A point mutation in Escherichia coli DNA helicase II renders the enzyme nonfunctional in two DNA repair pathways. Evidence for initiation of unwinding from a nick in vivo.
Biosynthetic errors and DNA damage introduce mismatches and lesions in DNA that can lead to mutations. These abnormalities are susceptible to correction by a number of DNA repair mechanisms, each of which requires a distinct set of proteins. Escherichia coli DNA helicase II has been demonstrated to function in two DNA repair pathways, methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair. To define further the role of UvrD in DNA repair a site-specific mutant was characterized. The mutation, uvrDQ251E, resides within helicase motif III, a conserved segment of amino acid homology found in a superfamily of prokaryotic and eukaryotic DNA helicases. The UvrD-Q251E protein failed to complement the mutator and ultraviolet light-sensitive phenotypes of a uvrD deletion strain indicating that the mutant protein is inactive in both mismatch repair and excision repair. Biochemical characterization revealed a significant defect in the ability of the mutant enzyme to initiate unwinding at a nick. The elongation phase of the unwinding reaction was nearly normal. Together, the biochemical and genetic data provide evidence that UvrD-Q251E is dysfunctional because the mutant protein fails to initiate unwinding at the nick(s) used to initiate excision and subsequent repair synthesis. These results provide direct evidence to support the notion that helicase II initiates unwinding from a nick in vivo in mismatch repair and excision repair.
Brosh
R M
RM
Department of Biology, University of North Carolina, Chapel Hill 27599-3280, USA.
Matson
S W
SW
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases