Mycobacteriophage Exploit NHEJ to Facilitate Genome Circularization

Pitcher, R. S.; Tonkin, Louise M.; Daley, James M.; Palmbos, Phillip L.; Green, Andrew J.; Velting, Tricia L.; Brzostek, Anna; Korycka-Machała, Małgorzata; Cresawn, Steve; Dziadek, Jarosław; Hatfull, Graham F.; Wilson, Thomas E.; Doherty, Aidan J.

doi:10.1016/j.molcel.2006.07.009

Cited by 44 publications

(31 citation statements)

References 32 publications

(68 reference statements)

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“…A subset of bacteria, including B. subtilis, contains homologs of the Ku and LigD proteins, which mediate NHEJ in bacteria. These gene products have been shown to provide resistance to ionizing radiation or desiccation in several bacterial species, including B. subtilis (26,55,56,(59)(60)(61)(62)86).…”

Section: Discussionmentioning

confidence: 99%

“…Antibiotic concentrations were used as described previously (12,76). Where indicated for ionizing radiation treatment prior to microscopy, 10-ml portions of mid-exponential-phase cultures were placed in 50-ml conical tubes and exposed to a 60 Co gamma irradiator for a dose of 100 or 20 Gy, as indicated.…”

Section: Methodsmentioning

confidence: 99%

“…For microscopy, E. coli strains were grown in M63 minimal medium [ 4 ] at 30°C until mid-exponential growth (optical density at 600 nm of 0.5). For ionizing radiation treatment, 10 ml of cells were placed in a 50-ml conical tube for exposure to gamma rays from the same 60 Co source. After irradiation, cells were placed on agarose pads containing 1% agarose in M63 medium.…”

Section: Methodsmentioning

confidence: 99%

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Comparison of Responses to Double-Strand Breaks betweenEscherichia coliandBacillus subtilisReveals Different Requirements for SOS Induction

et al. 2009

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DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in both Escherichia coli and Bacillus subtilis. In E. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell. E. coli strains incapable of SOS induction were sensitive to ionizing radiation. In striking contrast, we found that in B. subtilis both ionizing radiation and a site-specific double-strand break causes induction of prophage PBSX and SOS gene expression in only a small subpopulation of cells. These results show that double-strand breaks provoke global SOS induction in E. coli but not in B. subtilis. Remarkably, RecA-GFP focus formation was nearly identical following ionizing radiation challenge in both E. coli and B. subtilis, demonstrating that formation of RecA-GFP foci occurs in response to double-strand breaks but does not require or result in SOS induction in B. subtilis. Furthermore, we found that B. subtilis cells incapable of inducing SOS had near wild-type levels of survival in response to ionizing radiation. Moreover, B. subtilis RecN contributes to maintaining low levels of SOS induction during double-strand break repair. Thus, we found that the contribution of SOS induction to double-strand break repair differs substantially between E. coli and B. subtilis.Both prokaryotes and eukaryotes respond to DNA damage, in part by altering the global expression profile of hundreds of gene products that function in a variety of cellular pathways (23,34,37,87). In Escherichia coli and Bacillus subtilis the transcriptional response to DNA-damaging agents has been well characterized (11,23,31,37,67). In both organisms, the highly conserved RecA and LexA proteins control the induction of the protective SOS response following DNA damage (for reviews, see references 83 and 88). RecA positively regulates SOS, while LexA represses this response. RecA binds excess single-stranded DNA (ssDNA) forming a nucleoprotein filament (for a review, see reference 24). The RecA/ssDNA nucleoprotein filament allows for RecA to activate LexA's latent protease activity, resulting in LexA autocleavage and subsequent derepression of the SOS regulon (for a review, see reference 83). In E. coli, the SOS regulon is comprised of 57 genes (for a review, see reference 77). These genes have been identified as part of the SOS regulon because they have a conserved LexA binding site(s) or because their transcription fails to respond to DNA damage in a strain bearing a noncleavable derivative of LexA [lexA(Ind Ϫ )] (for reviews, see references 77 and 23).In B. subtilis, RecA regulates the expression of approximately 600 genes following DNA damage or replication fork arrest (37). The majority of these genes belong to the prophages PBSX and SP␤ or are induced in response to the expression of these prophage genes (37). In addition, RecA appears to regulate 26 operons (63 genes) through B. subtilis LexA (11,37). As in E. col...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Comparison of Responses to Double-Strand Breaks betweenEscherichia coliandBacillus subtilisReveals Different Requirements for SOS Induction

