Conserved motifs II to 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 helicase II of Escherichia coli that have high homology among a large family of proteins involved in DNA metabolism. To address the functional importance of motifs II to VI, we employed site-directed mutagenesis to replace the charged amino acid residues in each motif with alanines. Cells carrying these mutant alleles exhibited higher UV and methyl methanesulfonate sensitivity, increased rates of spontaneous mutagenesis, and elevated levels of homologous recombination, indicating defects in both the excision repair and mismatch repair pathways. In addition, we also changed the highly conserved tyrosine(600) in motif VI to phenylalanine (uvrD309, Y600F). This mutant displayed a moderate increase in UV sensitivity but a decrease in spontaneous mutation rate, suggesting that DNA helicase II may have different functions in the two DNA repair pathways. Furthermore, a mutation in domain IV (uvrD307, R284A) significantly reduced the viability of some E. coli K-12 strains at 30 degrees C but not at 37 degrees C. The implications of these observations are discussed.
Zhang
G
G
Department of Genetics, University of Georgia, Athens 30602, USA.
Deng
E
E
Baugh
L R
LR
Hamilton
C M
CM
Maples
V F
VF
Kushner
S R
SR
D000251:Adenosine Triphosphatases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D000595:Amino Acid Sequence; D003227:Conjugation, Genetic; D017124:Conserved Sequence; D004265:DNA Helicases; D004252:DNA Mutational Analysis; D004260:DNA Repair / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004926:Escherichia coli / Q000201:enzymology*; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011995:Recombination, Genetic / Q000235:genetics; D014466:Ultraviolet Rays / Q000009:adverse effects
D008741:Methyl Methanesulfonate; D000251:Adenosine Triphosphatases; D004265:DNA Helicases