BRCA1/BRC-1 and SMC-5/6 regulate DNA repair pathway engagement during Caenorhabditis elegans meiosis.

The preservation of genome integrity during sperm and egg development is vital for reproductive success. During meiosis, the tumor suppressor BRCA1/BRC-1 and structural maintenance of chromosomes 5/6 (SMC-5/6) complex genetically interact to promote high fidelity DNA double strand break (DSB) repair, but the specific DSB repair outcomes these proteins regulate remain unknown. Using genetic and cytological methods to monitor resolution of DSBs with different repair partners in Caenorhabditis elegans, we demonstrate that both BRC-1 and SMC-5 repress intersister crossover recombination events. Sequencing analysis of conversion tracts from homolog-independent DSB repair events further indicates that BRC-1 regulates intersister/intrachromatid noncrossover conversion tract length. Moreover, we find that BRC-1 specifically inhibits error prone repair of DSBs induced at mid-pachytene. Finally, we reveal functional interactions of BRC-1 and SMC-5/6 in regulating repair pathway engagement: BRC-1 is required for localization of recombinase proteins to DSBs in smc-5 mutants and enhances DSB repair defects in smc-5 mutants by repressing theta-mediated end joining (TMEJ). These results are consistent with a model in which some functions of BRC-1 act upstream of SMC-5/6 to promote recombination and inhibit error-prone DSB repair, while SMC-5/6 acts downstream of BRC-1 to regulate the formation or resolution of recombination intermediates. Taken together, our study illuminates the coordinated interplay of BRC-1 and SMC-5/6 to regulate DSB repair outcomes in the germline.

Automated and customizable quantitative image analysis of whole Caenorhabditis elegans germlines.

Arranged in a spatial-temporal gradient for germ cell development, the adult germline of Caenorhabditis elegans is an excellent system for understanding the generation, differentiation, function, and maintenance of germ cells. Imaging whole C. elegans germlines along the distal-proximal axis enables powerful cytological analyses of germ cell nuclei as they progress from the pre-meiotic tip through all the stages of meiotic prophase I. To enable high-content image analysis of whole C. elegans gonads, we developed a custom algorithm and pipelines to function with image processing software that enables: (1) quantification of cytological features at single nucleus resolution from immunofluorescence images; and (2) assessment of these individual nuclei based on their position within the germline. We show the capability of our quantitative image analysis approach by analyzing multiple cytological features of meiotic nuclei in whole C. elegans germlines. First, we quantify double-strand DNA breaks (DSBs) per nucleus by analyzing DNA-associated foci of the recombinase RAD-51 at single-nucleus resolution in the context of whole germline progression. Second, we quantify the DSBs that are licensed for crossover repair by analyzing foci of MSH-5 and COSA-1 when they associate with the synaptonemal complex during meiotic prophase progression. Finally, we quantify P-granule composition across the whole germline by analyzing the colocalization of PGL-1 and ZNFX-1 foci. Our image analysis pipeline is an adaptable and useful method for researchers spanning multiple fields using the C. elegans germline as a model system.

Elevated Temperatures Cause Transposon-Associated DNA Damage in C. elegans Spermatocytes.

Sexually reproducing organisms use meiosis to generate haploid gametes and faithfully transmit their genome to the next generation. In comparison to oogenesis in many organisms, spermatogenesis is particularly sensitive to small temperature fluctuations, and spermatocytes must develop within a very narrow isotherm [1-4]. Although failure to thermoregulate spermatogenetic tissue and prolonged exposure to elevated temperatures are linked to male infertility in several organisms, the mechanisms of temperature-induced male infertility have not been fully elucidated [5]. Here, we show that upon exposure to a brief 2°C temperature increase, Caenorhabditis elegans spermatocytes exhibit up to a 25-fold increase in double-strand DNA breaks (DSBs) throughout meiotic prophase I and a concurrent reduction in male fertility. We demonstrate that these heat-induced DSBs in spermatocytes are independent of the endonuclease SPO-11. Further, we find that the production of these heat-induced DSBs in spermatocytes correlate with heat-induced mobilization of Tc1/mariner transposable elements, which are known to cause DSBs and alter genome integrity [6, 7]. Moreover, we define the specific sequences and regions of the male genome that preferentially experience these heat-induced de novo Tc1 insertions. In contrast, oocytes do not exhibit changes in DSB formation or Tc1 transposon mobility upon temperature increases. Taken together, our data suggest spermatocytes are less tolerant of higher temperatures because of an inability to effectively repress the movement of specific mobile DNA elements that cause excessive DNA damage and genome alterations, which can impair fertility.