The mechanism of degradation of duplex deoxyribonucleic acid by the recBC enzyme of Escherichia coli K-12.

Reaction intermediates formed during the degradation of duplex DNA by the recBC DNase have been characterized by sedimentation velocity, isopyknic centrifugation, electron microscopy, and susceptibility to various deoxyribonucleases which are specific for single-stranded DNA. It has been concluded that the intermediates are of two classes: (a) duplex molecules with single-stranded tails of several thousand nucleotides at the 3’ and the 5’ termini and (b) singlestranded fragments several hundred nucleotides in length. To explain the presence of these intermediates, even in synchronized reactions with DNA molecules in molar excess over enzyme, a reaction scheme for the degradation of duplex DNA is proposed. According to this scheme, the DNase unwinds the strands of the duplex from an end and clips off fragments several hundred nucleotides long from one strand, while remaining bound to the terminus of the undegraded strand. When a single-stranded tail of up to several thousand nucleotides is formed in this way, the enzyme switches strands and degrades the tail from its terminus, yielding a shortened, wholly duplex molecule. The cycle is then repeated until the molecule is entirely digested. The scheme recognizes the processive nature of the degradation of duplex DNA by this enzyme and hypothesizes that the ATP hydrolysis which accompanies DNA degradation is required for the unwinding reaction.