Methods for Studying the DNA Damage Response in the Caenorhabdatis elegans Germ Line

Craig, Ashley; Moser, Sandra; Bailly, Aymeric; Gartner, Anton

doi:10.1016/b978-0-12-394620-1.00011-4

Cited by 98 publications

(116 citation statements)

References 66 publications

Supporting

Mentioning

113

Contrasting

Order By: Relevance

“…To monitor apoptotic germ cells, we first quantified in vivo germ cell corpses in the proximal zone of the gonad arm (see Fig. 2A) by utilizing differential interference contrast (DIC) microscopy and staining with the DNA dye acridine orange21. In wild type animals, one to four apoptotic cells were detected (Fig.…”

Section: Resultsmentioning

confidence: 99%

A novel approach using C. elegans DNA damage-induced apoptosis to characterize the dynamics of uptake transporters for therapeutic drug discoveries

Papaluca

Ramotar

2016

Sci Rep

View full text Add to dashboard Cite

Organic cation transporter (OCT) function is critical for cellular homeostasis. C. elegans lacking OCT-1 displays a shortened lifespan and increased susceptibility to oxidative stress. We show that these phenotypes can be rescued by downregulating the OCT-1 paralogue, OCT-2. Herein, we delineate a biochemical pathway in C. elegans where uptake of genotoxic chemotherapeutics such as doxorubicin and cisplatin, and subsequent DNA damage-induced apoptosis of germ cells, are dependent exclusively upon OCT-2. We characterized OCT-2 as the main uptake transporter for doxorubicin, as well as a number of other therapeutic agents and chemical compounds, some identified through ligand-protein docking analyses. We provide insights into the conserved features of the structure and function and gene regulation of oct-1 and oct-2 in distinct tissues of C. elegans. Importantly, our innovative approach of exploiting C. elegans uptake transporters in combination with defective DNA repair pathways will have broad applications in medicinal chemistry.

show abstract

Section: Resultsmentioning

confidence: 99%

A novel approach using C. elegans DNA damage-induced apoptosis to characterize the dynamics of uptake transporters for therapeutic drug discoveries

Papaluca

Ramotar

2016

Sci Rep

View full text Add to dashboard Cite

show abstract

“…Developmental arrest in unstressed larvae was assayed as previously described (Craig et al 2012). Briefly, 100 L1 larvae of a given genotype were plated onto center-seeded 3-cm dishes in triplicate.…”

Section: Developmental Arrest Assaymentioning

confidence: 99%

Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans

et al. 2016

View full text Add to dashboard Cite

Homologous recombination (HR) repairs cytotoxic DNA double-strand breaks (DSBs) with high fidelity. Deficiencies in HR result in genome instability. A key early step in HR is the search for and invasion of a homologous DNA template by a single-stranded RAD-51 nucleoprotein filament. The Shu complex, composed of a SWIM domain-containing protein and its interacting RAD51 paralogs, promotes HR by regulating RAD51 filament dynamics. Despite Shu complex orthologs throughout eukaryotes, our understanding of its function has been most extensively characterized in budding yeast. Evolutionary analysis of the SWIM domain identified Caenorhabditis elegans sws-1 as a putative homolog of the yeast Shu complex member Shu2. Using a CRISPR-induced nonsense allele of sws-1, we show that sws-1 promotes HR in mitotic and meiotic nuclei. sws-1 mutants exhibit sensitivity to DSBinducing agents and fail to form mitotic RAD-51 foci following treatment with camptothecin. Phenotypic similarities between sws-1 and the two RAD-51 paralogs rfs-1 and rip-1 suggest that they function together. Indeed, we detect direct interaction between SWS-1 and RIP-1 by yeast two-hybrid assay that is mediated by the SWIM domain in SWS-1 and the Walker B motif in RIP-1. Furthermore, RIP-1 bridges an interaction between SWS-1 and RFS-1, suggesting that RIP-1 facilitates complex formation with SWS-1 and RFS-1. We propose that SWS-1, RIP-1, and RFS-1 compose a C. elegans Shu complex. Our work provides a new model for studying Shu complex disruption in the context of a multicellular organism that has important implications as to why mutations in the human RAD51 paralogs are associated with genome instability.KEYWORDS homologous recombination; RAD51 paralog; Shu complex; camptothecin; helq-1 D NA double-strand breaks (DSBs) are extremely cytotoxic lesions that threaten genome integrity. DSBs arise from both endogenous sources, such as replicative damage, or exogenous sources, such as ionizing radiation (IR) and chemotherapeutic agents. To ensure the maintenance of the genome, DSBs need to be repaired by high-fidelity repair pathways, the most robust of which is homologous recombination (HR), in which DNA from a sister chromatid or homologous chromosome provides a repair template. Initial processing of DSB ends by resection forms 39 singlestranded DNA (ssDNA) overhangs that are coated with the ssDNA-binding protein RPA. The exchange of RPA for the recombinase enzyme RAD51 facilitates the homology search and strand invasion of homologous DNA templates to form displacement loop structures. Subsequent stabilization of HR intermediates then requires removal of RAD51 from the doublestranded DNA to allow access to the DNA polymerization machinery. Given the central role of the RAD51 filament in HR, its assembly and disassembly are tightly regulated to ensure the fidelity of repair (Krejci et al. 2012;Jasin and Rothstein 2013;Heyer 2015).Key mediators of RAD51 filament assembly are the RAD51 paralogs. In humans, there are six RAD51 paralogs: RAD51B, RAD51C, RA...

