Michael S. Glickman: Double-Strand Break Repair in Mycobacteria: Novel pathways of repair and response

A new model system for prokaryotic DNA repair

The Glickman lab, in close collaboration with the Shuman lab, is broadly interested in the pathways and regulation of double-strand DNA break repair in mycobacteria. Our goals in the DNA repair arena are twofold: 1) to understand the pathways and regulation of double-strand break (DSB) repair in mycobacteria as a new model system for prokaryotic repair and 2) to use the knowledge gathered to understand the role of DSB repair in M. tuberculosis pathogenesis. The textbook model of double-strand DNA break repair in bacteria is derived predominantly from studies in E. coli, which elaborates homologous recombination (HR) as its major pathway of DSB repair. The presence of a single pathway of DSB repair in bacteria reinforced a general notion of bacterial cells as “simple” in comparison to their eukaryotic counterparts, which elaborate three DSB repair pathways. These three pathways, HR, nonhomologous end-joining (NHEJ), and single-strand annealing (SSA) have distinct protein mediators, repair DSBs in varying phases of the cell cycle, and have different requirements for homology donors or repeats. The NHEJ pathway was thought absent from prokaryotes. However, the identification of orthologs of the Ku protein, a central mediator of eukaryotic NHEJ, in bacterial proteomes suggested that some bacteria may encode an NHEJ pathway.

We demonstrated the existence of NHEJ in bacteria, first using a plasmid recircularization assay that simultaneously determined NHEJ efficiency and fidelity.(1)(2) These experiments immediately revealed an important feature of mycobacterial NHEJ: the pathway is mutagenic through addition of templated and nontemplated nucleotides during repair, a feature that we traced directly to an autonomous primase-polymerase domain of the LigD enzyme.(2)(3)(4) We have shown the importance of the NHEJ pathway for repair of DSBs in nonreplicating mycobacteria(5) (reminiscent of its function in eukaryotic cells), the influence of end configuration on NHEJ mediated repair(6), the existence of a “backup” NHEJ pathway that uses yet another ATP dependent DNA ligase(2)(6)(7), and the function of NHEJ in repairing homing-endonuclease-induced DSBs in the chromosome.(5)(8) A novel feature of the mycobacterial NHEJ system is the multifunctional LigD enzyme, which contains three autonomous enzymatic modules. LigD-Pol has a preference for ribonucleotide addition over dNTPs.(2)Similarly, the third domain of LigD, PE, is a 3’ end-healing enzyme that will resect ribonucleotides.(3)(6)Taken together, our results strongly indicate that the mutagenic mycobacterial NHEJ system, acting through the three catalytic modules of LigD, uses ribonucleotides to repair DSBs that arise during states of nonreplication, when dNTP pools are likely to be limiting.

The NHEJ system has potentially great relevance to the pathogenesis of M. tuberculosis infection. The hallmark of Mtb infection is a long period of clinical latency characterized by a lack of bacterial replication. It is well known that the products of mammalian immunity damage pathogen chromosomal DNA and that diverse bacterial pathogens rely on DNA repair systems to survive within the host. In this context, NHEJ would be potentially critical to repair DSBs that arise during TB latency, a hypothesis that we will explore in the future and is consistent with the characteristics of the NHEJ pathway elucidated by our studies.

Discovery of novel HR components and the SSA pathway of mycobacteria

We have also expanded our scope to examine the full complement of DSB repair pathways encoded by mycobacteria and the more global responses of mycobacteria to DNA damage. We developed a reporter system to assay three DSB repair pathways after cleavage of the chromosome with the homing endonuclease I-SceI 8. Use of this system in mycobacteria lacking DNA repair factors has led to several surprising conclusions about mycobacterial DSB repair, including : 1) Mycobacteria encode a single-strand annealing (SSA) pathway, which can mediate repair of DSBs that arise between repetitive sequences with consequent deletion of the intervening sequence; 2) RecBCD, the central resection nuclease for HR in E. coli, has no role in HR in mycobacteria and instead is dedicated to the SSA pathway(8); and 3) mycobacteria use a novel resection nuclease, AdnAB, in the HR pathway.(8)(9) These findings elucidate a plethora of DSB repair options in mycobacteria, including two pathways that can repair breaks without a homology donor (NHEJ and SSA), both of which are mutagenic.

Pathogenesis: Role in pathogenesis and genome diversification

Our studies to date have elucidated two new DNA repair pathways of mycobacteria, several new functions for DNA repair proteins in mycobacteria, and strong evidence that ribonucleotides may be the preferred nucleotide for the NHEJ repair machinery when repairing DSBs. We are poised to extend these findings to understand the role of each pathway of DSB repair in pathogenesis and genome diversification in vivo. The NHEJ and SSA pathways generate distinct mutational spectrums: NHEJ produces insertions and deletions, whereas SSA produces a deletion when the break arises between repeated sequences. Both of these mutation types are evident in sequenced genomes of clinical TB isolates, including antibiotic resistance mutations, indicating that these pathways may contribute to genome diversification during infection.

