Verena Jantsch

DNA lesions that persist in mitosis threaten genome stability. These lesions recruit TOPBP1-CIP2A, a complex crucial to tether and process damaged DNA on mitotic chromosomes. Importantly, CIP2A is synthetic lethal in BRCA1/2 mutant cancer cells. However, mechanistic insight into the function of CIP2A is lacking. Here we provide first structural insights into full-length CIP2A and reveal how CIP2A dimers, 46 nm in length, assemble via conserved C-terminal motifs into dumbbell-like tetramers. DNA repair signatures demonstrate that CIP2A tetramerization is crucial for mitotic DNA repair by polymerase theta (POLQ). Tetramerization of CIP2A is needed to recruit POLQ to mitotic DNA lesions and to prevent micronucleation upon replication-born DNA lesions. Tetramerization-defective CIP2A mutants specifically decrease survival of BRCA1/2 mutant cancer cells in a dominant negative manner. Combined, we demonstrate that CIP2A tetramers are crucial components of a mitotic DNA-tethering and repair scaffold, which processes replication-mediated DNA lesions that persist in mitosis.

Meiotic crossovers depend on the accurate processing of programmed DNA double-strand breaks by homologous recombination, which involves generating by resection the single-stranded DNA ends required for the subsequent strand invasion step. How regulatory kinases control DNA end resection in animal meiosis remains poorly understood. Here, we identify the Caenorhabditis elegans Dbf4-dependent kinase (DDK) complex, composed of CDC–7 and DBF–4, and identify CDK–12 as a candidate substrate. DDK promotes efficient meiotic DNA end resection, while crossover formation remains largely preserved in its absence. Loss of DDK function leads to SPO-11-dependent chromosome bridges at diakinesis that arise from recombination intermediates formed early in recombination. In addition to chromosome bridges, we observed elevated heterologous recombination that occurs independently of the canonical or alternative end-joining pathways. Interestingly, CDC–7 and DBF–4 localize dynamically during prophase I and load onto DNA in a hierarchical manner that depends on the BRC–1—BRD–1 complex. Epistasis analysis placed CDC–7 and CDK–12 within the DNA–2-dependent branch of long-range DNA resection. Together, our findings reveal an unidentified DDK/CDK–12/DNA–2 signaling axis that promotes efficient meiotic DNA resection to avoid the generation of aberrant recombination outcomes.

Proteins undergo dynamic conformational rearrangements and interactions that are central to their biological functions. Quantitative crosslinking mass spectrometry enables the analysis of those dynamics and molecular interactions, but rigorous confidence assessment and empirical validation strategies for quantitative measurements remain underdeveloped, and integrated analysis of complementary structural features, including monolinks and protein–RNA adducts, remains limited. Here we present a data-independent acquisition (DIA)-based framework for quantitative crosslinking mass spectrometry (DIA-QCLMS) that combines optimized acquisition strategies, crosslink-aware spectral libraries and empirical false-discovery-rate (FDR) validation. The workflow supports crosslinks, monolinks and protein–RNA adducts and integrates spectral-library generation from two crosslinking search engines (xiSEARCH and MS Annika). To enable robust confidence assessment in DIA data, we developed a four-state target–decoy spectral library strategy that explicitly models target–target, target–decoy, decoy–target and decoy–decoy crosslink spectra. Experimental entrapment datasets enabled empirical validation of confidence estimation, whereas benchmarking with PhoX-crosslinked Cas9 demonstrated improved quantitative completeness and reproducibility compared with data-dependent acquisition. Application of the workflow to the ATP-dependent RNA helicase UAP56 (DDX39B) resolved ligand-dependent changes in intramolecular restraints, residue accessibility and candidate RNA-contact sites associated with the transition from an open to a clamped conformation. These results establish DIA-QCLMS as a scalable framework for quantitative structural proteomics and provide practical strategies for confidence-controlled analysis of dynamic protein interactions and conformational states.

