Biswas I, Hsieh P
Genetics & Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1810, USA.
J Biol Chem. 1996 Mar 1;271(9):5040-8. doi: 10.1074/jbc.271.9.5040.
Recognition of mispaired or unpaired bases during DNA mismatch repair is carried out by the MutS protein family. Here, we describe the isolation and characterization of a thermostable MutS homolog from Thermus aquaticus YT-1. Sequencing of the mutS gene predicts an 89.3-kDa polypeptide sharing extensive amino acid sequence homology with MutS homologs from both prokaryotes and eukaryotes. Expression of the T. aquaticus mutS gene in Escherichia coli results in a dominant mutator phenotype. Initial biochemical characterization of the thermostable MutS protein, which was purified to apparent homogeneity, reveals two thermostable activities, an ATP hydrolysis activity in which ATP is hydrolyzed to ADP and Pi and a specific DNA mismatch binding activity with affinities for heteroduplex DNAs containing either an insertion/deletion of one base or a GT mismatch. The ATPase activity exhibits a temperature optimum of approximately 80 degrees C. Heteroduplex DNA binding by the T. aquaticus MutS protein requires Mg2+ and occurs over a broad temperature range from 0 degrees C to at least 70 degrees C. The thermostable MutS protein may be useful for further biochemical and structural studies of mismatch binding and for applications involving mutation detection.
DNA错配修复过程中对错配或未配对碱基的识别是由MutS蛋白家族完成的。在此，我们描述了从嗜热栖热菌YT-1中分离和鉴定一种热稳定的MutS同源物。mutS基因的测序预测了一种89.3 kDa的多肽，它与原核生物和真核生物的MutS同源物具有广泛的氨基酸序列同源性。嗜热栖热菌mutS基因在大肠杆菌中的表达导致显性突变表型。纯化至表观均一的热稳定MutS蛋白的初步生化特性揭示了两种热稳定活性，一种是ATP水解活性，其中ATP被水解为ADP和Pi，另一种是对含有一个碱基插入/缺失或GT错配的异源双链DNA具有亲和力的特异性DNA错配结合活性。ATP酶活性的最适温度约为80℃。嗜热栖热菌MutS蛋白与异源双链DNA的结合需要Mg2+，且在从0℃到至少70℃的宽温度范围内发生。这种热稳定的MutS蛋白可能有助于对错配结合进行进一步的生化和结构研究，以及用于涉及突变检测的应用。
