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 b een unclear, since M(r) of 250,000-655,000 have been previously report ed. We have isolated two oligomeric forms of the enzyme, one (monomeri c) 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) approximate to 3 30,000) can form a stable initiation complex on the end of ds DNA. Dep ending on the nature of the ds end, K-D estimates ranged from approxim ate to 0.1 nM to approximate to 0.7 nM in the presence of Mg2+ ions, w hich 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 p roposed that uses repeated conformational changes between two states o f a complex between DNA and a dimeric form of the enzyme. Our results make such a model unlikely for RecBCD enzyme.