Crystal Structure of Human Thymine DNA Glycosylase Bound to DNA Elucidates Sequence-Specific Mismatch Recognition

Journal Article · Tue May 19 04:00:00 EDT 2009 · Proc. Nat. Acad. Sci. 105:8890,2008

OSTI ID:953103

Maiti, A; Morgan, M T; Pozharski, E; Drohat, A C

Cytosine methylation at CpG dinucleotides produces m{sup 5}CpG, an epigenetic modification that is important for transcriptional regulation and genomic stability in vertebrate cells. However, m{sup 5}C deamination yields mutagenic G{center_dot}T mispairs, which are implicated in genetic disease, cancer, and aging. Human thymine DNA glycosylase (hTDG) removes T from G{center_dot}T mispairs, producing an abasic (or AP) site, and follow-on base excision repair proteins restore the G{center_dot}C pair. hTDG is inactive against normal A{center_dot}T pairs, and is most effective for G{center_dot}T mispairs and other damage located in a CpG context. The molecular basis of these important catalytic properties has remained unknown. Here, we report a crystal structure of hTDG (catalytic domain, hTDG{sup cat}) in complex with abasic DNA, at 2.8 {angstrom} resolution. Surprisingly, the enzyme crystallized in a 2:1 complex with DNA, one subunit bound at the abasic site, as anticipated, and the other at an undamaged (nonspecific) site. Isothermal titration calorimetry and electrophoretic mobility-shift experiments indicate that hTDG and hTDG{sup cat} can bind abasic DNA with 1:1 or 2:1 stoichiometry. Kinetics experiments show that the 1:1 complex is sufficient for full catalytic (base excision) activity, suggesting that the 2:1 complex, if adopted in vivo, might be important for some other activity of hTDG, perhaps binding interactions with other proteins. Our structure reveals interactions that promote the stringent specificity for guanine versus adenine as the pairing partner of the target base and interactions that likely confer CpG sequence specificity. We find striking differences between hTDG and its prokaryotic ortholog (MUG), despite the relatively high (32%) sequence identity.

• Research Organization: Stanford Linear Accelerator Center (SLAC)
• Sponsoring Organization: USDOE
• DOE Contract Number: AC02-76SF00515
• OSTI ID: 953103
• Report Number(s): SLAC-REPRINT-2009-181
• Journal Information: Proc. Nat. Acad. Sci. 105:8890,2008, Journal Name: Proc. Nat. Acad. Sci. 105:8890,2008 Journal Issue: 26 Vol. 105; ISSN 0027-8424; ISSN PNASA6
• Country of Publication: United States
• Language: English

Related Subjects

36 MATERIALS SCIENCE
ADENINES
CALORIMETRY
CRYSTAL STRUCTURE
CYTOSINE
DEAMINATION
DNA
ENZYMES
EXCISION REPAIR
GENETICS
GUANINE
IN VIVO
KINETICS
METHYLATION
NEOPLASMS
Other
OTHER
PROTEINS
SPECIFICITY
STOICHIOMETRY
THYMINE
TITRATION
VERTEBRATES