ATP-dependent partitioning of the DNA template into supercoiled domains by Escherichia coli UvrAB.
The helicase action of the Escherichia coli UvrAB complex on a covalently closed circular DNA template was monitored using bacterial DNA topoisomerase I, which specifically removes negative supercoils. In the presence of E. coli DNA topoisomerase I and ATP, the UvrAB complex gradually introduced positive supercoils into the input relaxed plasmid DNA template. Positive supercoils were not produced when E. coli DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I or when both E. coli and eukaryotic DNA topoisomerases I were added simultaneously. These results suggest that like other DNA helix-tracking processes, the ATP-dependent action of the UvrAB complex on duplex DNA simultaneously generates both positive and negative supercoils, which are not constrained by protein binding but are torsionally strained. The supercoiling activity of UvrAB on UV-damaged DNA was also studied using UV-damaged plasmid DNA and a mutant UvrA protein that lacks the 40 C-terminal amino acids and is defective in preferential binding to UV-damaged DNA. UvrAB was found to preferentially supercoil the UV-damaged DNA template, whereas the mutant protein supercoiled UV-damaged and undamaged DNA with equal efficiency. Our results therefore suggest that the DNA helix-tracking activity of UvrAB may be involved in searching and/or prepriming the damaged DNA for UvrC incision. A possible role of supercoiled domains in the incision process is discussed.
Koo
H S
HS
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205-2185.
Claassen
L
L
Grossman
L
L
Liu
L F
LF
D000255:Adenosine Triphosphate / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004269:DNA, Bacterial / Q000302:isolation & purification; D004278:DNA, Superhelical / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D009690:Nucleic Acid Conformation; D010957:Plasmids; D013698:Templates, Genetic
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D004706:Endodeoxyribonucleases; C036385:endodeoxyribonuclease uvrABC; D004264:DNA Topoisomerases, Type I