DNA连接酶
dna连接酶
DNA
泛素连接酶
生物化学
生物
结扎测序
DNA钳
分子生物学
化学
泛素
基因组文库
核糖核酸
逆转录酶
基因
基序列
作者
Aidan J. Doherty,Timothy R. Dafforn
标识
DOI:10.1006/jmbi.1999.3423
摘要
Phage T7 DNA ligase seals nicked DNA substrates and is a representative member of the ATP-dependent class of DNA ligases. Although the catalytic mechanism of DNA ligases has been delineated, little is known about the nature of nick recognition by these enzymes. Here, we show that T7 ligase discriminates, at the nick-binding step, between nicks containing either a 5'-phosphate or a 5'-OH. T7 ligase binds preferentially to phosphorylated nicks and catalyses the sealing reaction. We also show using DNA footprinting studies, that T7 ligase binds asymmetrically to nicks as a monomer, with the protein interface covering between 12 and 14 bp of DNA. Based on molecular modelling studies we propose a structural model of the ligase-DNA complex consistent with these and other data. Using photo-crosslinking and site-directed mutagenesis we have identified two residues, K238 and K240, critical for the transadenylation and nick-sealing reactions. Sequence conservation and structural analysis supports the premise that these two lysine residues are critical for both nucleotide binding and DNA nick recognition. The implications of these results on the ligation mechanism are discussed.
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