The recBC Deoxyribonuclease of Escherichia coli K-12

Abstract The behavior of the recBC DNase of Escherichia coli with a variety of substrates has been investigated. Duplex circular DNA is resistant to digestion, is not a cofactor for the DNA-dependent ATPase, and does not effectively compete for enzyme with substrate DNAs. Treatment of duplex circles with ultraviolet light or x-rays does not render them susceptible to the enzyme nor does nicking with pancreatic DNase. In the latter case the nicks are neither sealed nor translocated. Removal of the phosphomonoester group from a nick has no effect, but localized denaturation or removal of approximately five nucleotides at a nick renders duplex circles susceptible to digestion. The enzyme appears to digest linear DNA by a processive mechanism in which the rate is limited by the initiation of digestion of a molecule. It shows no apparent polarity of degradation, nor does it appear to be affected by the presence or absence of terminal phosphate groups. The original terminal nucleotides are dispersed among all classes of the products in a limit digest, so they do not constitute a special type of product. Linear DNA treated with ultraviolet light or x-rays is degraded by the enzyme, but RNA-DNA hybrid is resistant. The latter substrate can serve as cofactor for the DNA-dependent ATPase, however, effectively uncoupling the two activities. At ATP concentrations of 1 mm or greater the enzyme generates early in the reaction large, sedimentable DNA fragments, as well as nonsedimentable products. The large fragments are primarily duplex, but contain single-stranded regions at 5' termini and probably at 3' termini at roughly equal frequency.