DNA double-strand break repair in Caenorhabditis elegans

Faithful repair of DNA double-strand breaks (DSBs) is vital for animal development, as inappropriate repair can cause gross chromosomal alterations that result in cellular dysfunction, ultimately leading to cancer, or cell death. Correct processing of DSBs is not only essential for maintaining genomic integrity, but is also required in developmental programs, such as gametogenesis, in which DSBs are deliberately generated. Accordingly, DSB repair deficiencies are associated with various developmental disorders including cancer predisposition and infertility. To avoid this threat, cells are equipped with an elaborate and evolutionarily well-conserved network of DSB repair pathways. In recent years, Caenorhabditis elegans has become a successful model system in which to study DSB repair, leading to important insights in this process during animal development. This review will discuss the major contributions and recent progress in the C. elegans field to elucidate the complex networks involved in DSB repair, the impact of which extends well beyond the nematode phylum

DNA replication determines timing of mitosis by restricting CDK1 and PLK1 activation

To maintain genome stability, cells need to replicate their DNA before dividing. The kinases CDK1 and PLK1 drive mitotic entry and become active when bulk DNA synthesis is completed at the S/G2 transition. Here, we have tested the hypothesis that DNA replication controls activation of mitotic kinases. Using an optimized double-degron system, we find that human cells unable to initiate DNA replication in S-phase promptly activate CDK1 and PLK1 and prematurely enter mitosis. In the presence of DNA replication, inhibition of CHK1 and p38 leads to premature activation of CDK1 and PLK1. While CDK2 activity promotes DNA replication, activation of CDK1 in S-phase induces severe replication stress. We propose that mitotic kinase activation is governed by a CDK2- and DNA replication-dependent feed-forward loop that ensures timely cell division while preserving genome stability. DNA replication thus functions as a break that coordinates cell cycle activities and determines cell cycle duration

Actionable cancer vulnerability due to translational arrest, p53 aggregation and ribosome biogenesis stress evoked by the disulfiram metabolite CuET.

We would like to thank M.Oren (Weizmann Institute of Science) for kindly providing the MDM2 antibodies, the core facility for Bioinformatics and Expression Analysis (BEA, Karolinska, Huddinge) for assisting in massive parallel sequencing and computational infrastructure, as well as E Dratkiewicz, AS Nilsson, and JF Martinez for excellent technical assistance.Drug repurposing is a versatile strategy to improve current therapies. Disulfiram has long been used in the treatment of alcohol dependency and multiple clinical trials to evaluate its clinical value in oncology are ongoing. We have recently reported that the disulfiram metabolite diethyldithiocarbamate, when combined with copper (CuET), targets the NPL4 adapter of the p97VCP segregase to suppress the growth of a spectrum of cancer cell lines and xenograft models in vivo. CuET induces proteotoxic stress and genotoxic effects, however important issues concerning the full range of the CuET-evoked tumor cell phenotypes, their temporal order, and mechanistic basis have remained largely unexplored. Here, we have addressed these outstanding questions and show that in diverse human cancer cell models, CuET causes a very early translational arrest through the integrated stress response (ISR), later followed by features of nucleolar stress. Furthermore, we report that CuET entraps p53 in NPL4-rich aggregates leading to elevated p53 protein and its functional inhibition, consistent with the possibility of CuET-triggered cell death being p53-independent. Our transcriptomics profiling revealed activation of pro-survival adaptive pathways of ribosomal biogenesis (RiBi) and autophagy upon prolonged exposure to CuET, indicating potential feedback responses to CuET treatment. The latter concept was validated here by simultaneous pharmacological inhibition of RiBi and/or autophagy that further enhanced CuET's tumor cytotoxicity, using both cell culture and zebrafish in vivo preclinical models. Overall, these findings expand the mechanistic repertoire of CuET's anti-cancer activity, inform about the temporal order of responses and identify an unorthodox new mechanism of targeting p53. Our results are discussed in light of cancer-associated endogenous stresses as exploitable tumor vulnerabilities and may inspire future clinical applications of CuET in oncology, including combinatorial treatments and focus on potential advantages of using certain validated drug metabolites, rather than old, approved drugs with their, often complex, metabolic profiles.This work was funded by the following grants: the Swedish Cancer Society (grant number: 170176), the Swedish Research Council (VR-MH 2014-46602-117891-30), Novo Nordisk Foundation (NNF20OC0060590), Danish National Research Foundation (project CARD, DNRF 125), the Danish Cancer Society (R204-A12617-B153), DFF 1026-00241B (all granted to JB), and the Grant agency of the Czech Republic: GACR 20-28685S (granted to ZS and MM). Open access funding provided by Karolinska Institute.S

COM-1 promotes homologous recombination during Caenorhabditis elegans meiosis by antagonizing Ku-mediated non-homologous end joining.

