DNA double-strand break repair inPenaeus monodonis predominantly dependent on homologous recombination

Abstract

DNA double-strand breaks (DSBs) are mostly repaired by nonhomologous end joining (NHEJ) and homologous recombination (HR) in higher eukaryotes. In contrast, HR-mediated DSB repair is the major double-strand break repair pathway in lower order organisms such as bacteria and yeast. Penaeus monodon, commonly known as black tiger shrimp, is one of the economically important crustaceans facing large-scale mortality due to exposure to infectious diseases. The animals can also get exposed to chemical mutagens under the culture conditions as well as in wild. Although DSB repair mechanisms have been described in mammals and some invertebrates, its mechanism is unknown in the shrimp species. In the present study, we show that HR-mediated DSB repair is the predominant mode of repair in P. monodon. Robust repair was observed at a temperature of 30 °C, when 2 µg of cell-free extract derived from hepatopancreas was used for the study. Although HR occurred through both reciprocal recombination and gene conversion, the latter was predominant when the bacterial colonies containing recombinants were evaluated. Unlike mammals, NHEJ-mediated DSB repair was undetectable in P. monodon. However, we could detect evidence for an alternative mode of NHEJ that uses microhomology, termed as microhomology-mediated end joining (MMEJ). Interestingly, unlike HR, MMEJ was predominant at lower temperatures. Therefore, the results suggest that, while HR is major DSB repair pathway in shrimp, MMEJ also plays a role in ensuring the continuity and stability of the genome.

Characterization of DNA double‐strand break repair pathways in diffuse large B cell lymphoma

“…The construction of plasmids pTO223 and pTO231 was described previously . The supercoiled plasmids were purified and used for the study as described earlier . The plasmid DNA used for HR assay was either circular DNA (circular‐circular; pTO223 + pTO231) or circular‐linear (pTO223 + pTO231/SalI).…”

“…Cell free extracts from DLBCL cell line (SUDHL8) and pre‐B cell leukemia cell lines NALM6 and REH were prepared in ice cold conditions as described earlier . Briefly, approximately 2 × 10 8 cells were washed with cold PBS and lysed using hypotonic buffer (10 mM Tris‐HCl [pH 8.0], 1 mM EDTA, and 5 mM DTT) followed by homogenization.…”

“…The NHEJ assay was performed on different DNA substrates as described previously . Cell‐free extract prepared from cancer cell lines (2 μg) was incubated with radiolabelled DNA substrates (40 nM) or increasing concentrations of DNA (20, 40, 80, and 120 nM) for 1 h at 37°C in 1× NHEJ buffer (30 mM HEPES‐KOH (pH 8), 7.5 mM MgCl 2 , 1 mM DTT, 2 mM ATP, 50 μM dNTPs, and 10 μg/mL BSA).…”

“…In vitro HR assay using two independent plasmids was performed as described previously . Two independent plasmids were generated from pTO221 by introducing two independent mutations to the neomycin resistant gene .…”

“…Restriction analyses of plasmids recovered from recombinant colonies were done to distinguish two molecular mechanisms that result in functional neomycin gene, viz, gene conversion, and reciprocal exchange . Recombinant plasmid DNA was subjected to EcoRI/SalI (for reciprocal exchange) and SalI/HindIII (for gene conversion) restriction digestion.…”