Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecBlike nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (k cat 415 s ؊1 ), and the affinity of the motor for the ssDNA cofactor increases 140-fold as DNA length is extended from 12 to 44 nucleotides. Using a streptavidin displacement assay, we demonstrate that AdnAB is a 3 3 5 translocase on ssDNA. AdnAB binds stably to DSB ends. In the presence of ATP, the motor unwinds the DNA duplex without requiring an ssDNA loading strand. We integrate these findings into a model of DSB unwinding in which the "leading" AdnB and "lagging" AdnA motor domains track in tandem, 3 to 5, along the same DNA single strand. This contrasts with RecBCD, in which the RecB and RecD motors track in parallel along the two separated DNA single strands. The effects of 5 and 3 terminal obstacles on ssDNA cleavage by wild-type AdnAB suggest that the AdnA nuclease receives and processes the displaced 5 strand, while the AdnB nuclease cleaves the displaced 3 strand. We present evidence that the distinctive "molecular ruler" function of the ATP-dependent single strand DNase, whereby AdnAB measures the distance from the 5-end to the sites of incision, reflects directional pumping of the ssDNA through the AdnAB motor into the AdnB nuclease. These and other findings suggest a scenario for the descent of the RecBCD-and AddABtype DSB-processing machines from an ancestral AdnAB-like enzyme.DNA repair systems play an important role in bacterial pathogenesis, by allowing disease-causing bacteria to withstand DNAdamaging mediators of host innate immune systems (1-6). Mycobacteria, including the agent of human tuberculosis, have two distinct pathways to repair DNA double-strand breaks (DSBs), 2 the most lethal form of DNA damage: (i) a RecA-dependent homologous recombination (HR) system and (ii) a non-homologous end-joining (NHEJ) system driven by dedicated DNA ligases and the end-binding protein Ku (7-11). In HR, an intact copy of the broken chromosome segment, typically a newly replicated sister chromatid, serves as a template for DNA synthesis across the break, ultimately yielding a faithfully restored chromosome with no mutations. By contrast, NHEJ protects the bacterial chromosome against DSBs during quiescent states, when there is no sister chromatid available to direct HR (12, 13). The recent finding that dormant mycobacteria develop into spores containing a single chromosome (14) suggests a role for NHEJ in spore viability under conditions of clastogenic stress, as demonstrated for the classic spore-forming bacterium, Bacillus subtilis (15, 16). The mycobacterial NHEJ mechanism, unlike that of HR, is conspicuously mutagenic (9, 11).Nucleolytic resection of DSB ends is an essentia...