RESUMEN
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.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Mycobacterium tuberculosis/fisiología , ARN Ribosómico/genética , Tuberculosis/microbiología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/metabolismo , Daño del ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ratones , Viabilidad Microbiana , Datos de Secuencia Molecular , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genética , Estrés Oxidativo , Regiones Promotoras Genéticas , ARN Ribosómico/metabolismo , Alineación de Secuencia , Factores de Transcripción/metabolismo , Transcripción Genética , Regulación hacia ArribaRESUMEN
Mycobacterial UvrD2 is a DNA-dependent ATPase with 3' to 5' helicase activity. UvrD2 is an atypical helicase, insofar as its N-terminal ATPase domain resembles the superfamily I helicases UvrD/PcrA, yet it has a C-terminal HRDC domain, which is a feature of RecQ-type superfamily II helicases. The ATPase and HRDC domains are connected by a CxxC-(14)-CxxC tetracysteine module that defines a new clade of UvrD2-like bacterial helicases found only in Actinomycetales. By characterizing truncated versions of Mycobacterium smegmatis UvrD2, we show that whereas the HRDC domain is not required for ATPase or helicase activities in vitro, deletion of the tetracysteine module abolishes duplex unwinding while preserving ATP hydrolysis. Replacing each of the CxxC motifs with a double-alanine variant AxxA had no effect on duplex unwinding, signifying that the domain module, not the cysteines, is crucial for function. The helicase activity of a truncated UvrD2 lacking the tetracysteine and HRDC domains was restored by the DNA-binding protein Ku, a component of the mycobacterial NHEJ system and a cofactor for DNA unwinding by the paralogous mycobacterial helicase UvrD1. Our findings indicate that coupling of ATP hydrolysis to duplex unwinding can be achieved by protein domains acting in cis or trans. Attempts to disrupt the M. smegmatis uvrD2 gene were unsuccessful unless a second copy of uvrD2 was present elsewhere in the chromosome, indicating that UvrD2 is essential for growth of M. smegmatis.
Asunto(s)
Adenosina Trifosfato/química , Proteínas Bacterianas/química , ADN Helicasas/química , Mycobacterium smegmatis/enzimología , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Mycobacterium smegmatis/genética , Estructura Terciaria de Proteína/genética , Eliminación de SecuenciaRESUMEN
Bacterial nonhomologous end joining (NHEJ) is a recently described DNA repair pathway best characterized in mycobacteria. Bacterial NHEJ proteins LigD and Ku have been analyzed biochemically, and their roles in linear plasmid repair in vivo have been verified genetically; yet the contributions of NHEJ to repair of chromosomal DNA damage are unknown. Here we use an extensive set of NHEJ- and homologous recombination (HR)-deficient Mycobacterium smegmatis strains to probe the importance of HR and NHEJ in repairing diverse types of chromosomal DNA damage. An M. smegmatis Delta recA Delta ku double mutant has no apparent growth defect in vitro. Loss of the NHEJ components Ku and LigD had no effect on sensitivity to UV radiation, methyl methanesulfonate, or quinolone antibiotics. NHEJ deficiency had no effect on sensitivity to ionizing radiation in logarithmic- or early-stationary-phase cells but was required for ionizing radiation resistance in late stationary phase in 7H9 but not LB medium. In addition, NHEJ components were required for repair of I-SceI mediated chromosomal double-strand breaks (DSBs), and in the absence of HR, the NHEJ pathway rapidly mutates the chromosomal break site. The molecular outcomes of NHEJ-mediated chromosomal DSB repair involve predominantly single-nucleotide insertions at the break site, similar to previous findings using plasmid substrates. These findings demonstrate that prokaryotic NHEJ is specifically required for DSB repair in late stationary phase and can mediate mutagenic repair of homing endonuclease-generated chromosomal DSBs.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Mycobacterium/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Rotura Cromosómica/efectos de los fármacos , Rotura Cromosómica/efectos de la radiación , Cromosomas Bacterianos/efectos de los fármacos , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/efectos de la radiación , Daño del ADN , ADN Ligasas/genética , ADN Ligasas/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Rayos gamma , Metilmetanosulfonato/farmacología , Viabilidad Microbiana/efectos de los fármacos , Viabilidad Microbiana/genética , Viabilidad Microbiana/efectos de la radiación , Modelos Genéticos , Mutación , Mycobacterium/efectos de los fármacos , Mycobacterium/efectos de la radiación , Fenotipo , Quinolonas/farmacología , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Proteínas de Saccharomyces cerevisiae , Rayos UltravioletaRESUMEN
Mycobacterium tuberculosis and other bacterial pathogens have a Ku-dependent nonhomologous end joining pathway of DNA double-strand break repair. Here we identify mycobacterial UvrD1 as a novel interaction partner for Ku in a genome-wide yeast two-hybrid screen. UvrD1 per se is a vigorous DNA-dependent ATPase but a feeble DNA helicase. Ku stimulates UvrD1 to catalyze ATP-dependent unwinding of 3'-tailed DNAs. UvrD1, Ku, and DNA form a stable ternary complex in the absence of ATP. The Ku binding determinants are located in the distinctive C-terminal segment of UvrD1. A second mycobacterial paralog, UvrD2, is a vigorous Ku-independent DNA helicase. Ablation of UvrD1 sensitizes Mycobacterium smegmatis to killing by ultraviolet and ionizing radiation and to a single chromosomal break generated by I-SceI endonuclease. The physical and functional interactions of bacterial Ku and UvrD1 highlight the potential for cross-talk between components of nonhomologous end joining and nucleotide excision repair pathways.
