Cloning and Expression of Human G/T Mismatch-specific Thymine-DNA Glycosylase

Hydrolytic deamination of 5-methylcytosine leads to the formation of G/T mismatches. We have shown previously that these G/T mispairs are corrected to G/C pairs by a mismatch-specific thymine-DNA glycosylase, TDG, which we subsequently purified from human cells. Here we describe the cloning of the h...

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Bibliographic Details
Published in:The Journal of biological chemistry 1996-05, Vol.271 (22), p.12767-12774
Main Authors: Neddermann, P, Gallinari, P, Lettieri, T, Schmid, D, Truong, O, Hsuan, J J, Wiebauer, K, Jiricny, J
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Base Sequence
Blotting, Western
Cell-Free System
Cloning, Molecular
Deoxyribonuclease (Pyrimidine Dimer)
DNA Repair
DNA, Complementary
Endodeoxyribonucleases - genetics
Endodeoxyribonucleases - metabolism
Escherichia coli - genetics
HeLa Cells
Humans
Mice
Molecular Sequence Data
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Substrate Specificity
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Summary:Hydrolytic deamination of 5-methylcytosine leads to the formation of G/T mismatches. We have shown previously that these G/T mispairs are corrected to G/C pairs by a mismatch-specific thymine-DNA glycosylase, TDG, which we subsequently purified from human cells. Here we describe the cloning of the human cDNA encoding TDG. We have identified two distinct cDNA species that differ by 100 nucleotides at the 3′-untranslated region. These cDNAs contain a 410-amino acid open reading frame that encodes a 46-kDa polypeptide. The G/T glycosylase, expressed both in vitro and in Escherichia coli , migrated in denaturing polyacrylamide gels with an apparent size of 60 kDa. The substrate specificity of the recombinant protein corresponded to that of the cellular enzyme, and polyclonal antisera raised against the recombinant protein neutralized both activities. We therefore conclude that the cDNA described below encodes human TDG. Data base searches identified a serendipitously cloned mouse cDNA sequence that could be shown to encode the murine TDG homologue. No common amino acid sequence motifs between the G/T-specific enzyme and other DNA glycosylases could be found, suggesting that TDG belongs to a new class of base-excision repair enzymes.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.271.22.12767