Helicase motifs V and VI of the Escherichia coli UvrB protein of the UvrABC endonuclease are essential for the formation of the preincision complex.
The UvrB protein is a subunit of the UvrABC endonuclease which is involved in the repair of a large variety of DNA lesions. We have 91 isolated random uvrB mutants which are impaired in the repair of UV-damage in vivo. These mutants were classified on the basis of the ability to form normal levels of protein and the position of the mutations in the gene. The amino acid substitutions in the N-terminal part or in the C-terminal part of the UvrB protein are exclusively found in the conserved boxes of the so-called "helicase motifs" present in these parts of the protein, indicating that these motifs are essential for UvrB function. The proteins of four C-terminal mutants were purified: two mutants in motif V (E514K and G509S), one mutant in motif VI (R544H) and a double mutant in both motifs (E514K + R541H). In vitro experiments with these mutant proteins show that the helicase motifs V and VI are involved in the induction of ATP hydrolysis in the presence of (damaged) DNA and in the strand-displacement activity of the UvrA2B complex as is observed in a helicase assay. Furthermore, our results suggest that this strand-displacement activity is correlated to a local unwinding, which seems to be used to form the UvrB-DNA preincision complex.
Laboratory of Molecular Genetics, Leiden Institute of Chemistry, The Netherlands.
G F
GF
Moolenaar
R
R
Visse
M
M
Ortiz-Buysse
N
N
Goosen
P
P
van de Putte
D000255:Adenosine Triphosphate / Q000737:chemistry; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism*; D017931:DNA Primers; D004260:DNA Repair; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009154:Mutation
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; C058093:UvrB protein, E coli; D000255:Adenosine Triphosphate; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC; D004265:DNA Helicases