ATPase activity of UvrB protein form Thermus thermophilus HB8 and its interaction with DNA.
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.
Kato
R
R
Department of Biology, Faculty of Science, Osaka University, Osaka, Japan.
Yamamoto
N
N
Kito
K
K
Kuramitsu
S
S
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004265:DNA Helicases; D017931:DNA Primers; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D016963:Thermus thermophilus / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; C068631:UvrA protein, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases