Takamatsu S, Kato R, Kuramitsu S
Department of Biology, Faculty of Science, Osaka University, Toyonaka, Japan.
Nucleic Acids Res. 1996 Feb 15;24(4):640-7. doi: 10.1093/nar/24.4.640.
The mutS gene, implicated in DNA mismatch repair, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence encoded a 819-amino acid protein with a molecular mass of 91.4 kDa. Its predicted amino acid sequence showed 56 and 39% homology with Escherichia coli MutS and human hMsh2 proteins, respectively. The T.thermophilus mutS gene complemented the hypermutability of the E.coli mutS mutant, suggesting that T.thermophilus MutS protein was active in E.coli and could interact with E.coli MutL and/or MutH proteins. The T.thermophilus mutS gene product was overproduced in E.coli and then purified to homogeneity. Its molecular mass was estimated to be 91 kDa by SDS-PAGE but approx. 330 kDa by size-exclusion chromatography, suggesting that T.thermophilus MutS protein was a tetramer in its native state. Circular dichroic measurements indicated that this protein had an alpha-helical content of approx. 50%, and that it was stable between pH 1.5 and 12 at 25 degree C and was stable up to 80 degree C at neutral pH. Thermus thermophilus MutS protein hydrolyzed ATP to ADP and Pi, and its activity was maximal at 80 degrees C. The kinetic parameters of the ATPase activity at 65 degrees C were Km = 130 microM and Kcat = 0.11 s(-1). Thermus thermophilus MutS protein bound specifically with G-T mismatched DNA even at 60 degrees C.
参与DNA错配修复的mutS基因是从嗜热栖热菌HB8中克隆得到的。其核苷酸序列编码一个819个氨基酸的蛋白质，分子量为91.4 kDa。预测的氨基酸序列与大肠杆菌MutS和人类hMsh2蛋白的同源性分别为56%和39%。嗜热栖热菌mutS基因弥补了大肠杆菌mutS突变体的高突变性，这表明嗜热栖热菌MutS蛋白在大肠杆菌中具有活性，并且能够与大肠杆菌MutL和/或MutH蛋白相互作用。嗜热栖热菌mutS基因产物在大肠杆菌中过量表达，然后纯化至均一。通过SDS-PAGE估计其分子量为91 kDa，但通过尺寸排阻色谱法估计约为330 kDa，这表明嗜热栖热菌MutS蛋白在天然状态下是四聚体。圆二色性测量表明，该蛋白质的α-螺旋含量约为50%，并且在25℃下pH 1.5至12之间稳定，在中性pH下高达80℃时稳定。嗜热栖热菌MutS蛋白将ATP水解为ADP和Pi，其活性在80℃时最大。在65℃下ATPase活性的动力学参数为Km = 130 μM和Kcat = 0.11 s(-1)。嗜热栖热菌MutS蛋白即使在60℃时也能与G-T错配DNA特异性结合。
