Control of T cell antigen reactivity via programmed TCR downregulation.

The T cell antigen receptor (TCR) is unique in that its affinity for ligand is unknown before encounter and can vary by orders of magnitude. How the immune system regulates individual T cells that display very different reactivity to antigen remains unclear. Here we found that activated CD4(+) T cells, at the peak of clonal expansion, persistently downregulated their TCR expression in proportion to the strength of the initial antigen recognition. This programmed response increased the threshold for cytokine production and recall proliferation in a clone-specific manner and ultimately excluded clones with the highest antigen reactivity. Thus, programmed downregulation of TCR expression represents a negative feedback mechanism for constraining T cell effector function with a suitable time delay to thereby allow pathogen control while avoiding excess inflammatory damage.

Converting cancer therapies into cures: lessons from infectious diseases.

During the past decade, cancer drug development has shifted from a focus on cytotoxic chemotherapies to drugs that target specific molecular alterations in tumors. Although these drugs dramatically shrink tumors, the responses are temporary. Research is now focused on overcoming drug resistance, a frequent cause of treatment failure. Here we reflect on analogous challenges faced by researchers in infectious diseases. We compare and contrast the resistance mechanisms arising in cancer and infectious diseases and discuss how approaches for overcoming viral and bacterial infections, such as HIV and tuberculosis, are instructive for developing a more rational approach for cancer therapy. In particular, maximizing the effect of the initial treatment response, which often requires synergistic combination therapy, is foremost among these approaches. A remaining challenge in both fields is identifying drugs that eliminate drug-tolerant "persister" cells (infectious disease) or tumor-initiating/stem cells (cancer) to prevent late relapse and shorten treatment duration.

Mycobacterial Mutagenesis and Drug Resistance Are Controlled by Phosphorylation- and Cardiolipin-Mediated Inhibition of the RecA Coprotease.

Infection with Mycobacterium tuberculosis continues to cause substantial human mortality, in part because of the emergence of antimicrobial resistance. Antimicrobial resistance in tuberculosis is solely the result of chromosomal mutations that modify drug activators or targets, yet the mechanisms controlling the mycobacterial DNA-damage response (DDR) remain incompletely defined. Here, we identify RecA serine 207 as a multifunctional signaling hub that controls the DDR in mycobacteria. RecA S207 is phosphorylated after DNA damage, which suppresses the emergence of antibiotic resistance by selectively inhibiting the LexA coprotease function of RecA without affecting its ATPase or strand exchange functions. Additionally, RecA associates with the cytoplasmic membrane during the mycobacterial DDR, where cardiolipin can specifically inhibit the LexA coprotease function of unmodified, but not S207 phosphorylated, RecA. These findings reveal that RecA S207 controls mutagenesis and antibiotic resistance in mycobacteria through phosphorylation and cardiolipin-mediated inhibition of RecA coprotease function.

TOX is a critical regulator of tumour-specific T cell differentiation.

Tumour-specific CD8 T cell dysfunction is a differentiation state that is distinct from the functional effector or memory T cell states1-6. Here we identify the nuclear factor TOX as a crucial regulator of the differentiation of tumour-specific T (TST) cells. We show that TOX is highly expressed in dysfunctional TST cells from tumours and in exhausted T cells during chronic viral infection. Expression of TOX is driven by chronic T cell receptor stimulation and NFAT activation. Ectopic expression of TOX in effector T cells in vitro induced a transcriptional program associated with T cell exhaustion. Conversely, deletion of Tox in TST cells in tumours abrogated the exhaustion program: Tox-deleted TST cells did not upregulate genes for inhibitory receptors (such as Pdcd1, Entpd1, Havcr2, Cd244 and Tigit), the chromatin of which remained largely inaccessible, and retained high expression of transcription factors such as TCF-1. Despite their normal, 'non-exhausted' immunophenotype, Tox-deleted TST cells remained dysfunctional, which suggests that the regulation of expression of inhibitory receptors is uncoupled from the loss of effector function. Notably, although Tox-deleted CD8 T cells differentiated normally to effector and memory states in response to acute infection, Tox-deleted TST cells failed to persist in tumours. We hypothesize that the TOX-induced exhaustion program serves to prevent the overstimulation of T cells and activation-induced cell death in settings of chronic antigen stimulation such as cancer.

Mycobacterial helicase Lhr abets resistance to DNA crosslinking agents mitomycin C and cisplatin.

