Monomeric RecBCD enzyme binds and unwinds DNA.
RecBCD enzyme is a multifunctional nuclease that is essential for the major pathway of homologous genetic recombination in Escherichia coli. It has a potent helicase activity that uses ATP hydrolysis to unwind very long stretches of DNA. The functional form of RecBCD enzyme has been unclear, since M(r) of 250,000-655,000 have been previously reported. We have isolated two oligomeric forms of the enzyme, one (monomeric) containing a single copy of the RecB, RecC, and RecD polypeptides, and the other (dimeric) containing two copies of each polypeptide. We show here that the monomeric form of the enzyme (M(r) approximately 330,000) can form a stable initiation complex on the end of ds DNA. Depending on the nature of the ds end, KD estimates ranged from approximately 0.1 nM to approximately 0.7 nM in the presence of Mg2+ ions, which enhanced but was not required for binding. We further showed that the complex of monomeric RecBCD enzyme and a ds DNA end was competent to unwind DNA. A general model for the action of helicases has been proposed that uses repeated conformational changes between two states of a complex between DNA and a dimeric form of the enzyme. Our results make such a model unlikely for RecBCD enzyme.
Taylor
A F
AF
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104, USA.
Smith
G R
GR
D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; C468322:exodeoxyribonuclease V, E coli; D004265:DNA Helicases