Nonhomologous end joining of complementary and noncomplementary DNA termini in mouse testicular extracts

Raghavan, Sathees C.; Raman, Mercy J.

doi:10.1016/j.dnarep.2004.04.007

Cited by 23 publications

(29 citation statements)

References 62 publications

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“…The majority of the joined products observed by us were consistent with earlier reports (23,43,50). In contrast to some of the earlier reports, we could also see the intramolecular circularization (23,45,51,52). Although, by using T7 exonuclease, we could unambiguously prove formation of circular products (Fig.…”

Section: Discussionsupporting

confidence: 91%

“…EJ Assay-EJ reactions were performed as described earlier with modifications (43,45). Reactions were done in a volume of 10 l by mixing 4 nM end-labeled DNA substrate in EJ buffer containing 30 mM HEPES-KOH (pH 7.9), 7.5 mM MgCl 2 , 1 mM DTT, 2 mM ATP, 50 M dNTPs, 2 mM EDTA, 0.1 M cold strand and 0.1 g of BSA at 37°C for 2 or 4 h. 5 g of cell-free extracts were used per reaction, unless mentioned otherwise.…”

Section: Methodsmentioning

confidence: 99%

“…However, irrespective of cell lines, the joining efficiency was higher for sticky ends. This could be easily explained because these ends could be ligated with or without further modification (43,45). However, in the case of noncompatible ends, the joining efficiency was very low, even in the cells where compatible end joining was higher.…”

Section: Efficiency Of Ej Varies Between Cancer Cell Lines But the Mmentioning

confidence: 99%

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Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells

Kumar

Kari

Choudhary

et al. 2010

Journal of Biological Chemistry

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Cancer cells are often associated with secondary chromosomal rearrangements, such as deletions, inversions, and translocations, which could be the consequence of unrepaired/misrepaired DNA double strand breaks (DSBs). Nonhomologous DNA end joining is one of the most common pathways to repair DSBs in higher eukaryotes. By using oligomeric DNA substrates mimicking various endogenous DSBs in a cell-free system, we studied end joining (EJ) in different cancer cell lines. We found that the efficiency of EJ varies among cancer cells; however, there was no remarkable difference in the mechanism and expression of EJ proteins. Interestingly, cancer cells with lower levels of EJ possessed elevated expression of BCL2 and vice versa. Removal of BCL2 by immunoprecipitation or protein fractionation led to elevated EJ. More importantly, we show that overexpression of BCL2 or the addition of purified BCL2 led to the down-regulation of EJ. Further, we found that BCL2 interacts with KU proteins both in vitro and in vivo. Hence, our results suggest that EJ in cancer cells could be negatively regulated by the anti-apoptotic protein, BCL2, and this may contribute toward increased chromosomal abnormalities in cancer.

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Section: Discussionsupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Section: Efficiency Of Ej Varies Between Cancer Cell Lines But the Mmentioning

confidence: 99%

See 1 more Smart Citation

Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells

Kumar

Kari

Choudhary

et al. 2010

Journal of Biological Chemistry

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“…Nevertheless, the study showed that this activity is present in all the three major germ cell stages, suggesting a significant contribution of HRmediated DSB repair during spermatogenesis in preserving genomic integrity. Studies in our laboratory have also established the existence of efficient NHEJ repair in testicular extracts and the end joining process, 23,24 as well as extracts of enriched germ cells at these three stages have been observed to be proficient in NHEJ (K. Sharma and M. J. Raman, unpublished data).…”

Section: Multiple Dsb Repair Pathways In Germ Cellsmentioning

confidence: 86%

“…22 However, efficient NHEJ activity has been demonstrated in mouse male germ cell extracts. 23,24 The NHEJ process being potentially error prone, the conservative HRR should be a predominant DSB repair pathway in germ cells, especially in spermatocytes, the major cell type that undergoes meiotic recombination. However, no study has so far been carried out in mammalian male germ cells to evaluate the role of the homologous recombination pathway in the DSB repair process.…”

Section: Introductionmentioning

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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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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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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Nonhomologous end joining of complementary and noncomplementary DNA termini in mouse testicular extracts

Cited by 23 publications

References 62 publications

Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells

Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells

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

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