Mycobacterium smegmatis Lhr exemplifies a novel clade of helicases composed of an N-terminal ATPase/helicase domain (Lhr-Core) and a large C-terminal domain (Lhr-CTD) that nucleates a unique homo-tetrameric quaternary structure. Expression of Lhr, and its operonic neighbor Nei2, is induced in mycobacteria exposed to mitomycin C (MMC). Here we report that lhr deletion sensitizes M. smegmatis to killing by DNA crosslinkers MMC and cisplatin but not to killing by monoadduct-forming alkylating agent methyl methanesulfonate or UV irradiation. Testing complementation of MMC and cisplatin sensitivity by expression of Lhr mutants in Δlhr cells established that: (i) Lhr-CTD is essential for DNA repair activity, such that Lhr-Core does not suffice; (ii) ATPase-defective mutant D170A/E171A fails to complement; (iii) ATPase-active, helicase-defective mutant W597A fails to complement and (iv) alanine mutations at the CTD-CTD interface that interdict homo-tetramer formation result in failure to complement. Our results instate Lhr's ATP-driven motor as an agent of inter-strand crosslink repair in vivo, contingent on Lhr's tetrameric quaternary structure. We characterize M. smegmatis Nei2 as a monomeric enzyme with AP ß-lyase activity on single-stranded DNA. Counter to previous reports, we find Nei2 is inactive as a lyase at a THF abasic site and has feeble uracil glycosylase activity.

Single-Cell Transcriptional Profiling Reveals Signatures of Helper, Effector, and Regulatory MAIT Cells during Homeostasis and Activation.

Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that recognize microbial vitamin B metabolites and have emerging roles in infectious disease, autoimmunity, and cancer. Although MAIT cells are identified by a semi-invariant TCR, their phenotypic and functional heterogeneity is not well understood. Here we present an integrated single cell transcriptomic analysis of over 76,000 human MAIT cells during early and prolonged Ag-specific activation with the MR1 ligand 5-OP-RU and nonspecific TCR stimulation. We show that MAIT cells span a broad range of homeostatic, effector, helper, tissue-infiltrating, regulatory, and exhausted phenotypes, with distinct gene expression programs associated with CD4+ or CD8+ coexpression. During early activation, MAIT cells rapidly adopt a cytotoxic phenotype characterized by high expression of GZMB, IFNG and TNF In contrast, prolonged stimulation induces heterogeneous states defined by proliferation, cytotoxicity, immune modulation, and exhaustion. We further demonstrate a FOXP3 expressing MAIT cell subset that phenotypically resembles conventional regulatory T cells. Moreover, scRNAseq-defined MAIT cell subpopulations were also detected in individuals recently exposed to Mycobacterium tuberculosis, confirming their presence during human infection. To our knowledge, our study provides the first comprehensive atlas of human MAIT cells in activation conditions and defines substantial functional heterogeneity, suggesting complex roles in health and disease.

CarD is an essential regulator of rRNA transcription required for Mycobacterium tuberculosis persistence.

Mycobacterium tuberculosis is arguably the world's most successful infectious agent because of its ability to control its own cell growth within the host. Bacterial growth rate is closely coupled to rRNA transcription, which in E. coli is regulated through DksA and (p)ppGpp. The mechanisms of rRNA transcriptional control in mycobacteria, which lack DksA, are undefined. Here we identify CarD as an essential mycobacterial protein that controls rRNA transcription. Loss of CarD is lethal for mycobacteria in culture and during infection of mice. CarD depletion leads to sensitivity to killing by oxidative stress, starvation, and DNA damage, accompanied by failure to reduce rRNA transcription. CarD can functionally replace DksA for stringent control of rRNA transcription, even though CarD associates with a different site on RNA polymerase. These findings highlight a distinct molecular mechanism for regulating rRNA transcription in mycobacteria that is critical for M. tuberculosis pathogenesis.

The C-Terminal Acid Phosphatase Module of the RNase HI Enzyme RnhC Controls Rifampin Sensitivity and Light-Dependent Colony Pigmentation of Mycobacterium smegmatis.

