Photoaffinity labeling of the recBCD enzyme of Escherichia coli with 8-azidoadenosine 5'-triphosphate.

The recB and recD subunits of the recBCD enzyme (exonuclease V) from Escherichia coli were covalently photolabeled with the ATP photoaffinity analogue [alpha-32P]8-azido-ATP. The labeling was specific for ATP binding sites by the following criteria. Saturation occurs at high 8-azido-ATP concentrations with dissociation constants of 30 and 120 microM for the recD and recB subunits, respectively; ATP strongly inhibits the photolabeling; 8-azido-ATP is hydrolyzed by the recBCD enzyme and supports its double-stranded DNA exonuclease activity; and the label is largely confined to two peptides obtained by tryptic digestion of the photolabeled holoenzyme; one is derived from the recB subunit and the other from the recD subunit.