NHEJ protects mycobacteria in stationary phase against the harmful effects of desiccation

Pitcher, R. S.; Green, Andrew J.; Brzostek, Anna; Korycka-Machała, Małgorzata; Dziadek, Jarosław; Doherty, Aidan J.

doi:10.1016/j.dnarep.2007.02.009

Cited by 86 publications

(89 citation statements)

References 20 publications

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“…5). Similarly, sensitivity to drying is higher in E. coli, Mycobacterium smegmatis, and spores of Bacillus subtilis carrying an inactivated recA gene than in the respective parent strains (3,34,44).…”

Section: Discussionmentioning

confidence: 99%

Acinetobacter baumannii RecA Protein in Repair of DNA Damage, Antimicrobial Resistance, General Stress Response, and Virulence

et al. 2011

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RecA is the major enzyme involved in homologous recombination and plays a central role in SOS mutagenesis. In Acinetobacter spp., including Acinetobacter baumannii, a multidrug-resistant bacterium responsible for nosocomial infections worldwide, DNA repair responses differ in many ways from those of other bacterial species. In this work, the function of A. baumannii RecA was examined by constructing a recA mutant. Alteration of this single gene had a pleiotropic effect, showing the involvement of RecA in DNA damage repair and consequently in cellular protection against stresses induced by DNA damaging agents, several classes of antibiotics, and oxidative agents. In addition, the absence of RecA decreased survival in response to both heat shock and desiccation. Virulence assays in vitro (with macrophages) and in vivo (using a mouse model) similarly implicated RecA in the pathogenicity of A. baumannii. Thus, the data strongly suggest a protective role for RecA in the bacterium and indicate that inactivation of the protein can contribute to a combined therapeutic approach to controlling A. baumannii infections.

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Section: Discussionmentioning

confidence: 99%

Acinetobacter baumannii RecA Protein in Repair of DNA Damage, Antimicrobial Resistance, General Stress Response, and Virulence

et al. 2011

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“…There are several reasons why it may be favorable to incorporate RNA during DSB end joining. NHEJ is the DSB-repair pathway of choice for nondividing cells (10), where NTPs are much more abundant than dNTPs in the available nucleotide pools (33). Another distinct possibility, driven by the availability of ribonucleotides, is that NHEJ repair pathways evolved to incorporate RNA rather than DNA selectively into repaired DSBs to demarcate the boundaries of any alterations made to the repaired sites.…”

Section: Pe Resection Of Strand-displacement Intermediates During Nhejmentioning

confidence: 99%

“…Most bacterial organisms also possess an NHEJ complex, composed of a more limited set of proteins, including ligase D (LigD) and Ku (8,9), which is required for DSB repair in stationary phase (10). The mycobacterial LigD gene encodes a multifunctional enzyme which, along with Ku, is capable of remodeling and ligating a broad variety of DSBs (8,11).…”

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confidence: 99%

Ribonucleolytic resection is required for repair of strand displaced nonhomologous end-joining intermediates

Bartlett

Brissett

Doherty

2013

Proc. Natl. Acad. Sci. U.S.A.