RNase H enzymes participate in various processes that require processing of RNA-DNA hybrids, including DNA replication, transcription, and ribonucleotide excision from DNA. Mycobacteria encode multiple RNase H enzymes, and prior data indicate that RNase HI activity is essential for mycobacterial viability. However, the additional roles of mycobacterial RNase Hs are unknown, including whether RNase HII (RnhB and RnhD) excises chromosomal ribonucleotides misincorporated during DNA replication and whether individual RNase HI enzymes (RnhA and RnhC) mediate additional phenotypes. We find that loss of RNase HII activity in Mycobacterium smegmatis (through combined deletion of rnhB/rnhD) or individual RNase HI enzymes does not affect growth, hydroxyurea sensitivity, or mutagenesis, whereas overexpression (OE) of either RNase HII severely compromises bacterial viability. We also show that deletion of rnhC, which encodes a protein with an N-terminal RNase HI domain and a C-terminal acid phosphatase domain, confers sensitivity to rifampin and oxidative stress as well as loss of light-induced carotenoid pigmentation. These phenotypes are due to loss of the activity of the C-terminal acid phosphatase domain rather than the RNase HI activity, suggesting that the acid phosphatase activity may confer rifampin resistance through the antioxidant properties of carotenoid pigment production. IMPORTANCE Mycobacteria encode multiple RNase H enzymes, with RNase HI being essential for viability. Here, we examine additional functions of RNase H enzymes in mycobacteria. We find that RNase HII is not involved in mutagenesis but is highly toxic when overexpressed. The RNase HI enzyme RnhC is required for tolerance to rifampin, but this role is surprisingly independent of its RNase H activity and is instead mediated by an autonomous C-terminal acid phosphatase domain. This study provides new insights into the functions of the multiple RNase H enzymes of mycobacteria.

Division of labor between SOS and PafBC in mycobacterial DNA repair and mutagenesis.

DNA repair systems allow microbes to survive in diverse environments that compromise chromosomal integrity. Pathogens such as Mycobacterium tuberculosis must contend with the genotoxic host environment, which generates the mutations that underlie antibiotic resistance. Mycobacteria encode the widely distributed SOS pathway, governed by the LexA repressor, but also encode PafBC, a positive regulator of the transcriptional DNA damage response (DDR). Although the transcriptional outputs of these systems have been characterized, their full functional division of labor in survival and mutagenesis is unknown. Here, we specifically ablate the PafBC or SOS pathways, alone and in combination, and test their relative contributions to repair. We find that SOS and PafBC have both distinct and overlapping roles that depend on the type of DNA damage. Most notably, we find that quinolone antibiotics and replication fork perturbation are inducers of the PafBC pathway, and that chromosomal mutagenesis is codependent on PafBC and SOS, through shared regulation of the DnaE2/ImuA/B mutasome. These studies define the complex transcriptional regulatory network of the DDR in mycobacteria and provide new insight into the regulatory mechanisms controlling the genesis of antibiotic resistance in M. tuberculosis.

A multilayered repair system protects the mycobacterial chromosome from endogenous and antibiotic-induced oxidative damage.

Oxidative damage to DNA is a threat to the genomic integrity and coding accuracy of the chromosomes of all living organisms. Guanine is particularly susceptible to oxidation, and 8-oxo-dG (OG), when produced in situ or incorporated by DNA polymerases, is highly mutagenic due to mispairing with adenine. In many bacteria, defense against OG depends on MutT enzymes, which sanitize OG in the nucleotide pool, and the MutM/Y system, which counteracts OG in chromosomal DNA. In Escherichia coli, antibiotic lethality has been linked to oxidative stress and the downstream consequences of OG processing. However, in mycobacteria, the role of these systems in genomic integrity and antibiotic lethality is not understood, in part because mycobacteria encode four MutT enzymes and two MutMs, suggesting substantial redundancy. Here, we definitively probe the role of OG handling systems in mycobacteria. We find that, although MutT4 is the only MutT enzyme required for resistance to oxidative stress, this effect is not due to OG processing. We find that the dominant system that defends against OG-mediated mutagenesis is MutY/MutM1, and this system is dedicated to in situ chromosomal oxidation rather than correcting OG incorporated by accessory polymerases (DinB1/DinB2/DinB3/DnaE2). In addition, we uncover that mycobacteria resist antibiotic lethality through nucleotide sanitization by MutTs, and in the absence of this system, accessory DNA polymerases and MutY/M contribute to antibiotic-induced lethality. These results reveal a complex, multitiered system of OG handling in mycobacteria with roles in oxidative stress resistance, mutagenesis, and antibiotic lethality.

Bacterial immunotherapy for cancer induces CD4-dependent tumor-specific immunity through tumor-intrinsic interferon-γ signaling.

