Novel mechanistic insights into the role of Mer2 as the keystone of meiotic DNA break formation

In meiosis, DNA double strand break (DSB) formation by Spo11 initiates recombination and enables chromosome segregation. Numerous factors are required for Spo11 activity, and couple the DSB machinery to the development of a meiosis-specific “axis-tethered loop” chromosome organization. Through in vitro reconstitution and budding yeast genetics we here provide architectural insight into the DSB machinery by focussing on a foundational DSB factor, Mer2. We characterise the interaction of Mer2 with the histone reader Spp1, and show that Mer2 directly associates to nucleosomes, likely highlighting a contribution of Mer2 to tethering DSB factors to chromatin. We reveal the biochemical basis of Mer2 association with Hop1, a HORMA domain-containing chromosomal axis factor. Finally, we identify a conserved region within Mer2 crucial for DSB activity, and show that this region of Mer2 interacts with the DSB factor Mre11. In combination with previous work, we establish Mer2 as a keystone of the DSB machinery by bridging key protein complexes involved in the initiation of meiotic recombination.

Sumo-mediated recruitment allows timely function of the Yen1 nuclease in mitotic cells

The modification of DNA damage response proteins with Sumo is an important mechanism to orchestrate a timely and orderly recruitment of repair factors to damaged sites. After replication stress and double-strand break formation a number of repair factors are Sumoylated and interact with other Sumoylated factors, including the nuclease Yen1. Yen1 plays a critical role to ensure genome stability and unperturbed chromosome segregation by removing covalently linked DNA intermediates that are formed by homologous recombination. Here we show how this important role of Yen1 is dependent on interactions mediated by non-covalent binding to Sumoylated partners. Mutations in the motifs that allow Sumo-mediated recruitment of Yen1 impair its ability to resolve DNA intermediates and result in increased genome instability and chromosome missegregation.

Heterologous synapsis in C. elegans is regulated by meiotic double-strand breaks and crossovers

Alignment of the parental chromosomes during meiotic prophase is key to the formation of genetic exchanges, or crossovers, and consequently to the successful production of gametes. In almost all studied organisms, alignment involves synapsis: the assembly of a conserved inter-chromosomal interface called the synaptonemal complex (SC). While the SC usually synapses homologous sequences, it can assemble between heterologous sequences. However, little is known about the regulation of heterologous synapsis. Here we study the dynamics of heterologous synapsis in the nematode C. elegans. We characterize two experimental scenarios: SC assembly onto a folded-back chromosome that cannot pair with its homologous partner; and synapsis of pseudo-homologs, a fusion chromosome partnering with an unfused chromosome half its size. We observed elevated levels of heterologous synapsis when the number of meiotic double-strand breaks or crossovers were reduced, indicating that the promiscuity of synapsis is regulated by break formation or repair. By manipulating the levels of breaks and crossovers, we infer both chromosome-specific and nucleus-wide regulation on heterologous synapsis. Finally, we identify differences between the two conditions, suggesting that attachment to the nuclear envelope plays a role in regulating heterologous synapsis.

The HMCES DNA-protein cross-link functions as a constitutive DNA repair intermediate

The HMCES protein forms a covalent DNA-protein cross-link (DPC) with abasic (AP) sites in ssDNA, and the resulting HMCES-DPC is thought to suppress double-strand break formation in S phase. However, the dynamics of HMCES cross-linking and whether any DNA repair pathways normally include an HMCES-DPC intermediate remain unknown. Here, we show that an HMCES-DPC forms efficiently on the AP site generated during replication-coupled DNA interstrand cross-link (ICL) repair. We use this system to show that HMCES cross-links form on DNA after the replicative CMG helicase has passed over the AP site, and that HMCES is subsequently removed by the SPRTN protease. The HMCES-DPC suppresses DSB formation, slows translesion synthesis (TLS) past the AP site, and introduces a bias for insertion of deoxyguanosine opposite the AP site. These data show that HMCES-DPCs can form as constitutive intermediates in replication-coupled repair, and they suggest a general model of how HMCES protects AP sites during DNA replication.

Natural Merosesquiterpenes Activate the DNA Damage Response via DNA Strand Break Formation and Trigger Apoptotic Cell Death in p53-Wild-type and Mutant Colorectal Cancer

Colorectal cancer (CRC) is a frequently occurring malignant disease with still low survival rates, highlighting the need for novel therapeutics. Merosesquiterpenes are secondary metabolites from marine sponges, which might be useful as antitumor agents. To address this issue, we made use of a compound library comprising 11 isolated merosesquiterpenes. The most cytotoxic compounds were smenospongine > ilimaquinone ≈ dactylospontriol, as shown in different human CRC cell lines. Alkaline Comet assays and γH2AX immunofluorescence microscopy demonstrated DNA strand break formation in CRC cells. Western blot analysis revealed an activation of the DNA damage response with CHK1 phosphorylation, stabilization of p53 and p21, which occurred both in CRC cells with p53 knockout and in p53-mutated CRC cells. This resulted in cell cycle arrest followed by a strong increase in the subG1 population, indicative of apoptosis, and typical morphological alterations. In consistency, cell death measurements showed apoptosis following exposure to merosesquiterpenes. Gene expression studies and analysis of caspase cleavage revealed mitochondrial apoptosis via BAX, BIM, and caspase-9 as the main cell death pathway. Interestingly, the compounds were equally effective in p53-wild-type and p53-mutant CRC cells. Finally, the cytotoxic activity of the merosesquiterpenes was corroborated in intestinal tumor organoids, emphasizing their potential for CRC chemotherapy.

Sex without crossing over in the yeastSaccharomycodes ludwigii

Meiotic recombination is a ubiquitous function of sexual reproduction throughout eukaryotes. Here we report that recombination is extremely suppressed during meiosis in the yeast speciesSaccharomycodes ludwigii. DNA double-strand break formation, processing and repair are required for normal meiosis but do not lead to crossing over. Although the species has retained an intact meiotic gene repertoire, genetic and population analyses suggest the exceptionally rare occurrence of meiotic crossovers. We propose thatSd. ludwigiihas followed a unique evolutionary trajectory that possibly derives fitness benefits from the combination of frequent fertilization within the meiotic tetrad with the absence of meiotic recombination.

Targeting the nucleotide salvage factor DNPH1 sensitizes BRCA-deficient cells to PARP inhibitors

Mutations in the BRCA1 or BRCA2 tumor suppressor genes predispose individuals to breast and ovarian cancer. In the clinic, these cancers are treated with inhibitors that target poly(ADP-ribose) polymerase (PARP). We show that inhibition of DNPH1, a protein that eliminates cytotoxic nucleotide 5-hydroxymethyl-deoxyuridine (hmdU) monophosphate, potentiates the sensitivity of BRCA-deficient cells to PARP inhibitors (PARPi). Synthetic lethality was mediated by the action of SMUG1 glycosylase on genomic hmdU, leading to PARP trapping, replication fork collapse, DNA break formation, and apoptosis. BRCA1-deficient cells that acquired resistance to PARPi were resensitized by treatment with hmdU and DNPH1 inhibition. Because genomic hmdU is a key determinant of PARPi sensitivity, targeting DNPH1 provides a promising strategy for the hypersensitization of BRCA-deficient cancers to PARPi therapy.