Role of Archaeal HerA Protein in the Biology of the Bacterium Thermus thermophilus

Blesa, Alba; Quintáns, Nieves G.; Baquedano, Ignacio; Mata, Carlos P.; Castón, José R.; Berenguer, José

doi:10.3390/genes8050130

Cited by 8 publications

(7 citation statements)

References 30 publications

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“…HR is probably the mechanism for genome repair, since Thermus, apparently, does not have a nonhomologous end joining pathway, the informational content of the genome is not degraded (near normal viability), and RecA is important for genome integrity in this organism (Castan et al, 2003). The HB27 strain does have RecJ (Yamagata, 2001) and RecQ genes (Brüggemann and Chen, 2006), together with the RecFOR pathway (Chaudhary et al, 2020), and other DNA repair proteins typical of archaea, like HerA (Blesa et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

“…HR is probably the mechanism for genome repair, since Thermus , apparently, does not have a non-homologous end joining pathway, the informational content of the genome is not degraded (near normal viability), and RecA is important for genome integrity in this organism ( Castan et al, 2003 ). The HB27 strain does have RecJ ( Yamagata, 2001 ) and RecQ genes ( Brüggemann and Chen, 2006 ), together with the RecFOR pathway ( Chaudhary et al, 2020 ), and other DNA repair proteins typical of archaea, like HerA ( Blesa et al, 2017 ). In the absence of AddAB, this set of genes could by itself support an active HR system that continuously repairs the genome and keeps its functionality, while allowing efficient transformation with plasmid and linear DNA, as can be observed in the mutant strains assayed.…”

Section: Discussionmentioning

confidence: 99%

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Deletion of the primase-polymerases encoding gene, located in a mobile element in Thermus thermophilus HB27, leads to loss of function mutation of addAB genes

et al. 2022

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DNA primase-polymerases (Ppol) have been shown to play active roles in DNA repair and damage tolerance, both in prokaryotes and eukaryotes. The ancestral thermophilic bacterium Thermus thermophilus strain HB27 encodes a Ppol protein among the genes present in mobile element ICETh2, absent in other T. thermophilus strains. Using different strategies we ablated the function of Ppol in HB27 cells, either by knocking out the gene through insertional mutagenesis, markerless deletion or through abolition of its catalytic activity. Whole genome sequencing of this diverse collection of Ppol mutants showed spontaneous loss of function mutation in the helicase-nuclease AddAB in every ppol mutant isolated. Given that AddAB is a major player in recombinational repair in many prokaryotes, with similar activity to the proteobacterial RecBCD complex, we have performed a detailed characterization of the ppol mutants in combination with addAB mutants. The results show that knockout addAB mutants are more sensitive to DNA damage agents than the wild type, and present a dramatic three orders of magnitude increase in natural transformation efficiencies with both plasmid and lineal DNA, whereas ppol mutants show defects in plasmid stability. Interestingly, DNA-integrity comet assays showed that the genome of all the ppol and/or addAB mutants was severely affected by widespread fragmentation, however, this did not translate in neat loss of viability of the strains. All these data support that Ppol appears to keep in balance the activity of AddAB as a part of the DNA housekeeping maintenance in T. thermophilus HB27, thus, playing a key role in its genome stability.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Deletion of the primase-polymerases encoding gene, located in a mobile element in Thermus thermophilus HB27, leads to loss of function mutation of addAB genes

et al. 2022

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“…Protein sequence identities between archaeal NurA-HerA and bacterial NurA-HerA were less than 20% [ 22 ]. The biological functions of bacterial NurA-HerA remain controversial, considering that the deletion of nurA or herA gene exhibit modest or improved resistance to gamma irradiation, ultraviolet (UV) radiation, and mitomycin C (MMC) treatment in D. radiodurans or Thermus thermophiles [ 22 – 24 ]. The structural and biochemical characterizations of bacterial NurA-HerA are not yet fully studied.…”

Section: Introductionmentioning

confidence: 99%

“…The first bacterial NurA structure from Thermotoga maritima (tmNurA, PDB ID: 1ZUP) has been deposited in the protein structure database but with little characterization. The negatively stained assembly of T. thermophilus HerA (ttHerA) was performed by electron microscopy (EM), and ttHerA was observed as a hexameric ring-like structure [ 24 ]. However, detailed structural and biochemical information of bacterial NurA-HerA to interpret the mechanisms of its DNA end resection process is not yet available.…”

