Academic literature on the topic 'Microhomology-mediated end joining (MMEJ)'

DNA repair pathways are important for the maintenance of genomic integrity and critical to the survival and health of an organism. In order to effectively eliminate damaged segments of DNA, DNA repair mechanisms require the separation of the two complementary strands?or a helicase. While the enzyme BLM helicase is implicated in several DNA damage repair mechanisms, its role in non-homologous repair of DNA double strand breaks through microhomology-mediated end joining (MMEJ) is of concern in this study as previous studies have suggested that BLM helicase is necessary for MMEJ. Under this, it was hypothesized that cells lacking BLM helicase would not undergo MMEJ as readily as those with the protein, thereby containing less DNA degradation fragments. This was to be determined by performing DNA degradation assays on cell lysates lacking and not lacking the BLM helicase protein and comparing results from the two sample pools, noting up regulation or down regulation of MMEJ in BLM knockdown samples. Thus far, optimization of the BLM helicase RNAi transfection process was accomplished to successfully produce knockdown of BLM helicase expression in the two experimental cell lines. Future studies regarding this experiment involve performing the degradation assays to test the aforementioned hypothesis.

Microhomology-mediated end joining (MMEJ) is a class of DNA repair used to join DNA ends following a double-strand break (DSB). It represents an unfavorable and error-prone pathway because it results in deletion of nucleotides between microhomologies, as well as the deletion of one microhomology. This study utilized a DNA repair assay to measure degradation prior to end rejoining in nuclear extracts. Our results were consistent with previous studies which found that ATM plays a regulatory role in repressing nuclease degradation of DSB ends and that BLM is a required component in the MMEJ pathway. MMEJ repair products were also found to utilize GC rich microhomologies and preferred sites for rejoining. Variation among extracts from the same cell line suggest that additional trials of the repair assay must be conducted to ensure that the differences in CF, AT, and BLM nuclear extracts are the result of protein composition differences rather than variation in extract preparation.

ATM is the defective kinase in the neurodegenerative disorder ataxia telangiectasia. This kinase is associated with DNA double-strand break (DSB) repair and cell cycle control. Our laboratory previously demonstrated elevated levels of deletions and error-prone double-strand break repair via microhomology-mediated end joining (MMEJ) in ATM-deficient (A-T) extracts when compared to controls (wtATM+). To assess the involvement of enhanced nuclease activities in A-T extracts we studied the stability of DNA duplex substrates in A-T and control nuclear extracts under DSB repair conditions. We observed a marked shift in detection from full-length products to shorter products in A-T extracts. Addition of purified ATM to A-T nuclear extracts restored full-length product detection. This repression of degradation by ATM was dependent on its kinase activities. These results demonstrated a role for ATM in suppressing the degradation of DNA ends possibly through inhibiting nucleases implicated in MMEJ such as Mre11. Therefore, we assessed DNA end-stability in Mre11-depleted nuclear extracts and in extracts treated with the Mre11 nuclease inhibitor, Mirin. This resulted in decreased DNA degradation in both control and A-T extracts. Knockdown of Mre11 levels also led to an enhancement of DNA end-stability in nuclear extracts. Examining MMEJ levels by employing an in vivo reporter assay system revealed a decline in this pathway in Mre11-knockdown cells and in those treated with Mirin. These results signify a role for the Mre11 nuclease in MMEJ in mammalian cells and indicate a regulatory function for ATM in the control of error-prone DSB repair and preservation of DNA end-stability at a break.

The genome is constantly subjected to spontaneous damage from exogenous and endogenous damaging agents. Of all DNA lesions, DNA double strand breaks (DSB) are the most genotoxic as they may lead to loss of large parts of the chromosome, potentiate chromosomal rearrangements, and ultimately result in the development of cancer. The eukaryotic Mrell/RadSO/Nbsl (MRN) complex is a key player in the reversal of DSBs and has been linked to roles in DSB detection, checkpoint signalling, homologous recombination and non-homologous end joining (NHEJ). Hypomorphic mutations within MRN components confer genome instability and susceptibility to cancer in humans. Using a Xenopus egg extract system I have demonstrated a role for the MRN complex in an alternative DNA end joining pathway, distinct from classical NHEJ, termed microhomology-mediated end joining.

