A dual role for mycobacterial RecO in RecA-dependent homologous recombination and RecA-independent single-strand annealing

Gupta, Richa; Ryzhikov, Mikhail; Королева, О. В.; Unciuleac, Mihaela-Carmen; Shuman, Stewart; Korolev, Sergey; Glickman, Michael S.

doi:10.1093/nar/gks1298

Cited by 34 publications

(62 citation statements)

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“…The SSA pathway of mycobacteria repairs DSBs that arise between repeats, with consequent deletion of the DNA between the repeats. SSA is independent of the strand exchange protein RecA but requires the helicase-nuclease RecBCD and the strand-annealing protein RecO (9,10). …”

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“…The RecO protein has an N-terminal OB (oligonucleotide binding) domain and can bind to ssDNA or dsDNA (23). D. radiodurans and Mycobacterium RecO have an additional tetracysteine zinc-binding domain; zinc binding has been shown to stimulate annealing of SSB-coated cDNA strands by these RecO proteins (10,24). DNA strand annealing activity has also been demonstrated for other bacterial RecO proteins, which do not require zinc but instead rely on an interaction with the SSB C terminus, an interaction that the mycobacterial and Deinococcus RecO proteins have relinquished (10,25,26).…”

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“…D. radiodurans and Mycobacterium RecO have an additional tetracysteine zinc-binding domain; zinc binding has been shown to stimulate annealing of SSB-coated cDNA strands by these RecO proteins (10,24). DNA strand annealing activity has also been demonstrated for other bacterial RecO proteins, which do not require zinc but instead rely on an interaction with the SSB C terminus, an interaction that the mycobacterial and Deinococcus RecO proteins have relinquished (10,25,26). In addition to strand annealing, RecO can promote RecA nucleation and filament formation on SSB-coated ssDNA, but this activity requires RecR (27,28 (31,32).…”

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RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria

2015

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Mycobacteria encode three DNA double-strand break repair pathways: (i) RecA-dependent homologous recombination (HR), (ii) Ku-dependent nonhomologous end joining (NHEJ), and (iii) RecBCD-dependent single-strand annealing (SSA). Mycobacterial HR has two presynaptic pathway options that rely on the helicase-nuclease AdnAB and the strand annealing protein RecO, respectively. Ablation of adnAB or recO individually causes partial impairment of HR, but loss of adnAB and recO in combination abolishes HR. RecO, which can accelerate annealing of single-stranded DNA in vitro, also participates in the SSA pathway. The functions of RecF and RecR, which, in other model bacteria, function in concert with RecO as mediators of RecA loading, have not been examined in mycobacteria. Here, we present a genetic analysis of recF and recR in mycobacterial recombination. We find that RecF, like RecO, participates in the AdnAB-independent arm of the HR pathway and in SSA. In contrast, RecR is required for all HR in mycobacteria and for SSA. The essentiality of RecR as an agent of HR is yet another distinctive feature of mycobacterial DNA repair. N ature has evolved mechanisms to repair the structurally diverse DNA lesions that arise during chromosomal replication and from exogenous mutagens. Double-strand breaks (DSBs) are especially deleterious, insofar as a single chromosomal DSB can be lethal to a cell if not repaired prior to cell division. The major mechanisms of DSB repair are conserved, at least in their general principles, from bacteria to humans. This parallel is especially relevant for mycobacteria, which implement the same three distinct DSB repair pathways operative in eukarya: homologous recombination (HR), nonhomologous end joining (NHEJ), and singlestrand annealing (SSA). Mycobacterial NHEJ relies on the end-binding protein Ku, DNA ligase LigD (a multifunctional ligase-polymerase-3=-phosphoesterase), DNA ligase LigC1, and two additional polymerases (PolD1 and PolD2) (1-9). The SSA pathway of mycobacteria repairs DSBs that arise between repeats, with consequent deletion of the DNA between the repeats. SSA is independent of the strand exchange protein RecA but requires the helicase-nuclease RecBCD and the strand-annealing protein RecO (9,10). IMPORTANCE This study clarifies the molecular requirements for homologous recombination in mycobacteria. Specifically, we demonstrate that RecF and RecR play important roles in both theThe central agent of homology search in the HR pathways of all organisms is a strand exchange protein that polymerizes on the single-strand DNA (ssDNA) resulting from end resection at a DSB. In bacteria, the strand exchange protein is RecA; in eukarya, it is Rad51. RecA nucleation on ssDNA is regulated in order to prevent inappropriate nucleoprotein filament formation, which can be toxic (11). The presence of single-strand binding protein (SSB) on ssDNA prevents RecA loading unless a "mediator" protein or protein complex catalyzes the exchange of SSB for RecA to create the RecA nucleoprotein filam...

