Kato R, Yamamoto N, Kito K, Kuramitsu S
Department of Biology, Faculty of Science, Osaka University, Osaka, Japan.
J Biol Chem. 1996 Apr 19;271(16):9612-8. doi: 10.1074/jbc.271.16.9612.
Many living organisms remove wide range of DNA lesions from their genomes by the nucleotide excision repair system. The uvrB gene, which plays an essential role in the prokaryotic excision repair, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence was determined, and the deduced amino acid sequence showed it possessed a helicase motif, including a nucleotide-binding consensus sequence (Walker's A-type motif), which was also conserved in other UvrB proteins. The prokaryotic UvrB proteins and eukaryotic DNA repair helicases (Rad3 and XP-D) were classified into different groups by molecular phylogenetic analysis. The T. thermophilus uvrB gene product was overproduced in Escherichia coli and purified to apparent homogeneity. The purified T. thermophilus UvrB protein was stable up to 80 degrees C at neutral pH. T. thermophilus UvrB protein showed ATPase activity at its physiological temperature, whereas the E. coli UvrB protein alone has not been shown to exhibit detectable ATPase activity. The values of K(m) and k(cat) for the ATPase activity were 4.2 mM and 0.32 s-1 without DNA, and 4.0 mM and 0.46 s-1 with single-stranded DNA, respectively. This suggests that T. thermophilus UvrB protein could interact with single-stranded DNA in the absence of UvrA protein.
许多生物通过核苷酸切除修复系统从其基因组中去除多种DNA损伤。在原核生物切除修复中起关键作用的uvrB基因，是从嗜热栖热菌HB8中克隆出来的。测定了其核苷酸序列，推导的氨基酸序列表明它具有一个解旋酶基序，包括一个核苷酸结合共有序列（沃克A型基序），该序列在其他UvrB蛋白中也保守。通过分子系统发育分析，原核生物UvrB蛋白和真核生物DNA修复解旋酶（Rad3和XP-D）被分为不同的组。嗜热栖热菌uvrB基因产物在大肠杆菌中过量表达并纯化至表观均一。纯化的嗜热栖热菌UvrB蛋白在中性pH下高达80℃仍稳定。嗜热栖热菌UvrB蛋白在其生理温度下显示ATP酶活性，而单独的大肠杆菌UvrB蛋白尚未显示出可检测到的ATP酶活性。ATP酶活性的K(m)值和k(cat)值在无DNA时分别为4.2 mM和0.32 s-1，在有单链DNA时分别为4.0 mM和0.46 s-1。这表明嗜热栖热菌UvrB蛋白在没有UvrA蛋白的情况下可以与单链DNA相互作用。