Bacillus Calmette-Guérin (BCG) immunotherapy for bladder cancer is the only bacterial cancer therapy approved for clinical use. Although presumed to induce T cell-mediated immunity, whether tumor elimination depends on bacteria-specific or tumor-specific immunity is unknown. Herein we show that BCG-induced bladder tumor elimination requires CD4 and CD8 T cells, although augmentation or inhibition of bacterial antigen-specific T cell responses does not alter the efficacy of BCG-induced tumor elimination. In contrast, BCG stimulates long-term tumor-specific immunity that primarily depends on CD4 T cells. We demonstrate that BCG therapy results in enhanced effector function of tumor-specific CD4 T cells, mainly through enhanced production of IFN-γ. Accordingly, BCG-induced tumor elimination and tumor-specific immune memory require tumor cell expression of the IFN-γ receptor, but not MHC class II. Our findings establish that a bacterial immunotherapy for cancer is capable of inducing tumor immunity, an antitumor effect that results from enhanced function of tumor-specific CD4 T cells, and ultimately requires tumor-intrinsic IFN-γ signaling, via a mechanism that is distinct from other tumor immunotherapies.

Mycobacterium tuberculosis protease MarP activates a peptidoglycan hydrolase during acid stress.

Mycobacterium tuberculosis (Mtb) can persist in the human host in a latent state for decades, in part because it has the ability to withstand numerous stresses imposed by host immunity. Prior studies have established the essentiality of the periplasmic protease MarP for Mtb to survive in acidified phagosomes and establish and maintain infection in mice. However, the proteolytic substrates of MarP that mediate these phenotypes were unknown. Here, we used biochemical methods coupled with supravital chemical probes that facilitate imaging of nascent peptidoglycan to demonstrate that during acid stress MarP cleaves the peptidoglycan hydrolase RipA, a process required for RipA's activation. Failure of RipA processing in MarP-deficient cells leads to cell elongation and chain formation, a hallmark of progeny cell separation arrest. Our results suggest that sustaining peptidoglycan hydrolysis, a process required for cell elongation, separation of progeny cells, and cell wall homeostasis in growing cells, may also be essential for Mtb's survival in acidic conditions.

Efficient 5-OP-RU-Induced Enrichment of Mucosa-Associated Invariant T Cells in the Murine Lung Does Not Enhance Control of Aerosol Mycobacterium tuberculosis Infection.

Mucosa-associated invariant T (MAIT) cells are an innate-like T cell subset in mammals that recognize microbial vitamin B metabolites presented by the evolutionarily conserved major histocompatibility complex class I (MHC I)-related molecule, MR1. Emerging data suggest that MAIT cells may be an attractive target for vaccine-induced protection against bacterial infections because of their rapid cytotoxic responses at mucosal services to a widely conserved bacterial ligand. In this study, we tested whether a MAIT cell priming strategy could protect against aerosol Mycobacterium tuberculosis infection in mice. Intranasal costimulation with the lipopeptide Toll-like receptor (TLR)2/6 agonist, Pam2Cys (P2C), and the synthetic MR1 ligand, 5-OP-RU, resulted in robust expansion of MAIT cells in the lung. Although MAIT cell priming significantly enhanced MAIT cell activation and expansion early after M. tuberculosis challenge, these MAIT cells did not restrict M. tuberculosis bacterial load. MAIT cells were depleted by the onset of the adaptive immune response, with decreased detection of granzyme B+ and gamma interferon (IFN-γ)+ MAIT cells relative to that in uninfected P2C/5-OP-RU-treated mice. Decreasing the infectious inoculum, varying the time between priming and aerosol infection, and testing MAIT cell priming in nitric oxide synthase 2 (NOS2)-deficient mice all failed to reveal an effect of P2C/5-OP-RU-induced MAIT cells on M. tuberculosis control. We conclude that intranasal MAIT cell priming in mice induces early MAIT cell activation and expansion after M. tuberculosis exposure, without attenuating M. tuberculosis growth, suggesting that MAIT cell enrichment in the lung is not sufficient to control M. tuberculosis infection.

Division of labor among Mycobacterium smegmatis RNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase.

