MUTATIONS IN MOTIF-II OF ESCHERICHIA-COLI DNA HELICASE-II RENDER THE ENZYME NONFUNCTIONAL IN BOTH MISMATCH REPAIR AND EXCISION-REPAIR WITH DIFFERENTIAL-EFFECTS ON THE UNWINDING REACTION

Site-directed mutagenesis has been employed to address the functional significance of the highly conserved aspartic and glutamic acid residu es present in the Walker B (also called motif II) sequence in Escheric hia coli DNA helicase II. Two mutant proteins, UvrDE221Q and UvrDD220N E221Q, were expressed and purified to apparent homogeneity. Biochemica l characterization of the DNA-dependent ATPase activity of each mutant protein demonstrated a k(cat) that was <0.5% of that of the wild-type protein, with no significant change in the apparent K-m for ATP. The E221Q mutant protein exhibited no detectable unwinding of either parti al duplex or blunt duplex DNA substrates. The D220NE221Q mutant, howev er, catalyzed unwinding of both partial duplex and blunt duplex substr ates, but at a greatly reduced rate compared with that of the wild-typ e enzyme. Both mutants were able to bind DNA. Thus, the motif II mutan ts E221Q and D220NE221Q were able to bind ATP and DNA to the same exte nt as wild-type helicase LI but demonstrate a significant reduction in ATP hydrolysis and helicase functions. The mutant uvrD alleles were a lso characterized by examining their abilities to complement the mutat or and UV light-sensitive phenotypes of a uvrD deletion mutant. Neithe r the uvrDE221Q nor the uvrDD220NE221Q allele, supplied on a plasmid, was able to complement either phenotype. Further genetic characterizat ion of the mutant uvrD alleles demonstrated that uvrDE221Q confers a d ominant negative growth phenotype; the uvrDD220NE221Q allele does not exhibit this effect, The observed difference in effect on viability ma y reflect the gene products' dissimilar kinetics for unwinding duplex DNA substrates in vitro.