Section: Introductionmentioning

confidence: 99%

Structural and DNA end resection study of the bacterial NurA-HerA complex

et al. 2023

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Background The nuclease NurA and the ATPase/translocase HerA play a vital role in repair of double-strand breaks (DSB) during the homologous recombination in archaea. A NurA-HerA complex is known to mediate DSB DNA end resection, leading to formation of a free 3′ end used to search for the homologous sequence. Despite the structures of individual archaeal types of NurA and HerA having been reported, there is limited information regarding the molecular mechanisms underlying this process. Some bacteria also possess homologs of NurA and HerA; however, the bacterial type of this complex, as well as the detailed mechanisms underlying the joining of NurA-HerA in DSB DNA end resection, remains unclear. Results We report for the first time the crystal structures of Deinococcus radiodurans HerA (drHerA) in the nucleotide-free and ADP-binding modes. A D. radiodurans NurA-HerA complex structure was constructed according to a low-resolution cryo-electron microscopy map. We performed site-directed mutagenesis to map the drNurA-HerA interaction sites, suggesting that their interaction is mainly mediated by ionic links, in contrast to previously characterized archaeal NurA-HerA interactions. The key residues responsible for the DNA translocation activity, DNA unwinding activity, and catalytic activities of the drNurA-HerA complex were identified. A HerA/FtsK-specific translocation-related motif (TR motif) that guarantees the processivity of double-stranded DNA (dsDNA) translocation was identified. Moreover, a mechanism for the translocation-regulated resection of the 5′ tail of broken dsDNA and the corresponding generation of a recombinogenic 3′ single-stranded DNA tail by the drNurA-HerA complex was elucidated. Conclusions Our work provides new insights into the mechanism underlying bacterial NurA-HerA-mediated DSB DNA end resection, and the way this complex digests the 5′ tail of a DNA duplex and provides long 3′ free end for strand invasion in the bacterial homologous recombination process.

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“…The available experimental data commented above lead to hypothesize a model for transjugation in which the restriction enzyme TthHB27I could cleave the donor DNA at different locations in the polyploid genome, whereas TdtA, putatively helped by NurA, could use these cuts as origins for transfer to the recipient strain. A concomitant need for DNA repair of the cleaved sites in the donor cell could underlie the requirement for HepA (TTC0147), which is a homologue of TdtA belonging to the HerA family of archaeal DNA helicases involved in DNA repair [ 18 ]. According to this model, recipient cells would receive different genome fragments from the donor cells simultaneously during transjugation, leading to the generation of genomic mosaicism among the progeny after integration by recombination, which is similar to those described for Mycobacterium spp [ 8 ] or Mycoplasma agalactiae [ 19 ].…”

Section: Introductionmentioning

confidence: 99%

Integrative and Conjugative Element ICETh1 Functions as a Pangenomic DNA Capture Module in Thermus thermophilus

et al. 2020

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Transjugation is an unconventional conjugation mechanism in Thermus thermophilus (Tth) that involves the active participation of both mating partners, encompassing a DNA secretion system (DSS) in the donor and an active natural competence apparatus (NCA) in the recipient cells. DSS is encoded within an integrative and conjugative element (ICETh1) in the strain Tth HB27, whereas the NCA is constitutively expressed in both mates. Previous experiments suggested the presence of multiple origins of transfer along the genome, which could generate genomic mosaicity among the progeny. Here, we designed transjugation experiments between two closely related strains of Tth with highly syntenic genomes, containing enough single nucleotide polymorphisms to allow precise parenthood analysis. Individual clones from the progeny were sequenced, revealing their origin as derivatives of our ICETh1-containing intended “donor” strain (HB27), which had acquired separate fragments from the genome of the ICETh1-free HB8 cells, which are our intended recipient. Due to the bidirectional nature of transjugation, only assays employing competence-defective HB27 derivatives as donors allowed the recovery of HB8-derived progeny. These results show a preference for a retrotransfer mechanism in transjugation in ICETh1-bearing strains, supporting an inter-strain gene-capture function for ICETh1. This function could benefit the donor-capable host by facilitating the acquisition of adaptive traits from external sources, ultimately increasing the open pangenome of Thermus, maximizing the potential repertoire of physiological and phenotypical traits related to adaptation and speciation.

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Role of Archaeal HerA Protein in the Biology of the Bacterium Thermus thermophilus

Cited by 8 publications

References 30 publications

Deletion of the primase-polymerases encoding gene, located in a mobile element in Thermus thermophilus HB27, leads to loss of function mutation of addAB genes

Deletion of the primase-polymerases encoding gene, located in a mobile element in Thermus thermophilus HB27, leads to loss of function mutation of addAB genes

Structural and DNA end resection study of the bacterial NurA-HerA complex

Integrative and Conjugative Element ICETh1 Functions as a Pangenomic DNA Capture Module in Thermus thermophilus

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