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RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria

2015

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“…M. smegmatis elaborates three genetically distinct DNA repair pathways: RecA-dependent HR, single-strand annealing (SSA), and nonhomologous end joining (NHEJ) (6). All mycobacterial HR uses the RecA strand exchange protein, whereas two subpathways utilize the AdnAB helicase-nuclease (6,7) or RecO (8), respectively. The SSA pathway requires RecBCD (6), which does not participate in HR in mycobacteria, and RecO (8).…”

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“…All mycobacterial HR uses the RecA strand exchange protein, whereas two subpathways utilize the AdnAB helicase-nuclease (6,7) or RecO (8), respectively. The SSA pathway requires RecBCD (6), which does not participate in HR in mycobacteria, and RecO (8).…”

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DNA Ligase C1 Mediates the LigD-Independent Nonhomologous End-Joining Pathway of Mycobacterium smegmatis

2014

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Chromosomal DNA is under constant attack from clastogens that threaten the coding capacity and stability of the genome. Among the wide variety of DNA lesions that the cell must process, double strand breaks (DSBs) of DNA represent a particularly lethal form of DNA damage that must be repaired for chromosome replication and transcription to proceed. The sources of DSBs include ionizing radiation (IR), replication across a singlestranded nick (1), DNA processing enzymes that generate DSBs, such as topoisomerases, and embedded ribonucleotides in chromosomal DNA (2-5). As such, the repair of DSBs is a critical function in all living organisms, including bacteria. The most intensely studied and universally distributed pathway of DSB repair is RecA-dependent homologous recombination (HR), which uses an intact chromosomal template to repair the DSB without mutation. RecA-dependent HR is the dominant and sole DSB repair pathway in Escherichia coli. However, additional pathways of DSB repair operate in other bacteria, including mycobacteria, such as Mycobacterium smegmatis and M. tuberculosis. M. smegmatis elaborates three genetically distinct DNA repair pathways: RecA-dependent HR, single-strand annealing (SSA), and nonhomologous end joining (NHEJ) (6). All mycobacterial HR uses the RecA strand exchange protein, whereas two subpathways utilize the AdnAB helicase-nuclease (6, 7) or RecO (8), respectively. The SSA pathway requires RecBCD (6), which does not participate in HR in mycobacteria, and RecO (8).Mycobacterial NHEJ can recircularize transformed linear plasmids (9-14), repair IR-induced DSBs in late-stationary-phase cells (15, 16), protect mycobacteria from desiccation (15), and seal homing endonuclease-induced DSBs (6,16). In all of these experimental systems, the NHEJ pathway requires the DNA end binding protein Ku and the polyfunctional DNA ligase LigD. Genetic ablation of ku or ligD reduces the efficiency of plasmid NHEJ by approximately 500-fold (10). Similarly, M. smegmatis ⌬Ku or ⌬ligD bacteria in late stationary phase but not log phase are sensitized to IR (16) and cannot repair I-SceI-induced chromosomal breaks by NHEJ (6). Ablation of ku or ligD also leads to reciprocal upregulation of the HR pathway in the I-SceI system, suggesting that the NHEJ and HR pathways compete for repair of DSBs in log-phase cells (6).The LigD protein has three autonomous enzymatic domains: polymerase (POL), phosphoesterase (PE), and ligase (LIG). LigD-POL is a primase-like polymerase that can add both templated and nontemplated deoxynucleoside triphosphates (dNTPs) or ribonucleoside triphosphates (rNTPs) to DNA substrates (13,17). Alanine substitution mutations at the diaspartate metal binding site of the polymerase (D136A/D138A) abolish polymerase activity in vitro (11). M. smegmatis expressing LigD-D136/138A cannot add nontemplated nucleotides to blunt end linear plasmid substrates, resulting in a rise in the fidelity of NHEJ with little change in the overall efficiency (10, 13). However, templated fill-in of 5=-overhang NHEJ...

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Double‐Strand DNA Break Repair in Mycobacteria

Gupta

Glickman

2016

Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria

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A dual role for mycobacterial RecO in RecA-dependent homologous recombination and RecA-independent single-strand annealing

Cited by 34 publications

References 42 publications

RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria

RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria

DNA Ligase C1 Mediates the LigD-Independent Nonhomologous End-Joining Pathway of Mycobacterium smegmatis

Double‐Strand DNA Break Repair in Mycobacteria

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