RNase H enzymes sense the presence of ribonucleotides in the genome and initiate their removal by incising the ribonucleotide-containing strand of an RNA:DNA hybrid. Mycobacterium smegmatis encodes four RNase H enzymes: RnhA, RnhB, RnhC and RnhD. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of RnhA. We report that RnhA (like RnhC characterized previously) is an RNase H1-type magnesium-dependent endonuclease with stringent specificity for RNA:DNA hybrid duplexes. Whereas RnhA does not incise an embedded mono-ribonucleotide, it can efficiently cleave within tracts of four or more ribonucleotides in duplex DNA. We gained genetic insights to the division of labor among mycobacterial RNases H by deleting the rnhA, rnhB, rnhC and rnhD genes, individually and in various combinations. The salient conclusions are that: (i) RNase H1 activity is essential for mycobacterial growth and can be provided by either RnhC or RnhA; (ii) the RNase H2 enzymes RnhB and RnhD are dispensable for growth and (iii) RnhB and RnhA collaborate to protect M. smegmatis against oxidative damage in stationary phase. Our findings highlight RnhC, the sole RNase H1 in pathogenic mycobacteria, as a candidate drug discovery target for tuberculosis and leprosy.

Homologous recombination mediated by the mycobacterial AdnAB helicase without end resection by the AdnAB nucleases.

Current models of bacterial homologous recombination (HR) posit that extensive resection of a DNA double-strand break (DSB) by a multisubunit helicase-nuclease machine (e.g. RecBCD, AddAB or AdnAB) generates the requisite 3' single-strand DNA substrate for RecA-mediated strand invasion. AdnAB, the helicase-nuclease implicated in mycobacterial HR, consists of two subunits, AdnA and AdnB, each composed of an N-terminal ATPase domain and a C-terminal nuclease domain. DSB unwinding by AdnAB in vitro is stringently dependent on the ATPase activity of the 'lead' AdnB motor translocating on the 3' ssDNA strand, but not on the putative 'lagging' AdnA ATPase. Here, we queried genetically which activities of AdnAB are pertinent to its role in HR and DNA damage repair in vivo by inactivating each of the four catalytic domains. Complete nuclease-dead AdnAB enzyme can sustain recombination in vivo, as long as its AdnB motor is intact and RecO and RecR are available. We conclude that AdnAB's processive DSB unwinding activity suffices for AdnAB function in HR. Albeit not excluding the agency of a backup nuclease, our findings suggest that mycobacterial HR can proceed via DSB unwinding and protein capture of the displaced 3'-OH single strand, without a need for extensive end-resection.

Reconstitution of a Mycobacterium tuberculosis proteostasis network highlights essential cofactor interactions with chaperone DnaK.

During host infection, Mycobacterium tuberculosis (Mtb) encounters several types of stress that impair protein integrity, including reactive oxygen and nitrogen species and chemotherapy. The resulting protein aggregates can be resolved or degraded by molecular machinery conserved from bacteria to eukaryotes. Eukaryotic Hsp104/Hsp70 and their bacterial homologs ClpB/DnaK are ATP-powered chaperones that restore toxic protein aggregates to a native folded state. DnaK is essential in Mycobacterium smegmatis, and ClpB is involved in asymmetrically distributing damaged proteins during cell division as a mechanism of survival in Mtb, commending both proteins as potential drug targets. However, their molecular partners in protein reactivation have not been characterized in mycobacteria. Here, we reconstituted the activities of the Mtb ClpB/DnaK bichaperone system with the cofactors DnaJ1, DnaJ2, and GrpE and the small heat shock protein Hsp20. We found that DnaJ1 and DnaJ2 activate the ATPase activity of DnaK differently. A point mutation in the highly conserved HPD motif of the DnaJ proteins abrogates their ability to activate DnaK, although the DnaJ2 mutant still binds to DnaK. The purified Mtb ClpB/DnaK system reactivated a heat-denatured model substrate, but the DnaJ HPD mutants inhibited the reaction. Finally, either DnaJ1 or DnaJ2 is required for mycobacterial viability, as is the DnaK-activating activity of a DnaJ protein. These studies lay the groundwork for strategies to target essential chaperone-protein interactions in Mtb, the leading cause of death from a bacterial infection.

Challenges for the MD Physician-Scientist Upon Entering the Lab: From the Grand to the Practical.

Physicians who seek to become laboratory-based physician-scientists, especially those without a PhD degree, face substantial challenges during the critical transition period between clinical and laboratory training. These challenges range from the complex process of adopting new logic structures and standards of proof to the more mundane but important challenges that accompany extending training in a new discipline when other career options are available. Discussion of these challenges can inform individual and institutional strategies to enhance entry and retention of physicians in the physician-scientist career path.

Control of biotin biosynthesis in mycobacteria by a pyruvate carboxylase dependent metabolic signal.

