A region near the C-terminal end of Escherichia coli DNA helicase II is required for single-stranded DNA binding.
The role of the C terminus of Escherichia coli DNA helicase II (UvrD), a region outside the conserved helicase motifs, was investigated by using three mutants: UvrDDelta107C (deletion of the last 107 C-terminal amino acids), UvrDDelta102C, and UvrDDelta40C. This region, which lacks sequence similarity with other helicases, may function to tailor UvrD for its specific in vivo roles. Genetic complementation assays demonstrated that mutant proteins UvrDDelta107C and UvrDDelta102C failed to substitute for the wild-type protein in methyl-directed mismatch repair and nucleotide excision repair. UvrDDelta40C protein fully complemented the loss of helicase II in both repair pathways. UvrDDelta102C and UvrDDelta40C were purified to apparent homogeneity and characterized biochemically. UvrDDelta102C was unable to bind single-stranded DNA and exhibited a greatly reduced single-stranded DNA-stimulated ATPase activity in comparison to the wild-type protein (kcat = 0.01% of the wild-type level). UvrDDelta40C was slightly defective for DNA binding and was essentially indistinguishable from wild-type UvrD when single-stranded DNA-stimulated ATP hydrolysis and helicase activities were measured. These results suggest a role for a region near the C terminus of helicase II in binding to single-stranded DNA.
Mechanic
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LE
Department of Biochemistry and Biophysics, Protein Engineering and Molecular Genetics Training Program, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Latta
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ME
Matson
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SW
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D009154:Mutation; D011487:Protein Conformation; D017384:Sequence Deletion
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C506746:UvrD protein, E coli; D004265:DNA Helicases