Deinococcus radiodurans is known for its extreme radioresistance. Comparative genomics identified a radiation-desiccation response (RDR) regulon comprising genes that are highly induced after DNA damage and containing a conserved motif (RDRM) upstream of their coding region. We demonstrated that the RDRM sequence is involved in cis-regulation of the RDR gene ddrB in vivo. Using a transposon mutagenesis approach, we showed that, in addition to ddrO encoding a predicted RDR repressor and irrE encoding a positive regulator recently shown to cleave DdrO in Deinococcus deserti, two genes encoding α-keto-glutarate dehydrogenase subunits are involved in ddrB regulation. In wild-type cells, the DdrO cell concentration decreased transiently in an IrrE-dependent manner at early times after irradiation. Using a conditional gene inactivation system, we showed that DdrO depletion enhanced expression of three RDR proteins, consistent with the hypothesis that DdrO acts as a repressor of the RDR regulon. DdrO-depleted cells loose viability and showed morphological changes evocative of an apoptotic-like response, including membrane blebbing, defects in cell division and DNA fragmentation. We propose that DNA repair and apoptotic-like death might be two responses mediated by the same regulators, IrrE and DdrO, but differently activated depending on the persistence of IrrE-dependent DdrO cleavage.

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The conserved ATPase, PCH-2/TRIP13, is required during both the spindle checkpoint and meiotic prophase. However, its specific role in regulating meiotic homolog pairing, synapsis and recombination has been enigmatic. Here, we report that this enzyme is required to proofread meiotic homolog interactions. We generated a mutant version of PCH-2 in C . elegans that binds ATP but cannot hydrolyze it: pch-2 E253Q . In vitro , this mutant can bind a known substrate but is unable to remodel it. This mutation results in some non-homologous synapsis and impaired crossover assurance. Surprisingly, worms with a null mutation in PCH-2’s adapter protein, CMT-1, the ortholog of p31 comet , localize PCH-2 to meiotic chromosomes, exhibit non-homologous synapsis and lose crossover assurance. The similarity in phenotypes between cmt-1 and pch-2 E253Q mutants suggest that PCH-2 can bind its meiotic substrates in the absence of CMT-1, in contrast to its role during the spindle checkpoint, but requires its adapter to hydrolyze ATP and remodel them.

D. radiodurans is one of the most radiation-resistant organisms known. This bacterium is able to cope with high levels of DNA lesions generated by exposure to extreme doses of ionizing radiation and to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Here, we identified partners of PprA, a radiation-induced Deinococcus-specific protein, previously shown to be required for radioresistance. Our study leads to three main findings: (i) PprA interacts with DNA gyrase after irradiation, (ii) treatment of cells with novobiocin results in defects in chromosome segregation that are aggravated by the absence of PprA, and (iii) PprA stimulates the decatenation activity of DNA gyrase. Our results extend the knowledge of how D. radiodurans cells survive exposure to extreme doses of gamma irradiation and point out the link between DNA repair, chromosome segregation, and DNA gyrase activities in the radioresistant D. radiodurans bacterium.

HU proteins have an important architectural role in nucleoid organization in bacteria. Compared with HU of many bacteria, HU proteins from Deinococcus species possess an N-terminal lysine-rich extension similar to the eukaryotic histone H1 C-terminal domain involved in DNA compaction. The single HU gene in Deinococcus radiodurans, encoding DrHU, is required for nucleoid compaction and cell viability. Deinococcus deserti contains three expressed HU genes, encoding DdHU1, DdHU2 and DdHU3. Here, we show that either DdHU1 or DdHU2 is essential in D. deserti. DdHU1 and DdHU2, but not DdHU3, can substitute for DrHU in D. radiodurans, indicating that DdHU3 may have a non-essential function different from DdHU1, DdHU2 and DrHU. Interestingly, the highly abundant DrHU and DdHU1 proteins, and also the less expressed DdHU2, are translated in Deinococcus from leaderless mRNAs, which lack a 59-untranslated region and, hence, the Shine-Dalgarno sequence. Unexpectedly, cloning the DrHU or DdHU1 gene under control of a strong promoter in an expression plasmid, which results in leadered transcripts, strongly reduced the DrHU and DdHU1 protein level in D. radiodurans compared with that obtained from the natural leaderless gene. We also show that the start codon position for DrHU and DdHU1 should be reannotated, resulting in proteins that are 15 and 4 aa residues shorter than initially reported. The expression level and start codon correction were crucial for functional characterization of HU in Deinococcus.

