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 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.
Biswas
I
I
Genetics & Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1810, USA.
Hsieh
P
P
D000251:Adenosine Triphosphatases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D017931:DNA Primers; D004260:DNA Repair; D004268:DNA-Binding Proteins; D004795:Enzyme Stability; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D006358:Hot Temperature; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013824:Thermus / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D004247:DNA; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli