IDENTIFICATION AND CHARACTERIZATION OF A THERMOSTABLE MUTS HOMOLOG FROM THERMUS-AQUATICUS

Recognition of mispaired or unpaired bases during DNA mismatch repair is carried out by the MutS protein family. Here, we describe the isola tion and characterization of a thermostable MutS homolog from Thermus aquaticus YT-1. Sequencing of the mutS gene predicts an 89.3-kDa polyp eptide sharing extensive amino acid sequence homology with MutS homolo gs from both prokaryotes and eukaryotes. Expression of the T. aquaticu s mutS gene in Escherichia coli results in a dominant mutator phenotyp e. Initial biochemical characterization of the thermostable MutS prote in, which was purified to apparent homogeneity, reveals two thermostab le activities, an ATP hydrolysis activity in which ATP is hydrolyzed t o ADP and P-i and a specific DNA mismatch binding activity with affini ties 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 tempe rature range from 0 degrees C to at least 70 degrees C. The thermostab le MutS protein may be useful for further biochemical and structural s tudies of mismatch binding and for applications involving mutation det ection.