Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay.
We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono- and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. The apparent Km values for recBCD enzyme helicase activity on linear M13 dsDNA molecules at 25 degrees C are 0.6 nM dsDNA molecules and 130 microM ATP, respectively. The apparent turnover number for unwinding is approximately 15 microM base pairs s-1 (microM recBCD enzyme)-1. When this rate is corrected for the observed stoichiometry of recBCD enzyme binding to dsDNA, kcat for helicase activity corresponds to an unwinding rate of approximately 250 base pairs of DNA s-1 (functional recBCD complex)-1 at 25 degrees C. At 37 degrees C, the apparent Km value for dsDNA molecules was the same as that at 25 degrees C, but the apparent turnover number became 56 microM base pairs s-1 (microM recBCD enzyme)-1 [or 930 base pairs s-1 (functional recBCD complex)-1 when corrected for observed stoichiometry]. With increasing NaCl concentration, kcat peaks at 100 mM, and the apparent Km value for dsDNA increases by 3-fold at 200 mM NaCl. In the presence of 5 mM calcium acetate, the apparent Km value is increased by 3-fold, and kcat decreased by 20-30%. We have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule.
Roman
L J
LJ
Department of Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611.
Kowalczykowski
S C
SC
D000085:Acetates / Q000494:pharmacology; D019342:Acetic Acid; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009994:Osmolar Concentration; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D000085:Acetates; D012965:Sodium Chloride; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D004264:DNA Topoisomerases, Type I; D019342:Acetic Acid