Identification of a conserved 5′-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair

Ory, Ana de; Nagler, Katja; Carrasco, Begoña; Raguse, Marina; Zafra, Olga; Moeller, Ralf; Vega, Miguel de

doi:10.1093/nar/gkw054

Cited by 21 publications

(26 citation statements)

References 64 publications

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“…However, no clear trend toward the co-occurrence of these two “variable” genes can be noticed in the 38 analysed genomes. In B. subtilis , the recent involvement of LigD Bsub and Ku Bsub in base excision repair along with their major role in NHEJ supports the third hypothesis (de Ory et al, 2014, 2016). …”

Section: Discussionmentioning

confidence: 62%

Multiple and Variable NHEJ-Like Genes Are Involved in Resistance to DNA Damage in Streptomyces ambofaciens

et al. 2016

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Non-homologous end-joining (NHEJ) is a double strand break (DSB) repair pathway which does not require any homologous template and can ligate two DNA ends together. The basic bacterial NHEJ machinery involves two partners: the Ku protein, a DNA end binding protein for DSB recognition and the multifunctional LigD protein composed a ligase, a nuclease and a polymerase domain, for end processing and ligation of the broken ends. In silico analyses performed in the 38 sequenced genomes of Streptomyces species revealed the existence of a large panel of NHEJ-like genes. Indeed, ku genes or ligD domain homologues are scattered throughout the genome in multiple copies and can be distinguished in two categories: the “core” NHEJ gene set constituted of conserved loci and the “variable” NHEJ gene set constituted of NHEJ-like genes present in only a part of the species. In Streptomyces ambofaciens ATCC23877, not only the deletion of “core” genes but also that of “variable” genes led to an increased sensitivity to DNA damage induced by electron beam irradiation. Multiple mutants of ku, ligase or polymerase encoding genes showed an aggravated phenotype compared to single mutants. Biochemical assays revealed the ability of Ku-like proteins to protect and to stimulate ligation of DNA ends. RT-qPCR and GFP fusion experiments suggested that ku-like genes show a growth phase dependent expression profile consistent with their involvement in DNA repair during spores formation and/or germination.

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Section: Discussionmentioning

confidence: 62%

Multiple and Variable NHEJ-Like Genes Are Involved in Resistance to DNA Damage in Streptomyces ambofaciens

et al. 2016

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“…Together, these findings establish that both LigC and LigD-dependent complexes contribute significantly to the repair of lesions produced by oxidative damage during stationary phase in mycobacteria. Notably, Ory et al 44 reported recently that Bacillus subtilis LigD possesses dRP-ase activity in vitro supporting this proposed role in excision repair. Further studies are required to define the exact nature of the oxidative lesions that these Prim-Pol pathways process in vivo and uncover the overlap and “cross-talk” that occurs between these, and other, stationary phase DNA repair pathways in mycobacteria.…”

Section: Discussionmentioning

confidence: 77%

DNA Ligase C and Prim-PolC participate in base excision repair in mycobacteria

et al. 2017

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Prokaryotic Ligase D is a conserved DNA repair apparatus processing DNA double-strand breaks in stationary phase. An orthologous Ligase C (LigC) complex also co-exists in many bacterial species but its function is unknown. Here we show that the LigC complex interacts with core BER enzymes in vivo and demonstrate that together these factors constitute an excision repair apparatus capable of repairing damaged bases and abasic sites. The polymerase component, which contains a conserved C-terminal structural loop, preferentially binds to and fills-in short gapped DNA intermediates with RNA and LigC ligates the resulting nicks to complete repair. Components of the LigC complex, like LigD, are expressed upon entry into stationary phase and cells lacking either of these pathways exhibit increased sensitivity to oxidising genotoxins. Together, these findings establish that the LigC complex is directly involved in an excision repair pathway(s) that repairs DNA damage with ribonucleotides during stationary phase.

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“…The region of Pa-LigD gene encoding PolDom (residues 533-840; [43]) was amplified by PCR using as substrate the recombinant plasmid pET16-PaLigD [44], a sense strand primer that introduced an NdeI restriction site substituting the original codon for Arg532 by a methionine codon, and an antisense primer that introduced a BamHI site. Both, plasmid pET16 and the PCR products were double digested with NdeI and BamHI and further ligated to give rise to plasmid pET16-Pa-PolDom.…”

Section: Expression and Purification Of Recombinant Pa-poldommentioning

confidence: 99%

Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom

Sánchez-Salvador

Vega

2020

Biomolecules

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The catalytic active site of the Polymerization Domain (PolDom) of bacterial Ligase D is designed to promote realignments of the primer and template strands and extend mispaired 3′ ends. These features, together with the preferred use of ribonucleotides (NTPs) over deoxynucleotides (dNTPs), allow PolDom to perform efficient double strand break repair by nonhomologous end joining when only a copy of the chromosome is present and the intracellular pool of dNTPs is depleted. Here, we evaluate (i) the role of conserved histidine and serine/threonine residues in NTP insertion, and (ii) the importance in the polymerization reaction of a conserved lysine residue that interacts with the templating nucleotide. To that extent, we have analyzed the biochemical properties of variants at the corresponding His651, Ser768, and Lys606 of Pseudomonas aeruginosa PolDom (Pa-PolDom). The results show that preferential insertion of NMPs is principally due to the histidine that also contributes to the plasticity of the active site to misinsert nucleotides. Additionally, Pa-PolDom Lys606 stabilizes primer dislocations. Finally, we show that the active site of PolDom allows the efficient use of 7,8-dihydro-8-oxo-riboguanosine triphosphate (8oxoGTP) as substrate, a major nucleotide lesion that results from oxidative stress, inserting with the same efficiency both the anti and syn conformations of 8oxoGMP.

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Identification of a conserved 5′-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair

Cited by 21 publications

References 64 publications

Multiple and Variable NHEJ-Like Genes Are Involved in Resistance to DNA Damage in Streptomyces ambofaciens

Multiple and Variable NHEJ-Like Genes Are Involved in Resistance to DNA Damage in Streptomyces ambofaciens

DNA Ligase C and Prim-PolC participate in base excision repair in mycobacteria

Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom

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