In January 2019, Hyeon-Sook Koo and her research group of the Institute of Life Science and Biotechnology, Yonsei University, published the latest research results in Elsevier's publication DNA Repair. This paper was entitled " Single-strand annealing mediates the conservative repair of double-strand DNA breaks in homologous recombination-defective germ cells of Caenorhabditis elegans" . Dr. Koo ordered the strain PHX786 rad-54(syb786) from SunyBiotech to generate G284R mutation，which serves as supportive materials to the paper.
The research contents of this paper are as follows:
Brief introduction
Double strand DNA breaks (DSBs) occur during meiotic homologous recombination and after the collision of replication forks with endogenous DNA damage. DSBs can also be induced by exposing cells to ionizing radiation (IR) or to chemical agents such as bleomycin and neocarzinostatin. DSBs are repaired through several competing repair pathways in eukaryotic cells, including the two major pathways of homologous recombination (HR) and nonhomologous end-joining (NHEJ) (Fig. 1). However, in the absence of the Ku protein of NHEJ, microhomology-mediated end-joining (MMEJ), also called alternative end-joining (Alt-EJ), joins DSB ends. Single-strand annealing (SSA) involving Rad52 and XPF is another minor pathway for DSB repair in eukaryotes , although it is the major pathway for DSB repair in the germline of fruit flies.
Highlights
l A missense mutation of a Rad54 homolog in C. elegans decreases ATPase activity.
l Rad54 in C. elegans promotes the dissociation of Rad51 from the synapsed DNA intermediates not the prior association of Rad51to double-strand DNA breaks.
l The single-strand annealing pathway actively repairs double-strand DNA breaks in the C. elegans germ cells defective in homologous recombination.
Fig. 1. Competition between DSB repair pathways in C. elegans germ cells.
Research method
In the present study, the author investigated competition between HR, NHEJ, and SSA pathways for DSB repair in the model organism Caenorhabditis elegans. They measured relative contributions of the three DSB repair pathways in the germ cells of C. elegans, and examined how the activities of other pathways are altered when the major pathway, HR, is inactivated. To manipulate DSB repair pathways, we used C. elegans strains deficient in the following genes: rad-54 in HR, lig-4 in NHEJ, and xpf-1 in SSA. They also generated double- and triple-deficient strains of rad-54, lig-4, and xpf-1, and compared their responses to DSBs. By analyzing these responses, they found that in the absence of HR, SSA, and NHEJ exert positive and negative effects, respectively, on the conservation of genomic DNA in the germ cells of C. elegans.
Conclusion
The GST-tagged wild-type RAD-54 containing the catalytic domain was found to have ATPase activity in vitro. In contrast, GST-tagged RAD-54 containing the G284R substitution showed a several-fold reduction in the activity of ATPase (Fig. 2). In the current research, they identified a G284R missense mutation in C. elegans RAD-54, which reduced the number of progeny only slightly under normal conditions (data not shown) but greatly increased the sensitivity of the germ cells to γ-rays.
Fig. 2. The C. elegans rad-54 missense (G284R) mutant is hypersensitive to ionizing radiation,
and a double deficiency of rad-54 and xpf-1 has synergistic effects on the sensitivity.
SunyBiotech is specializing in providing precise genome edited C. elegans. All strains generated by SunyBiotech will bare our nomenclature. PHX and syb is the strain designation and allele designation, respectively. We will continue working hard to provide high-quality services with higher efficiency.
