TERRA expression is regulated by the telomere-binding proteins POT-1 and POT-2 in Caenorhabditis elegans.

Several aspects of telomere biology are regulated by the telomeric repeat-containing RNA TERRA. While TERRA expression is conserved through evolution, species-specific mechanisms regulate its biogenesis and function. Here we report on the expression of TERRA in Caenorhabditis elegans. We show that C. elegans TERRA is regulated by the telomere-binding proteins POT-1 and POT-2 which repress TERRA in a telomere-specific manner. C. elegans TERRA transcripts are heterogeneous in length and form discrete nuclear foci, as detected by RNA FISH, in both postmitotic and germline cells; a fraction of TERRA foci localizes to telomeres. Interestingly, in germ cells, TERRA is expressed in all stages of meiotic prophase I, and it increases during pachytene, a stage in meiosis when homologous recombination is ongoing. We used the MS2-GFP system to study the spatiotemporal dynamics of single-telomere TERRA molecules. Single particle tracking revealed different types of motilities, suggesting complex dynamics of TERRA transcripts. Finally, we unveiled distinctive features of C. elegans TERRA, which is regulated by telomere shortening in a telomere-specific manner, and it is upregulated in the telomerase-deficient trt-1; pot-2 double mutant prior to activation of the alternative lengthening mechanism ALT. Interestingly, in these worms TERRA displays distinct dynamics with a higher fraction of fast-moving particles.

The topoisomerase 3 zinc finger domain cooperates with the RMI1 scaffold to promote stable association of the BTR complex to recombination intermediates in the Caenorhabditis elegans germline.

Homologous recombination is the predominant DNA repair pathway used in the gonad. Of the excess DNA double-strand breaks formed in meiosis, only a subset matures into crossovers, with the remainder repaired as non-crossovers. The conserved BTR complex (comprising Bloom helicase, topoisomerase 3 and RMI1/2 scaffold proteins) acts at multiple steps during recombination to dismantle joint DNA molecules, thereby mediating the non-crossover outcome and chromosome integrity. Furthermore, the complex displays a role at the crossover site that is less well understood. Besides catalytic and TOPRIM domains, topoisomerase 3 enzymes contain a variable number of carboxy terminal zinc finger (ZnF) domains. Here, we studied the Caenorhabditis elegans mutant, in which the single ZnF domain is deleted. In contrast to the gene disruption allele, the top-3-ZnF mutant is viable, with no replication defects; the allele appears to be a hypomorph. The TOP-3-ZnF protein is recruited into foci but the mutant has increased numbers of crossovers along its chromosomes, with minor defects in repressing heterologous recombination, and a marked delay in the maturation/processing of recombination intermediates after loading of the RAD-51 recombinase. The ZnF domain cooperates with the RMI1 homolog RMH-2 to stabilize association of the BTR complex with recombination intermediates and to prevent recombination between heterologous DNA sequences.

Caenorhabditis elegans RMI2 functional homolog-2 (RMIF-2) and RMI1 (RMH-1) have both overlapping and distinct meiotic functions within the BTR complex.

Homologous recombination is a high-fidelity repair pathway for DNA double-strand breaks employed during both mitotic and meiotic cell divisions. Such repair can lead to genetic exchange, originating from crossover (CO) generation. In mitosis, COs are suppressed to prevent sister chromatid exchange. Here, the BTR complex, consisting of the Bloom helicase (HIM-6 in worms), topoisomerase 3 (TOP-3), and the RMI1 (RMH-1 and RMH-2) and RMI2 scaffolding proteins, is essential for dismantling joint DNA molecules to form non-crossovers (NCOs) via decatenation. In contrast, in meiosis COs are essential for accurate chromosome segregation and the BTR complex plays distinct roles in CO and NCO generation at different steps in meiotic recombination. RMI2 stabilizes the RMI1 scaffolding protein, and lack of RMI2 in mitosis leads to elevated sister chromatid exchange, as observed upon RMI1 knockdown. However, much less is known about the involvement of RMI2 in meiotic recombination. So far, RMI2 homologs have been found in vertebrates and plants, but not in lower organisms such as Drosophila, yeast, or worms. We report the identification of the Caenorhabditis elegans functional homolog of RMI2, which we named RMIF-2. The protein shows a dynamic localization pattern to recombination foci during meiotic prophase I and concentration into recombination foci is mutually dependent on other BTR complex proteins. Comparative analysis of the rmif-2 and rmh-1 phenotypes revealed numerous commonalities, including in regulating CO formation and directing COs toward chromosome arms. Surprisingly, the prevalence of heterologous recombination was several fold lower in the rmif-2 mutant, suggesting that RMIF-2 may be dispensable or less strictly required for some BTR complex-mediated activities during meiosis.