Biotin is an essential cofactor utilized by all domains of life, but only synthesized by bacteria, fungi and plants, making biotin biosynthesis a target for antimicrobial development. To understand biotin biosynthesis in mycobacteria, we executed a genetic screen in Mycobacterium smegmatis for biotin auxotrophs and identified pyruvate carboxylase (Pyc) as required for biotin biosynthesis. The biotin auxotrophy of the pyc::tn strain is due to failure to transcriptionally induce late stage biotin biosynthetic genes in low biotin conditions. Loss of bioQ, the repressor of biotin biosynthesis, in the pyc::tn strain reverted biotin auxotrophy, as did reconstituting the last step of the pathway through heterologous expression of BioB and provision of its substrate DTB. The role of Pyc in biotin regulation required its catalytic activities and could be supported by M. tuberculosis Pyc. Quantitation of the kinetics of depletion of biotinylated proteins after biotin withdrawal revealed that Pyc is the most rapidly depleted biotinylated protein and metabolomics revealed a broad metabolic shift in wild type cells upon biotin withdrawal which was blunted in cell lacking Pyc. Our data indicate that mycobacterial cells monitor biotin sufficiency through a metabolic signal generated by dysfunction of a biotinylated protein of central metabolism.

An essential nonredundant role for mycobacterial DnaK in native protein folding.

Protein chaperones are essential in all domains of life to prevent and resolve protein misfolding during translation and proteotoxic stress. HSP70 family chaperones, including E. coli DnaK, function in stress induced protein refolding and degradation, but are dispensable for cellular viability due to redundant chaperone systems that prevent global nascent peptide insolubility. However, the function of HSP70 chaperones in mycobacteria, a genus that includes multiple human pathogens, has not been examined. We find that mycobacterial DnaK is essential for cell growth and required for native protein folding in Mycobacterium smegmatis. Loss of DnaK is accompanied by proteotoxic collapse characterized by the accumulation of insoluble newly synthesized proteins. DnaK is required for solubility of large multimodular lipid synthases, including the essential lipid synthase FASI, and DnaK loss is accompanied by disruption of membrane structure and increased cell permeability. Trigger Factor is nonessential and has a minor role in native protein folding that is only evident in the absence of DnaK. In unstressed cells, DnaK localizes to multiple, dynamic foci, but relocalizes to focal protein aggregates during stationary phase or upon expression of aggregating peptides. Mycobacterial cells restart cell growth after proteotoxic stress by isolating persistent DnaK containing protein aggregates away from daughter cells. These results reveal unanticipated essential nonredunant roles for mycobacterial DnaK in mycobacteria and indicate that DnaK defines a unique susceptibility point in the mycobacterial proteostasis network.

AdnAB: a new DSB-resecting motor-nuclease from mycobacteria.

The resection of DNA double-strand breaks (DSBs) in bacteria is a motor-driven process performed by a multisubunit helicase-nuclease complex: either an Escherichia coli-type RecBCD enzyme or a Bacillus-type AddAB enzyme. Here we identify mycobacterial AdnAB as the founder of a new family of heterodimeric helicase-nucleases with distinctive properties. The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal nuclease module. The AdnAB ATPase is triggered by dsDNA with free ends and the energy of ATP hydrolysis is coupled to DSB end resection by the AdnAB nuclease. The mycobacterial nonhomologous end-joining (NHEJ) protein Ku protects DSBs from resection by AdnAB. We find that AdnAB incises ssDNA by measuring the distance from the free 5' end to dictate the sites of cleavage, which are predominantly 5 or 6 nucleotides (nt) from the 5' end. The "molecular ruler" of AdnAB is regulated by ATP, which elicits an increase in ssDNA cleavage rate and a distal displacement of the cleavage sites 16-17 nt from the 5' terminus. AdnAB is a dual nuclease with a clear division of labor between the subunits. Mutations in the nuclease active site of the AdnB subunit ablate the ATP-inducible cleavages; the corresponding changes in AdnA abolish ATP-independent cleavage. Complete suppression of DSB end resection requires simultaneous mutation of both subunit nucleases. The nuclease-null AdnAB is a helicase that unwinds linear plasmid DNA without degrading the displaced single strands. Mutations of the phosphohydrolase active site of the AdnB subunit ablate DNA-dependent ATPase activity, DSB end resection, and ATP-inducible ssDNA cleavage; the equivalent mutations of the AdnA subunit have comparatively little effect. AdnAB is a novel signature of the Actinomycetales taxon. Mycobacteria are exceptional in that they encode both AdnAB and RecBCD, suggesting the existence of alternative end-resecting motor-nuclease complexes.