Asunto(s)
Proteínas Bacterianas/metabolismo , Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Reparación del ADN/fisiología , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/enzimología , Proteínas Bacterianas/genética , Roturas del ADN de Doble Cadena/efectos de la radiación , ADN Helicasas/genética , Reparación del ADN/efectos de la radiación , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Rayos gamma , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Unión Proteica/fisiología , Unión Proteica/efectos de la radiación , Estructura Terciaria de Proteína/fisiología , Proteínas de Saccharomyces cerevisiae , Rayos UltravioletaRESUMEN
DNA double-strand breaks (DSBs) can be repaired either via homologous recombination (HR) or nonhomologous end-joining (NHEJ). Both pathways are operative in eukaryotes, but bacteria had been thought to rely on HR alone. Here we provide direct evidence that mycobacteria have a robust NHEJ pathway that requires Ku and a specialized polyfunctional ATP-dependent DNA ligase (LigD). NHEJ of blunt-end and complementary 5'-overhang DSBs is highly mutagenic ( approximately 50% error rate). Analysis of the recombination junctions ensuing from individual NHEJ events highlighted the participation of several DNA end-remodeling activities, including template-dependent fill-in of 5' overhangs, nontemplated addition of single nucleotides at blunt ends, and nucleolytic resection. LigD itself has the template-dependent and template-independent polymerase functions in vitro that compose the molecular signatures of NHEJ in vivo. Another ATP-dependent DNA ligase (LigC) provides a backup mechanism for LigD-independent error-prone repair of blunt-end DSBs. We speculate that NHEJ allows mycobacteria to evade genotoxic host defense.
Asunto(s)
Antígenos Nucleares/metabolismo , Proteínas Bacterianas/metabolismo , ADN Ligasas/metabolismo , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Mycobacterium smegmatis/metabolismo , Recombinación Genética/genética , Secuencia de Bases , ADN/química , ADN/genética , ADN/metabolismo , Daño del ADN , ADN Ligasa (ATP) , ADN Ligasas/genética , Autoantígeno Ku , Mutación/genética , Mycobacterium smegmatis/genética , Unión Proteica , Moldes GenéticosRESUMEN
The execution of apoptosis or programmed cell death comprises both caspase-dependent and caspase-independent processes. Apoptosis inducing factor (AIF) was identified as a major player in caspase-independent cell death. It induces chromatin condensation and initial DNA cleavage via an unknown molecular mechanism. Here we report the crystal structure of human AIF at 1.8 A resolution. The structure reveals the presence of a strong positive electrostatic potential at the AIF surface, although the calculated isoelectric point for the entire protein is neutral. We show that recombinant AIF interacts with DNA in a sequence-independent manner. In addition, in cells treated with an apoptotic stimulus, endogenous AIF becomes co-localized with DNA at an early stage of nuclear morphological changes. Structure-based mutagenesis shows that DNA-binding defective mutants of AIF fail to induce cell death while retaining nuclear translocation. The potential DNA-binding site identified from mutagenesis also coincides with computational docking of a DNA duplex. These observations suggest that AIF-induced nuclear apoptosis requires a direct interaction with DNA.