BRCA1-BARD1 associate with the synaptonemal complex and pro-crossover factors and influence RAD-51 dynamics during Caenorhabditis elegans meiosis.

During meiosis, the maternal and paternal homologous chromosomes must align along their entire length and recombine to achieve faithful segregation in the gametes. Meiotic recombination is accomplished through the formation of DNA double-strand breaks, a subset of which can mature into crossovers to link the parental homologous chromosomes and promote their segregation. Breast and ovarian cancer susceptibility protein BRCA1 and its heterodimeric partner BARD1 play a pivotal role in DNA repair in mitotic cells; however, their functions in gametogenesis are less well understood. Here we show that localization of BRC-1 and BRD-1 (Caenorhabditis elegans orthologues of BRCA1 and BARD1) is dynamic during meiotic prophase I; they ultimately becoming concentrated at regions surrounding the presumptive crossover sites, co-localizing with the pro-crossover factors COSA-1, MSH-5 and ZHP-3. The synaptonemal complex and PLK-2 activity are essential for recruitment of BRC-1 to chromosomes and its subsequent redistribution towards the short arm of the bivalent. BRC-1 and BRD-1 form in vivo complexes with the synaptonemal complex component SYP-3 and the crossover-promoting factor MSH-5. Furthermore, BRC-1 is essential for efficient stage-specific recruitment/stabilization of the RAD-51 recombinase to DNA damage sites when synapsis is impaired and upon induction of exogenous damage. Taken together, our data provide new insights into the localization and meiotic function of the BRC-1-BRD-1 complex and highlight its essential role in DNA double-strand break repair during gametogenesis.

Separable Roles for a Caenorhabditis elegans RMI1 Homolog in Promoting and Antagonizing Meiotic Crossovers Ensure Faithful Chromosome Inheritance.

During the first meiotic division, crossovers (COs) between homologous chromosomes ensure their correct segregation. COs are produced by homologous recombination (HR)-mediated repair of programmed DNA double strand breaks (DSBs). As more DSBs are induced than COs, mechanisms are required to establish a regulated number of COs and to repair remaining intermediates as non-crossovers (NCOs). We show that the Caenorhabditis elegans RMI1 homolog-1 (RMH-1) functions during meiosis to promote both CO and NCO HR at appropriate chromosomal sites. RMH-1 accumulates at CO sites, dependent on known pro-CO factors, and acts to promote CO designation and enforce the CO outcome of HR-intermediate resolution. RMH-1 also localizes at NCO sites and functions in parallel with SMC-5 to antagonize excess HR-based connections between chromosomes. Moreover, RMH-1 also has a major role in channeling DSBs into an NCO HR outcome near the centers of chromosomes, thereby ensuring that COs form predominantly at off-center positions.

DNA topoisomerase 3 is required for efficient germ cell quality control.

An important quality control mechanism eliminates meiocytes that have experienced recombination failure during meiosis. The culling of defective oocytes in Caenorhabditis elegans meiosis resembles late oocyte elimination in female mammals. Here we show that topoisomerase 3 depletion generates DNA lesions in both germline mitotic and meiotic compartments that are less capable of triggering p53 (cep-1)-dependent apoptosis, despite the activation of DNA damage and apoptosis signaling. Elimination of nonhomologous, alternative end joining and single strand annealing repair factors (CKU-70, CKU-80, POLQ-1, and XPF-1) can alleviate the apoptosis block. Remarkably, the ability of single mutants in the other members of the Bloom helicase-topoisomerase-RMI1 complex to elicit apoptosis is not compromised, and depletion of Bloom helicase in topoisomerase 3 mutants restores an effective apoptotic response. Therefore, uncontrolled Bloom helicase activity seems to direct DNA repair toward normally not used repair pathways, and this counteracts efficient apoptosis. This implicates an as-yet undescribed requirement for topoisomerase 3 in mounting an effective apoptotic response to ensure germ cell quality control.