et al. 2009

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“…Seven of these were observed previously in viral genomes and metagenomes including thyA, def (PDF), dnaB, dnaG, xerC, ligD and dam (Scherzinger et al, 1977;Yonesaki, 1994;Subramanya et al, 1996;Huber and Waldor, 2002;Pitcher et al, 2006;Sullivan et al, 2010;Sharon et al, 2011). The two that are newly described for viruses include genes encoding an endonuclease (uvdE) and a phosphate transport protein (phoU).…”

Section: Co-localized With Viral Genes On Contigs (Includes Genes Idementioning

confidence: 92%

Depth-stratified functional and taxonomic niche specialization in the ‘core’ and ‘flexible’ Pacific Ocean Virome

2014

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Microbes drive myriad ecosystem processes, and their viruses modulate microbial-driven processes through mortality, horizontal gene transfer, and metabolic reprogramming by viral-encoded auxiliary metabolic genes (AMGs). However, our knowledge of viral roles in the oceans is primarily limited to surface waters. Here we assess the depth distribution of protein clusters (PCs) in the first large-scale quantitative viral metagenomic data set that spans much of the pelagic depth continuum (the Pacific Ocean Virome; POV). This established ‘core’ (180 PCs; one-third new to science) and ‘flexible’ (423K PCs) community gene sets, including niche-defining genes in the latter (385 and 170 PCs are exclusive and core to the photic and aphotic zones, respectively). Taxonomic annotation suggested that tailed phages are ubiquitous, but not abundant (<5% of PCs) and revealed depth-related taxonomic patterns. Functional annotation, coupled with extensive analyses to document non-viral DNA contamination, uncovered 32 new AMGs (9 core, 20 photic and 3 aphotic) that introduce ways in which viruses manipulate infected host metabolism, and parallel depth-stratified host adaptations (for example, photic zone genes for iron–sulphur cluster modulation for phage production, and aphotic zone genes for high-pressure deep-sea survival). Finally, significant vertical flux of photic zone viruses to the deep sea was detected, which is critical for interpreting depth-related patterns in nature. Beyond the ecological advances outlined here, this catalog of viral core, flexible and niche-defining genes provides a resource for future investigation into the organization, function and evolution of microbial molecular networks to mechanistically understand and model viral roles in the biosphere.

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“…Mycobacterium tuberculosis, the agent of human TB, and its avirulent cousin Mycobacterium smegmatis can avail themselves of two separate pathways to repair DSBs: (1) a RecA-dependent HR system (Pitcher et al 2007a;Stephanou et al 2007), and (2) a nonhomologous endjoining (NHEJ) system that depends on dedicated DNA ligases and the DNA end-binding protein Ku (Gong et al 2004(Gong et al , 2005Pitcher et al 2006;Aniukwu et al 2008). NHEJ helps protect the bacterial chromosome against DSBs during quiescent states, when there is no sister chromatid available to direct HR (for review, see Pitcher et al 2007b;Shuman and Glickman 2007).…”

mentioning

confidence: 99%

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

Sinha¹,

Unciuleac²,

Glickman³

et al. 2009

Genes Dev.

115

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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 helicasenucleases with distinctive properties. The AdnA and AdnB subunits are each composed of an N-terminal UvrDlike 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 59 end to dictate the sites of cleavage, which are predominantly 5 or 6 nucleotides (nt) from the 59 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 59 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 DNAdependent 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.[Keywords: DNA repair; helicase; double-strand breaks; ATP-dependent nuclease; molecular ruler] Supplemental material is available at http://www.genesdev.org.

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Mycobacteriophage Exploit NHEJ to Facilitate Genome Circularization

Cited by 44 publications

References 32 publications

Comparison of Responses to Double-Strand Breaks betweenEscherichia coliandBacillus subtilisReveals Different Requirements for SOS Induction

Comparison of Responses to Double-Strand Breaks betweenEscherichia coliandBacillus subtilisReveals Different Requirements for SOS Induction

Depth-stratified functional and taxonomic niche specialization in the ‘core’ and ‘flexible’ Pacific Ocean Virome

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

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