Rapid prophase chromosome movements ensure faithful alignment of the parental homologous chromosomes and successful synapsis formation during meiosis. These movements are driven by cytoplasmic forces transmitted to the nuclear periphery, where chromosome ends are attached through transmembrane proteins. During many developmental stages a specific genome architecture with chromatin nuclear periphery contacts mediates specific gene expression. Whether chromatin is removed from the nuclear periphery as a consequence of chromosome motions or by a specific mechanism is not fully understood. Here, we identify a mechanism to remove chromatin from the nuclear periphery through vaccinia related kinase (VRK-1)–dependent phosphorylation of Barrier to Autointegration Factor 1 (BAF-1) in Caenorhabditis elegans early prophase of meiosis. Interfering with chromatin removal delays chromosome pairing, impairs synapsis, produces oocytes with abnormal chromosomes and elevated apoptosis. Long read sequencing reveals deletions and duplications in offspring lacking VRK-1 underscoring the importance of the BAF-1–VRK-1 module in preserving genome stability in gametes during rapid chromosome movements.

Crossover (CO)-biased repair of meiotic DNA double-strand breaks is essential for proper chromosome segregation. However, only a subset of programmed induced DSBs is repaired as COs, while the rest is processed into non-COs. The Bloom-Topoisomerase 3–RMI1/2 complex is well documented to disassemble joint recombination intermediates into non-COs, but its pro-CO activities are less well understood. Here, we investigate how the pro-CO activities of the Caenorhabditis elegans Bloom helicase ortholog HIM-6 contribute to meiotic recombination by studying a catalytically inactive mutant. We show that HIM-6 helicase activity is required to provide a continuous flux of substrates for CO formation, probably via its unwinding activities, and that a structural role is sufficient to channel intermediates into the preferred pathway to generate correctly positioned COs. We provide evidence that the catalytic activity of Bloom helicase influences the geometry of the joint DNA molecules (double Holliday junctions (dHJ)). Localization of the signal for the dHJ-stabilizing complex MutSγ was more restricted, and epistasis experiments suggest that an altered geometry impedes the efficient processing of joint DNA molecules to generate CO-biased cleavage products.

Faithful meiotic segregation requires pairwise alignment of the homologous chromosomes and their synaptonemal complex (SC) mediated stabilization. Here, we investigate factors that promote and coordinate these events during C. elegans meiosis. We identify BRA-2 (BMP Receptor Associated family member 2) as an interactor of HIM-17, previously shown to promote double-strand break formation. We found that loss of bra-2 impairs synapsis elongation without affecting homolog recognition, chromosome movement or SC maintenance. Epistasis analyses reveal previously unrecognized activities for HIM-17 in regulating homolog pairing and SC assembly in a partially overlapping manner with BRA-2. We show that removing bra-2 or him-17 restores nuclear clustering, recruitment of PLK-2 at the nuclear periphery, and abrogation of ectopic synapsis in htp-1 mutants, suggesting intact CHK-2-mediated signaling and presence of a barrier that prevents SC polymerization in the absence of homology. Our findings shed light on the regulatory mechanisms ensuring faithful pairing and synapsis.

In this issue, El Mossadeq et al. (https://doi.org/10.1083/jcb.202403125) report that a structure forms around segregating chromosomes following meiosis I that shares features with the nuclear envelope in interphase but also has distinct, unique characteristics.

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.

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.

Transition from the stem/progenitor cell fate to meiosis is mediated by several redundant posttranscriptional regulatory pathways in Caenorhabditis elegans . Interfering with all three branches causes tumorous germ lines. SCF ^PROM-1 comprises one branch and mediates a scheduled degradation step at entry into meiosis. prom-1 mutants show defects in the timely initiation of meiotic prophase I events, resulting in high rates of embryonic lethality. Here, we identify the phosphatase PPM-1.D/Wip1 as crucial substrate for PROM-1. We report that PPM-1.D antagonizes CHK-2 kinase, a key regulator for meiotic prophase initiation, including DNA double-strand breaks, chromosome pairing, and synaptonemal complex formation. We propose that PPM-1.D controls the amount of active CHK-2 via both catalytic and noncatalytic activities; notably, noncatalytic regulation seems to be crucial at meiotic entry. PPM-1.D sequesters CHK-2 at the nuclear periphery, and programmed SCF ^PROM-1 –mediated degradation of PPM-1.D liberates the kinase and promotes meiotic entry.