Exonucleolytic cleavage of DNA by the recBC DNase is accompanied by a DNA-dependent ATP hydrolysis that ceases when the DNA has been digested to a limit. On the other hand, DNA that has been crosslinked by 4,5',8-trimethylpsoralen in the presence of 360-nm light remains an effective cofactor in the ATPase reaction, but is resistant to digestion by the enzyme. Psoralentreated DNA is degraded by pancreatic DNase, micrococcal nuclease, and Escherichia coli B restriction enzyme, but not by Neurospora crassa nuclease, suggesting that crosslinking did not grossly distort the duplex structure of the DNA. The psoralen-DNA is not a potent inhibitor, but competes with single-stranded DNA from bacteriophage fd for the recBC DNase to roughly the same extent as does normal duplex DNA. DNA treated with psoralen in the dark, exposed to 360-nm light in the absence of psoralen, or treated with the intercalating agents ethidium bromide, 9-aminoacridine, ICR-191, or actinomycin D, responds to the enzyme no differently from untreated DNA. However, DNA crosslinked with mitomycin C or nitrogen mustard behaves similarly to psoralen-treated DNA. The relationship of these findings to models for the function and control of the recBC ATPase and nuclease, and the advantages of psoralen as a DNA crosslinking agent, are discussed.The recB and recC genes of Escherichia coli control a deoxyribonuclease assumed to function in DNA recombination and rapair. This enzyme degrades duplex DNA exonucleolytically to short oligonucleotides and also catalyzes a DNA-dependent hydrolysis of ATP to ADP and Pi (1, 2). Furthermore, ATP-dependent DNases from other microorganisms have been reported that possess DNA-dependent ATPase activity (3-6). The ATPase reaction of these enzymes is curious in that up to 40 ATP molecules can be hydrolyzed per DNA phosphodiester bond broken, with the exact ratio depending upon the reaction conditions (3-7). In addition, there is no theoretical energy requirement for catalysis of the exothermic DNase reaction.To clarify the relation of ATP hydrolysis to the DNase reaction, we have been studying the action of the recBC nuclease on DNA substrates with well-defined localized perturbations or structural alterations. Cole has demonstrated that introduction of crosslinks into DNA with psoralen is considerably more lethal for E. coli mutants defective in repair or recombination than it is for the wild-type strains (8). Also, crosslinked DNA is an inefficient substrate in vitro for phage A exonuclease, the exonuclease activity of DNA polymerase I, Abbreviations: Psoralen-DNA, DNA that has been crosslinked by irradiation at 360 nm in the presence of psoralen (4,5'-8-trimethyl[furano-3',2':6,7-coumarin]); recBC ATPase or recBC DNase, the DNA-dependent ATPase and the DNase activities catalyzed by the enzyme controlled by the reeB and recC genes of E. coli. The DNase activity has also been referred to as exonuclease V (2). and other enzymes likely to be involved in recombination and repair (9). These findings prompted us to obse...