A plasmid system to monitor gene conversion and reciprocal recombination in vitro

Oppliger, Tanja; Würgler, Friedrich E.; Sengstag, Christian

doi:10.1016/0165-1161(93)90158-v

Cited by 11 publications

(11 citation statements)

References 19 publications

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“…[39][40][41] Hence, comparing these data, it can be concluded that the testicular extracts possess an efficient HR-mediated DSB repair activity that is 200-fold higher than that of mouse embryonic fibroblast extracts. The HR activity observed in testicular extracts when compared with that reported in a study on nuclear extract of Drosophila embryo, 27 in which the same plasmid construct as in the present study has been used, was seen to be comparable. However, creation of a single DSB in one of the substrates enhanced the frequency $11-fold in the case of Drosophila embryo extract, whereas only 3.2-fold enhancement was obtained with testicular extract.…”

Section: Hr Activity In Mouse Testicular Extractssupporting

confidence: 80%

“…27 As reported by them, the original construct was made by inserting a 1.5 Kb HindIII/SalI restriction fragment from pBR322::Tn5 containing the neo gene into the same restriction sites of the plasmid, pBLKS. The resultant plasmid (pTO221) was mutated differently creating two alleles; one by deleting a 245 bp NarI segment at the 5 0 of neo (pTO223, neoD2), and the other by deleting a 282 bp NaeI segment from the 3 0 (pTO231, neoD1).…”

Section: Plasmid Recombination Assaymentioning

confidence: 99%

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Homologous recombination‐mediated double‐strand break repair in mouse testicular extracts and comparison with different germ cell stages

Srivastava

Raman

2006

Cell Biochemistry & Function

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Homologous recombination (HR) is established as a significant contributor to double-strand break (DSB) repair in mammalian somatic cells; however, its role in mammalian germ cells has not been characterized, although being conservative in nature it is anticipated to be the major pathway in germ cells. The germ cell system has inherent limitations by which intact cell approaches are not feasible. The present study, therefore, investigates HR-mediated DSB repair in mouse germ cell extracts by using an in vitro plasmid recombination assay based on functional rescue of a neomycin (neo) gene. A significantly high-fold increase in neo+ (Kan(R)) colonies following incubation of two plasmid substrates (neo delta1 and neo delta2) with testicular extracts demonstrated the extracts' ability to catalyze intermolecular recombination. A significant enhancement in recombinants upon linearization of one of the plasmids suggested the existence of an HR-mediated DSB repair activity. Comparison of the activity at sequential developmental stages, spermatogonia, spermatocytes and spermatids revealed its presence at all the stages; spermatocyte being the most proficient stage. Further, restriction analysis of recombinant plasmids indicated the predominance of gene conversion in enriched spermatocytes (mostly pachytenes), in contrast to gonial and spermatid extracts that showed higher reciprocal exchange. In conclusion, this study demonstrates HR repair activity at all stages of male germ cells, suggesting an important role of HR-mediated DSB repair during mammalian spermatogenesis. Further, the observed preference of gene conversion over reciprocal exchange at spermatocyte stage correlates with the close association of gene conversion with the meiotic recombination program.

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Section: Hr Activity In Mouse Testicular Extractssupporting

confidence: 80%

Section: Plasmid Recombination Assaymentioning

confidence: 99%

Homologous recombination‐mediated double‐strand break repair in mouse testicular extracts and comparison with different germ cell stages

Srivastava

Raman

2006

Cell Biochemistry & Function

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“…Plasmids pTO223 and pTO231 were a kind gift from Dr Christian Sengstag, Switzerland. The construction of plasmids pTO223 and pTO231 was described previously . The supercoiled plasmids were purified and used for the study as described earlier .…”

Section: Methodsmentioning

confidence: 99%

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

Gopalakrishnan

Dahal

Radha

et al. 2018

Molecular Carcinogenesis

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Efficient DNA repair is indispensable for maintaining genomic integrity in humans. Cancer associated deletions and mutations are mainly due to misrepaired DNA double‐strand breaks (DSBs). Classical nonhomologous end joining (c‐NHEJ) and homologous recombination (HR) are two major DSB repair pathways in humans. An error prone, alternative NHEJ pathway that utilizes microhomology was also reported in cancer cells and to a lesser extent in normal cells. In the present study, we evaluated the efficiency of various DSB repair pathways in the most common lymphoma, the diffuse large B cell lymphoma (DLBCL). Here we show that DNA repair through c‐NHEJ pathway is limited in SUDHL8, a cell line derived from a DLBCL patient. Unlike c‐NHEJ, microhomology mediated end joining (MMEJ) was predominant at physiological temperature. Consistent with the observation, expression level of repair proteins such as LIGASE I, LIGASE III, PARP1, CtIP, and MRE11 was higher in DLBCL cells when compared to c‐NHEJ proteins. Further, inhibition of LIGASE I or MRE11, led to reduction in the efficiency of MMEJ in DLBCL cells. Besides, HR‐mediated DSB repair occurring through gene conversion was observed. Thus, our results reveal the predominance of MMEJ over c‐NHEJ in repairing DSBs in DLBCL cells, while error‐free repair through HR was also evident.

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“…In vitro HR assay was performed as described previously, using two independent plasmids 38 , 40 , 41 . Two plasmid constructs, pTO223 and pTO231, were generated by introducing mutations to neomycin resistant gene on pTO221 as described 40 .…”

Section: Methodsmentioning

confidence: 99%

“…Two plasmid constructs, pTO223 and pTO231, were generated by introducing mutations to neomycin resistant gene on pTO221 as described 40 . Briefly, pTO223 was created by deleting a 245-bp ( NarI ) segment from 5′ region of neomycin ( neoΔ2 ) gene and pTO231 was created by deleting 282 bp ( NaeI ) segment from 3′ of neomycin ( neoΔ1 ).…”

Section: Methodsmentioning

confidence: 99%

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

Srivastava

Dahal

Naidu

et al. 2017

DNA Res

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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.

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A plasmid system to monitor gene conversion and reciprocal recombination in vitro

Cited by 11 publications

References 19 publications

Homologous recombination‐mediated double‐strand break repair in mouse testicular extracts and comparison with different germ cell stages

Homologous recombination‐mediated double‐strand break repair in mouse testicular extracts and comparison with different germ cell stages

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

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

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