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18 19 The conserved ATPase, PCH-2/TRIP13, is required during both the spindle checkpoint and 20 meiotic prophase. However, it's specific role in regulating meiotic homolog pairing, synapsis and 21 recombination has been enigmatic. Here, we report that this enzyme is required to proofread 22 meiotic homolog interactions. We generated a mutant version of PCH-2 in C. elegans that binds 23 ATP but cannot hydrolyze it: pch-2 E253Q . In vitro, this mutant binds its substrates but is unable to 24 remodel them. This mutation results in non-homologous synapsis and loss of crossover 25 assurance. Surprisingly, worms with a null mutation in PCH-2's adapter protein, CMT-1, the 26 ortholog of p31 comet , localize PCH-2 to meiotic chromosomes, exhibit non-homologous synapsis 27 and lose crossover assurance. The similarity in phenotypes between cmt-1 and pch-2 E253Q 28 mutants indicate that PCH-2 can bind its meiotic substrates in the absence of in 29 contrast to its role during the spindle checkpoint, but requires its adapter to hydrolyze ATP and 30 remodel them. 31Introduction 32 33 Sexual reproduction relies on meiosis, the specialized cell division that generates haploid 34 gametes, such as sperm and eggs, from diploid progenitors so that fertilization restores diploidy. 35To ensure that gametes inherit the correct number of chromosomes, meiotic chromosome 36 segregation is exquisitely choreographed: Homologous chromosomes segregate in meiosis I 37 and sister chromatids segregate in meiosis II. Having an incorrect number of chromosomes, 38 also called aneuploidy, is associated with infertility, miscarriages and birth defects, underscoring 39 the importance of understanding this process to human health. 40 41 Events in meiotic prophase ensure proper chromosome segregation. During prophase, 42 homologous chromosomes undergo progressively intimate interactions that culminate in 43 synapsis and crossover recombination (reviewed in [1]). After homologs pair, a macromolecular 44 complex, called the synaptonemal complex (SC), is assembled between them in a process 45 called synapsis. Synapsis is a prerequisite for crossover recombination to generate the 46 linkages, or chiasmata, between homologous chromosomes that direct meiotic chromosome 47 segregation. Defects in any of these events can result in chromosome missegregation during 48 the meiotic divisions and gametes, and therefore embryos, with an incorrect number of 49 chromosomes. 50 51The conserved AAA-ATPase PCH-2 (Pch2 in budding yeast, PCH2 in Arabidopsis and TRIP13 52 in mice) is crucial to coordinate these events in meiotic prophase. In vitro and cytological 53 experiments indicate that it does this by using the energy of ATP hydrolysis to remodel meiotic 54HORMADs [2][3][4][5][6], chromosomal proteins that are essential for pairing, synapsis, recombination 55 and checkpoint function [7][8][9][10][11][12][13][14][15][16][17][18]. HORMADs are a protein family defined by the ability of a 56 domain, the HORMA domain, to adopt multiple conformations that specify pr...

Meiotic homolog synapsis is essential to ensure accurate segregation of chromosomes during meiosis. In C. elegans, proper regulation of synapsis and a checkpoint that monitors synapsis relies on the spindle checkpoint components, Mad1 and Mad2, and Pairing Centers (PCs), cis-acting loci that interact with the nuclear envelope to mobilize chromosomes within the nucleus. Here, we test what specific functions of Mad1 and Mad2 are required to regulate and monitor synapsis. We find that a mutation that prevents Mad1’s localization to the nuclear periphery abolishes the synapsis checkpoint but has no effect on Mad2’s localization to the nuclear periphery or synapsis. By contrast, a mutation that prevents Mad1’s interaction with Mad2 abolishes the synapsis checkpoint, delays synapsis and fails to localize Mad2 to the nuclear periphery. These data indicate that Mad1’s primary role in regulating synapsis is through control of Mad2 and that Mad2 can bind other factors at the nuclear periphery. We also tested whether Mad2’s ability to adopt a specific conformation associated with its activity during spindle checkpoint function is required for its role in meiosis. A mutation that prevents Mad2 from adopting its active conformer fails to localize to the nuclear periphery, abolishes the synapsis checkpoint and exhibits substantial defects in meiotic synapsis. Thus, Mad2, and its regulation by Mad1, is an important regulator of meiotic synapsis in C. elegans.