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°C at neutral pH. T. thermophilus UvrB protein showed ATPase activity at its physiological temperature, whereas the E. All living organisms have DNA repair systems to counteract the many forms of DNA damage due to sunlight, chemical agents, or ionizing radiation (1). If such damage is not repaired, mutagenesis or even cell death may occur. The DNA repair systems involve in situ repair (photoreactivation), base excision repair, nucleotide excision repair, mismatch repair, and recombinational repair (1). Of these, the nucleotide excision repair system can deal with a wide range of DNA lesions. In Escherichia coli of typical prokaryote, ABC excinuclease, which is encoded by the uvrA, B, and C genes, plays a major role in this system (2-5). The molecular mechanism of the nucleotide excision repair system, studied mainly in E. coli, is as follows. First, UvrA, a damage recognition protein, makes an UvrA 2 B complex with UvrB (6) and then this complex binds to the site of the DNA lesion, forming a UvrA 2 B-DNA complex (6, 7), in which, the DNA is unwound, creating a so-called "open complex." Next, UvrA dissociates from the complex and only UvrB is bound to the DNA. Then, UvrC becomes associated with the complex, forming an UvrBC-DNA complex (6). At this point, UvrB incises the 3Ј side of the DNA lesion and UvrC incises the 5Ј side (8). Finally, DNA helicase II, DNA polymerase I, and DNA ligase complete the repair (5).In the above model, UvrA recognizes the damaged site in the DNA and guides UvrB to it. However, Hsu et al. (9) showed that UvrB can bind to DNA in the absence of UvrA. Although this finding indicates that UvrB plays a central role in ABC excinuclease activity, the details of the reaction mechanism are not clear. In order to understand the molecular mechanism of this enzyme's action in detail, enzymatic studies and physicochemical approaches, including x-ray crystallography and nuclear magnetic resonance, are required.Thermus thermophilus HB8 is an aerobic, rod-shaped, nonsporulating, Gram-negative eubacterium, which can grow at temperatures over 75°C (10). Although hyper-thermophilic bacteria can grow at temperatures over 100°C (such as Pyrococcus fuliosus, 11), T. thermophilu...