Double strand break repair in mycobacteria Double strand break repair in mycobacteria DSBs are induced by host immunity or ROS from antibiotics, are sensed through mechanisms that are not understood, and generate a DNA damage response, some of which is transcriptional, but the full complement of signal amplification mechanisms have not been explored. We have defined three pathways of DSB repair with distinct molecular participants, mutational outcomes, and requirement for homologous DNA.

  1. Gong C., et al., Biochemical and genetic analysis of the four DNA ligases of mycobacteria. J Biol Chem, 2004. 279(20): p. 20594-606.
  2. Gong C., et al., Mechanism of nonhomologous end-joining in mycobacteria: a low-fidelity repair system driven by Ku, ligase D and ligase C. Nat Struct Mol Biol, 2005. 12(4): p. 304-12.
  3. Zhu H., et al., Atomic structure and nonhomologous end-joining function of the polymerase component of bacterial DNA ligase D. Proc Natl Acad Sci U S A, 2006. 103(6): p. 1711-6.
  4. Akey D., et al., Crystal structure and nonhomologous end-joining function of the ligase component of Mycobacterium DNA ligase D. J Biol Chem, 2006. 281(19): p. 13412-23.
  5. Shuman S. and Glickman M.S., Bacterial DNA repair by non-homologous end joining. Nat Rev Microbiol, 2007. 5(11): p. 852-61.
  6. Stephanou N.C., et al., Mycobacterial nonhomologous end joining mediates mutagenic repair of chromosomal double-strand DNA breaks. J Bacteriol, 2007. 189(14): p. 5237-46.
  7. Sinha K.M., et al., Mycobacterial UvrD1 is a Ku-dependent DNA helicase that plays a role in multiple DNA repair events, including double-strand break repair. J Biol Chem, 2007. 282(20): p. 15114-25.
  8.  Sinha K.M., et al., Domain requirements for DNA unwinding by mycobacterial UvrD2, an essential DNA helicase. Biochemistry, 2008. 47(36): p. 9355-64.
  9. Aniukwu J., Glickman M.S., and Shuman S., The pathways and outcomes of mycobacterial NHEJ depend on the structure of the broken DNA ends. Genes Dev, 2008. 22(4): p. 512-27.
  10. Sinha K.M., et al., AdnAB: a new DSB-resecting motor-nuclease from mycobacteria. Genes Dev, 2009. 23(12): p. 1423-37.
  11. Gupta R., et al., Mycobacteria exploit three genetically distinct DNA double-strand break repair pathways. Mol Microbiol, 2011. 79(2): p. 316-30.
  12. Stallings C.L., et al., Catalytic and non-catalytic roles for the mono-ADP-ribosyltransferase Arr in the mycobacterial DNA damage response. PLoS One, 2011. 6(7): p. e21807.
  13. Bhattarai H, Gupta R, Glickman MS. DNA ligase C1 mediates the LigD-independent nonhomologous end-joining pathway of Mycobacterium smegmatis. J Bacteriol. 2014 Oct;196(19):3366-76. doi: 10.1128/JB.01832-14. Epub 2014 Jun 23. PubMed PMID:24957619; PubMed Central PMCID: PMC4187670.
  14. Heaton BE, Barkan D, Bongiorno P, Karakousis PC, Glickman MS. Deficiency of double-strand DNA break repair does not impair Mycobacterium tuberculosis virulence in multiple animal models of infection. Infect Immun. 2014 Aug;82(8):3177-85. doi: 10.1128/IAI.01540-14. Epub 2014 May 19. PubMed PMID:24842925; PubMed Central PMCID: PMC4136208.
  15. Gupta R, Ryzhikov M, Koroleva O, Unciuleac M, Shuman S, Korolev S, Glickman MS. A dual role for mycobacterial RecO in RecA-dependent homologous recombination and RecA-independent single-strand annealing. Nucleic Acids Res. 2013 Feb 1;41(4):2284-95. doi: 10.1093/nar/gks1298. Epub 2013 Jan 7. PubMed PMID:23295671; PubMed Central PMCID: PMC3575820.
  16. Zhu H, Bhattarai H, Yan HG, Shuman S, Glickman MS. Characterization of Mycobacterium smegmatis PolD2 and PolD1 as RNA/DNA polymerases homologous to the  POL domain of bacterial DNA ligase D. Biochemistry. 2012 Dec 21;51(51):10147-58.  doi: 10.1021/bi301202e. Epub 2012 Dec 11. PubMed PMID:23198659; PubMed Centra PMCID: PMC3766730.