Maintenance of genomic integrity with high fidelity is of prime importance to any organism. An insult which may result in compromised genome integrity is prevented or its consequences are monitored by advanced cellular networks, collectively called the DNA damage response (DDR). Various DNA repair pathways, which are part of DDR, constantly correct the genome in the event of any undesirable change in the genetic material and prevent the transmission of any impairment to daughter cells. Non homologous DNA end joining (NHEJ) is the predominant DNA repair pathway associated with DDR in higher eukaryotes, correcting double-strand breaks (DSBs). Microhomology mediated end joining (MMEJ), an alternate mechanism to NHEJ also exists in cells, which is associated with erroneous joining of broken DNA, leading to mutagenesis. DDR is of paramount importance in cellular viability and therefore, any defects in DDR or the imbalance of repair pathways contribute to mutations, cellular transformations and various neurodegenerative and congenital abnormalities. Here, we investigate the DDR via NHEJ and MMEJ pathways during embryonic development in mice as well as in presence of an environmental pollutant, Endosulfan, in order to understand how physiological and exogenous factors condition the balance of repair pathways. Among various classes of pesticides known to cause side effects, organochlorine pesticides (OCPs) lead the list, possessing high transport potential, and a variety of toxic and untoward health effects. Endosulfan is a widely used organochlorine pesticide and is speculated to be detrimental to human health. However, very little is known about mechanism of its genotoxicity. Using in vivo and ex vivo model systems, we showed that exposure to Endosulfan induced reactive oxygen species (ROS) in a concentration dependent manner. Using an array of assays and equivalents of sub-lethal concentrations comparable to the detected level of Endosulfan in humans living in active areas of exposure, we demonstrated that ROS production by Endosulfan resulted in DNA double-strand breaks in mice, rats and human cells. In mice, the DNA damage was predominantly detected in type II pneumocytes of lung tissue; spermatogonial mother cells and primary spermatids of testes. Importantly, Endosulfan-induced DNA damage evoked DDR, which further resulted in elevated levels of classical NHEJ. However, sequence analyses of NHEJ junctions revealed that Endosulfan treatment resulted in extensive processing of broken DNA, culminating in increased and long junctional deletions, thereby favouring erroneous repair. We also find that exposure to Endosulfan led to significantly increased levels of MMEJ, which is a LIGASE III dependent, alternative, non classical repair pathway, encompassing long deletions and processing of DNA. Further, we show that the differential expression of proteins following exposure to Endosulfan correlated with activation of alternative DNA repair. At the physiological level, using mouse model system, we showed that exposure to Endosulfan affected physiology and cellular architecture of organs and tissues. Among all organs, damage to testes was extensive and it resulted in death of different testicular cell populations. We also found that the damage in testes resulted in qualitative and quantitative defects during spermatogenesis in a time dependent manner, increasing epididymal ROS levels and affecting sperm chromatin integrity. This further culminated in reduced number of epididymal sperms and actively motile sperms, which finally resulted in reduced fertility in male but not in female mice. Repair of DSBs is important for maintaining genomic integrity during the successful development of a fertilized egg into a whole organism. To date, the mechanism of DSB repair in post implantation embryos has been largely unknown except for the differential requirement of DNA repair genes in the course of development. These studies relied on null mutation analysis of animal phenotypes and therefore a quantitative understanding of repair pathways was absent. In the present study, using a cell free repair system derived from different embryonic stages of mice, we found that canonical NHEJ is predominant at 14.5 day of embryonic development. Interestingly, all types of DSBs tested were repaired by LIGASE IV/XRCC4 and Ku-dependent classical NHEJ. Characterization of end-joined junctions and expression studies further showed evidence for C-NHEJ. Strikingly, we observed non canonical end joining accompanied by DSB resection, dependent on microhomology and LIGASE III in 18.5-day embryos. Further we observed an elevated expression of CtIP, MRE11, and NBS1 at this stage, suggesting that it could act as a switch between classical and microhomology-mediated end joining at later stages of embryonic development. Keeping these observations in mind, we wondered if Endosulfan affected the differential regulation of DDR during development, similar to mice tissues. Upon analysing the effect of endosulfan on NHEJ/MMEJ at above mentioned stages of mouse embryonic development, we found that C-NHEJ efficiency remained low or unaltered while the efficiency of MMEJ was upregulated significantly, perturbing the repair balance during embryo development and hence facilitating mutagenic repair. Thus, our results establish the existence of both classical and non classical NHEJ pathways during the post implantation stages of mammalian embryonic development. Our studies also provide deeper insights into physiological and molecular events leading to male infertility upon Endosulfan exposure and its impact on impairing the differential regulation of DNA repair during embryonic development. Our findings suggest the plasticity of DNA repair pathways in physiological and pathological conditions and provide insights into mechanism of genome instability due to DNA repair imbalance, when exposed to environmental mutagens.