The Isolation and Characterization from Escherichia coli of an Adenosine Triphosphate-dependent Deoxyribonuclease Directed by rec B, C Genes

Abstract Extracts of wild type Escherichia coli contain a DNase which catalyzes the release of acid-soluble fragments from native DNA; this activity cannot be detected in rec B and C mutants. The properties of this enzyme have been studied with a fraction purified 200- to 600-fold. Only in the presence of ATP does the purified enzyme act on native DNA; with denatured DNA this ATP requirement is not absolute. The purified fraction requires Mg++ and is active over a broad pH range with an optimum of about 9.2. The exonuclease action of the purified enzyme fraction is due to two exonuclease activities. (a) An exonuclease, which we have called exonuclease V, acts on double-stranded DNA only in the presence of an activating nucleoside triphosphate, ATP and dATP being the most efficient. During DNA degradation, ATP is cleaved to ADP and Pi. The products formed during the degradation of native DNA are composed of 3'-hydroxyl- and 5'-phosphate-ended oligonucleotides, the average size of which decreases as the reaction proceeds. Exonuclease V attacks DNA from ends generated by double-stranded breakage and does not attack circular double-stranded DNA molecules with or without single-stranded breaks. Also associated with exonuclease V is an ATP-dependent cleavage of single-stranded DNA. (b) A 3'-exonuclease, acts on single-stranded DNA in the absence of ATP, and whose properties are distinguishable from those of exonuclease I, is responsible for the other exonuclease activity. The possible role of exonuclease V in DNA recombination is discussed.