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Cloning, sequencing, and expression of ruvB and characterization of RuvB proteins from two distantly related
1Department of Microbiology, Mount Sinai School of Medicine, New York 10029, USA.
Abstract:
The ruvB genes of the highly divergent thermophilic eubacteria Thermus thermophilus and Thermotoga maritima were cloned, sequenced, and expressed in Escherichia coli. Both thermostable RuvB proteins were purified to homogeneity. Like E. coli RuvB protein, both purified thermostable RuvB proteins showed strong double-stranded DNA-dependent ATPase activity at their temperature optima (> or = 70 degrees C). In the absence of ATP, T. thermophilus RuvB protein bound to linear double-stranded DNA with a preference for the ends. Addition of ATP or gamma-S-ATP destabilized the T. thermophilus RuvB-DNA complexes. Both thermostable RuvB proteins displayed helicase activity on supercoiled DNA. Expression of thermostable T. thermophilus RuvB protein in the E. coli ruvB recG mutant strain N3395 partially complemented the UV-sensitive phenotype, suggesting that T. thermophilus RuvB protein has a function similar to that of E. coli RuvB in vivo.