show abstract

“…A high degree of conservation is shared between Caenorhabditis elegans and humans in proteins and pathways involved in double-strand break repair (DSBR) and DNA damage response (DDR), so we take advantage of this genetically tractable multicellular nematode to understand the fundamental mechanisms of DSBR and DDR in the germline (O'Neil and Rose 2006;Craig et al 2012).…”

mentioning

confidence: 99%

New Insights into the Post-Translational Regulation of DNA Damage Response and Double-Strand Break Repair in Caenorhabditis elegans

Kim¹,

Colaiácovo

2015

Genetics

View full text Add to dashboard Cite

Although a growing number of studies have reported the importance of SUMOylation in genome maintenance and DNA double-strand break repair (DSBR), relevant target proteins and how this modification regulates their functions are yet to be clarified. Here, we analyzed SUMOylation of ZTF-8, the homolog of mammalian RHINO, to test the functional significance of this protein modification in the DSBR and DNA damage response (DDR) pathways in the Caenorhabditis elegans germline. We found that ZTF-8 is a direct target for SUMOylation in vivo and that its modification is required for DNA damage checkpoint induced apoptosis and DSBR. Non-SUMOylatable mutants of ZTF-8 mimic the phenotypes observed in ztf-8 null mutants, including reduced fertility, impaired DNA damage repair, and defective DNA damage checkpoint activation. However, while mutants for components acting in the SUMOylation pathway fail to properly localize ZTF-8, its localization is not altered in the ZTF-8 non-SUMOylatable mutants. Taken together, these data show that direct SUMOylation of ZTF-8 is required for its function in DSBR as well as DDR but not its localization. ZTF-8's human ortholog is enriched in the germline, but its meiotic role as well as its post-translational modification has never been explored. Therefore, our discovery may assist in understanding the regulatory mechanism of this protein in DSBR and DDR in the germline. KEYWORDS SUMOylation; DNA damage response; double-strand break repair; ZTF-8; Caenorhabditis elegans; germline M EIOSIS is a specialized cell division process by which a diploid cell produces haploid gametes that are essential for sexual reproduction. During meiosis, the homologous chromosomes contributed by each parent undergo genetic recombination that enhances genetic diversity in the progeny and generates chiasmata, which are essential to proper segregation at meiosis I. This process requires precise control of homologous chromosome pairing and genetic recombination to secure accurate chromosome segregation and transferring of genetic materials to the next generation. Therefore, defects in meiotic progression cause aneuploidy, which leads to reproductive failure and congenital birth defects as evidenced by 7-10% of chromosomally abnormal pregnancies in humans (Hunt and Hassold 2008).Although a higher frequency of deleterious consequences arise from meiotic defects, relatively fewer studies have focused on the mechanisms underlying accurate meiotic rather than mitotic cell divisions, especially in multicellular systems. A high degree of conservation is shared between Caenorhabditis elegans and humans in proteins and pathways involved in double-strand break repair (DSBR) and DNA damage response (DDR), so we take advantage of this genetically tractable multicellular nematode to understand the fundamental mechanisms of DSBR and DDR in the germline (O'Neil and Rose 2006;Craig et al. 2012).We previously showed that ztf-8 null mutants exhibit DNA damage sensitivity to g-irradiation (IR) and hydroxyurea (HU) and that ZTF...

show abstract

Methods for Studying the DNA Damage Response in the Caenorhabdatis elegans Germ Line

Cited by 98 publications

References 66 publications

A novel approach using C. elegans DNA damage-induced apoptosis to characterize the dynamics of uptake transporters for therapeutic drug discoveries

A novel approach using C. elegans DNA damage-induced apoptosis to characterize the dynamics of uptake transporters for therapeutic drug discoveries

Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans

New Insights into the Post-Translational Regulation of DNA Damage Response and Double-Strand Break Repair in Caenorhabditis elegans

Contact Info

Product

Resources

About