CONSERVED MOTIF-II TO MOTIF-VI OF DNA HELICASE-II FROM ESCHERICHIA-COLI ARE ALL REQUIRED FOR BIOLOGICAL-ACTIVITY

There are seven conserved motifs (IA, IB, and II to VI) in DNA helicas e II of Escherichia coli that have high homolog among a large family o f proteins involved in DNA metabolism. To address the functional impor tance of motifs II to VI, we employed site-directed mutagenesis to rep lace the charged amino acid residues in each motif with alanines. Cell s carrying these mutant alleles exhibited higher UV and methyl methane sulfonate sensitivity, increased rates of spontaneous mutagenesis, and elevated levels of homologous recombination, indicating defects in bo th the excision repair and mismatch repair pathways. In addition, we a lso changed the highly conserved tyrosine(600) in motif VI to phenylal anine (uvrD309, Y600F). this mutant displayed a moderate increase in U V sensitivity but a decrease in spontaneous mutation rate, suggesting that DNA helicase II may have different functions in the two DNA repai r pathways. Furthermore, a mutation in domain IV (uvrD307, R284A) sign ificantly reduced the viability of some E. coli K-12 strains at 30 deg rees C but not at 37 degrees C. The implications of these observations are discussed.