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

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

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

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

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

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

EXO-1 promotes DSB repair in germ cells.

<p>(A) Gene model of wild-type F45G2.3 (<i>exo-1</i>) with the position of its catalytic domain (gray) and below its truncation allele <i>tm1842</i>; a 559 bp deletion (purple) results in a premature stop (B) Percentage progeny survival of animals of the indicated genotype treated with the indicated dose of IR; values are the average of 3 independent experiments, error bars represent S.E.M. (C) RAD-51 immunostaining of mid-pachytene nuclei (zone 5) in <i>exo-1</i> deficient germlines; merge of RAD-51 (red) and DAPI signal (blue) (D) Representative picture of a diakinesis nucleus of <i>exo-1</i> deficient animals.</p

LIN-61 contributes to HR in mitotic cells, but is dispensable for meiotic HR.

<p>(A) DAPI-stained DNA bodies in diakinesis stage oocytes of animals mock treated (L4440) or depleted of SYP-2 by RNAi. (B) Time course of chromosomal fragmentation in response to 90 Gy dose of IR. In (A) and (B), the red arrowheads indicate chromosomal fragments and the inset number corresponds to the number of small fragments visible in the image. (C) Quantification of the chromosomal fragmentation. (D) Epistatic analysis of <i>brc-1</i> and <i>lin-61(pk2225)</i> IR sensitivity. L4 larvae were irradiated with the indicated dose. The percentage of viable embryos is plotted. Error bars represent s.d.</p