP,

abasic site;

TDG,

mismatch-specific thymine DNA glycosylase;

αFU,

2′-deoxy-2′-fluorouridine;

βFU,

1-(2′-deoxy-2′-fluoro β-d-arabinofuranosyl)-uracil;

diFU,

2′-deoxy-2′,2′-difluorouridine;

EMSA,

electrophoretic mobility shift assay;

ANPG,

3-methyladenine DNA glycosylase;

AlkA,

3-methyladenine DNA glycosylase II