Successful completion of meiosis requires the induction and faithful repair of DNA double-strand breaks (DSBs). DSBs can be repaired via homologous recombination (HR) or non-homologous end joining (NHEJ), yet only repair via HR can generate the interhomolog crossovers (COs) needed for meiotic chromosome segregation. Here we identify COM-1, the homolog of CtIP/Sae2/Ctp1, as a crucial regulator of DSB repair pathway choice during Caenorhabditis elegans gametogenesis. COM-1-deficient germ cells repair meiotic DSBs via the error-prone pathway NHEJ, resulting in a lack of COs, extensive chromosomal aggregation, and near-complete embryonic lethality. In contrast to its yeast counterparts, COM-1 is not required for Spo11 removal and initiation of meiotic DSB repair, but instead promotes meiotic recombination by counteracting the NHEJ complex Ku. In fact, animals defective for both COM-1 and Ku are viable and proficient in CO formation. Further genetic dissection revealed that COM-1 acts parallel to the nuclease EXO-1 to promote interhomolog HR at early pachytene stage of meiotic prophase and thereby safeguards timely CO formation. Both of these nucleases, however, are dispensable for RAD-51 recruitment at late pachytene stage, when homolog-independent repair pathways predominate, suggesting further redundancy and/or temporal regulation of DNA end resection during meiotic prophase. Collectively, our results uncover the potentially lethal properties of NHEJ during meiosis and identify a critical role for COM-1 in NHEJ inhibition and CO assurance in germ cells

EXO-1 is required for meiotic recombination in absence of COM-1.

<p>(A) Representative image of diplotene nuclei of <i>com-1 cku-80 exo-1</i> triple mutant animals that express a ZHP-3::GFP transgene (left: GFP signal only, right: merge of GFP and DAPI signal) (B) Representative picture of a diakinesis nucleus in <i>com-1 cku-80 exo-1</i> triple mutants germlines (C) RAD-51 immunostaining of mid-pachytene nuclei (zone 5) in <i>com-1 cku-80 exo-1</i> mutant germlines; merge of RAD-51 (red) and DAPI signal (blue) (D) Percentage progeny survival of animals of the indicated genotype; values are the average of 3 independent experiments*, error bars represent S.E.M. (E) Frequency distribution of DAPI-stained entities at diakinesis*. n = number of germlines analyzed. The <i>com-1 cku-80 exo-1</i> triple mutants occasionally showed >12 DAPI bodies due to chromosomal fragmentation. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003276#pgen-1003276-g007" target="_blank">Figure 7E</a> for quantification. Scale bars, 5 µm. *These experiments were performed in parallel to those depicted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003276#pgen-1003276-g001" target="_blank">Figure 1B and 1D</a>; reference values are depicted again here.</p

Loss of <i>lig-4</i> prevents chromosomal fusion in <i>com-1</i> mutants, but does not restore viability.

<p>(A) Two representative pictures of diakinesis nuclei of animals of the indicated genotype. White arrows point out chromosomal fragments (B) Percentage progeny survival; values are the average of 3 independent experiments, error bars represent S.E.M. (C) Percentage of diakinesis nuclei that show chromosomal fragments; n = number of germlines analyzed. Scale bars, 5 µm. *The difference between these genotypes was highly significant (p<0.001 by Fisher's exact test, two tailed).</p

LIN-61 is dispensable for DNA damage checkpoints in the germline.

<p>(A) Schematic diagram of the hermaphrodite germline. Cell cycle arrest (as in B–C) occurs in the mitotic zone and apoptosis (D–E) occurs at the bend of the germline. DTC, distal tip cell; TZ, transition zone. (B) Maximum projections of DAPI-stained mitotic nuclei 24 hours after irradiation with 60 Gy or mock-treatment. (C) Quantification of mitotic cell cycle arrest, error bars are s.d. (D) DIC images of pachytene stage nuclei 24 hours after irradiation with 60 Gy or mock-treatment. Arrowheads mark apoptotic corpses. (E) Quantification of apoptotic corpses per germline arm. Error bars represent s.d. (F) Quantification of <i>egl-1</i> mRNA by qRT-PCR, normalised to untreated wild types. Total RNA was isolated from mixed populations of developmentally staged young adults 24 hours after irradiation with 120 Gy, or mock treatment.</p

Model for meiotic recombination in <i>C. elegans</i>.

<p>In wild-type germlines, MRE-11 may create substrates at meiotic DSBs that allow COM-1 to efficiently remove Ku (and SPO-11). When COM-1 function is perturbed, MRE-11 mediated processing may still release SPO-11 bound oligos. However, MRE-11 activity alone is not sufficient to counteract Ku binding and prevent toxic NHEJ activity. Without COM-1 and Ku, SPO-11 is removed and EXO-1 promotes DNA end resection and allows the obligate COs to be formed. See text for further details.</p