Base Analog and Neighboring Base Effects on Substrate Specificity of Recombinant Human G:T Mismatch-Specific Thymine DNA-Glycosylase
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- 作者：Sibghat-Ullah ; Paola Gallinari ; Yao-Zhong Xu ; Myron F. Goodman ; Linda B. Bloom ; Josef Jiricny ; and Rufus S. Day ; III
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：October 1, 1996
- 年：1996
- 卷：35
- 期：39
- 页码：12926 - 12932
- 全文大小：458K
- 年卷期：v.35,no.39(October 1, 1996)
- ISSN：1520-4995
文摘
We studied the substrate specificity of the human G:Tmismatch-specific thymine glycosylasethat initiates the repair of G:T and G:U base mismatches to G:C basepairs. Such mismatches arise when5-methylcytosine or cytosine deaminate spontaneously (andhydrolytically) in DNA. Substrates were45-bp DNA heteroduplexes that bore single G:T, m6G:T,2,6-diaminopurine:T, 2-amino-6-(methylamino)purine:T, 2-aminopurine:T, and G:m4T mispairs. The bases 5' to thepoorly matched G were altered inselected G:T substrates to yield mispairs in four different contexts,ApG, CpG, GpG, and TpG. Therecombinant thymine glycosylase was incubated with the 45-bp DNAsubstrates, each labeled at the 5'-terminus of the strand containing the mismatched T. The DNAs werethen treated with 0.1 N NaOH tocatalyze phosphodiester bond breakage at the newly-generated AP sites,and the products were analyzedon DNA sequencing gels. As indicated by the amounts of the 20-ntincision product, the removal of thethymine base by the enzyme increased linearly between 0 and 40 min atwhich time the generation ofproduct from all substrates ceased, probably because of enzymeinactivation. The rate of incision wasgreatest (0.7 fmol/min) with DNA containing the G:T mispair followed bythe DNA containing the m6G:Tmispair (0.38 fmol/min) and the DNA with the2-amino-6-(methylamino)purine:T mispair (0.15 fmol/min); the extent of reaction was 90%, 40%, and 20% respectively.By contrast to previous findings withcell-free extracts, DNA substrates containing 2,6-diaminopurine:T,2-aminopurine:T, and G:m4T mispairswere not incised (<2%). The amount of incision of the 45-bp DNAsubstrates containing G:T mispairsin the CpG context was 3-12-fold greater than in the TpG, GpG, andApG contexts.