CLONING, SEQUENCING, AND EXPRESSION OF RUVB AND CHARACTERIZATION OF RUVB PROTEINS FROM 2 DISTANTLY RELATED THERMOPHILIC EUBACTERIA

The ruvB genes of the highly divergent thermophilic eubacteria Thermus thermophilus and Thermotoga maritima were cloned, sequenced, and expr essed in Escherichia call. Both thermostable RuvB proteins were purifi ed to homogeneity. Like E. coli RuvB protein, both purified thermostab le RuvB proteins showed strong double-stranded DNA-dependent ATPase ac tivity at their temperature optima (greater than or equal to 70 degree s C). In the absence of ATP, T. thermophilus RuvB protein bound to lin ear double-stranded DNA with a preference for the ends. Addition of AT P or gamma-S-ATP destabilized the T. thermophilus RuvB-DNA complexes. Both thermostable RuvB proteins displayed helicase activity on superco iled DNA. Expression of thermostable T. thermophilus RuvB protein in t he E. coli ruvB recG mutant strain N3395 partially complemented the W- sensitive phenotype, suggesting that T. thermophilus RuvB protein has a function similar to that off. coli RuvB in vivo.