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Nonhomologous end-joining (NHEJ) pathways repair DNA doublestrand breaks (DSBs) in eukaryotes and many prokaryotes, although it is not reported to operate in the third domain of life, archaea. Here, we describe a complete NHEJ complex, consisting of DNA ligase (Lig), polymerase (Pol), phosphoesterase (PE), and Ku from a mesophillic archaeon, Methanocella paludicola (Mpa). Mpa Lig has limited DNA nick-sealing activity but is efficient in ligating nicks containing a 3′ ribonucleotide. Mpa Pol preferentially incorporates nucleoside triphosphates onto a DNA primer strand, filling DNA gaps in annealed breaks. Mpa PE sequentially removes 3′ phosphates and ribonucleotides from primer strands, leaving a ligatable terminal 3′ monoribonucleotide. These proteins, together with the DNA end-binding protein Ku, form a functional NHEJ break-repair apparatus that is highly homologous to the bacterial complex. Although the major roles of Pol and Lig in break repair have been reported, PE's function in NHEJ has remained obscure. We establish that PE is required for ribonucleolytic resection of RNA intermediates at annealed DSBs. Polymerase-catalyzed strand-displacement synthesis on DNA gaps can result in the formation of nonligatable NHEJ intermediates. The function of PE in NHEJ repair is to detect and remove inappropriately incorporated ribonucleotides or phosphates from 3′ ends of annealed DSBs to configure the termini for ligation. Thus, PE prevents the accumulation of abortive genotoxic DNA intermediates arising from strand displacement synthesis that otherwise would be refractory to repair. D NA double-strand breaks (DSBs) are one of the most lethal forms of damage encountered by cells (1). Nonhomologous end joining (NHEJ) is the primary pathway for repairing DSBs in higher eukaryotes (2). NHEJ does not require the presence of a sister chromatid, in contrast to homologous recombination, and therefore can operate in quiescent cells. The direct repair and ligation of DSBs by NHEJ is considered to be more error prone because breaks are mended without the assistance of an intact DNA template.The higher eukaryotic NHEJ complex is composed primarily of the ligase IV, X-ray repair cross-complementing protein 4 (XRCC4), and XRCC4-like factor (XLF) complex (the LXX complex), DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), and Ku70/80 (1, 3). Ku and DNA-PKcs both assist in bridging the gap between the broken termini by promoting end synapsis. NHEJ repair of nonhomologous breaks requires remodeling of the DNA termini by processing enzymes, including polymerases (Pol λ and μ) and nucleases [Artemis and flap endonuclease 1 (Fen-1)], to prepare them for ligation (4-7). The LXX complex is recruited by Ku, which enables the ligation of the DNA ends.Most bacterial organisms also possess an NHEJ complex, composed of a more limited set of proteins, including ligase D (LigD) and Ku (8,9), which is required for DSB repair in stationary phase (10). The mycobacterial LigD gene encodes a multifunctional enzyme which, along with Ku, is ...

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“…In addition to their house-keeping NAD-dependent enzymes, many bacteria have one or more ATP-dependent DNA ligases, the evolutionary origin and cellular function of which have not been entirely determined [3]. A number of these accessory enzymes have been biochemically characterized and some are postulated to play a role in DNA repair [4][5][6][7] while others are suggested to be involved in competence and DNA uptake [8,9]. The genome of the pathogenic psychrophile Aliivibrio salmonicida encodes one such putative ATP-dependent DNA ligase [10].…”

Section: Introductionmentioning

confidence: 99%

Recombinant expression and purification of an ATP-dependent DNA ligase from Aliivibrio salmonicida

Williamson

Pedersen

2014

Protein Expression and Purification

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The genome of the psychrophilic fish-pathogen Aliivibrio salmonicida encodes a putative ATPdependent DNA ligase in addition to a housekeeping NAD-dependent enzyme. In order to study the structure and activity of the ATP dependent ligase in vitro we have undertaken its recombinant production and purification from an E. coli based expression system. Expression and purification of this protein presented two significant challenges. First, the gene product was moderately toxic to E. coli cells, second it was necessary to remove the large amounts of E. coli DNA present in bacterial lysates without contamination of the protein preparation by nucleases which might interfere with future assaying. The toxicity problem was overcome by fusion of the putative ligase to large solubility tags such as maltose-binding protein (MBP) or Glutathione-stransferase (GST), and DNA was removed by treatment with a nuclease which could be inhibited by reducing agents.As the A. salmonicida ATP-dependent DNA ligase gene encodes a predicted leader peptide, both the full-length and mature forms of the protein were produced. Both possessed ATP-dependent DNA ligase activity, but the truncated form was significantly more active. Here we detail the first reported production, purification and preliminary characterization of active Aliivibrio salmonicida ATPdependent DNA ligase. Highlights A putative periplasmic DNA ligase from Aliivibrio salmonicida was recombinantly produced in E. coli  Both the full-length and N-terminally truncated variants were expressed and purified  Toxicity of gene products to the host cells was overcame by fusion to large solubility tags  The truncated protein is more active in DNA ligation than the full-length version

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NHEJ protects mycobacteria in stationary phase against the harmful effects of desiccation

Cited by 86 publications

References 20 publications

Acinetobacter baumannii RecA Protein in Repair of DNA Damage, Antimicrobial Resistance, General Stress Response, and Virulence

Acinetobacter baumannii RecA Protein in Repair of DNA Damage, Antimicrobial Resistance, General Stress Response, and Virulence

Ribonucleolytic resection is required for repair of strand displaced nonhomologous end-joining intermediates

Recombinant expression and purification of an ATP-dependent DNA ligase from Aliivibrio salmonicida

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