Sheetal Sharma

Efficiency of nonhomologous DNA end joining varies among somatic tissues, despite similarity in mechanism

Failure to repair DNA double-strand breaks (DSBs) can lead to cell death or cancer. Although nonhomologous end joining (NHEJ) has been studied extensively in mammals, little is known about it in primary tissues. Using oligomeric DNA mimicking endogenous DSBs, NHEJ in cell-free extracts of rat tissues were studied. Results show that efficiency of NHEJ is highest in lungs compared to other somatic tissues. DSBs with compatible and blunt ends joined without modifications, while noncompatible ends joined with minimal alterations in lungs and testes. Thymus exhibited elevated joining, followed by brain and spleen, which could be correlated with NHEJ gene expression. However, NHEJ efficiency was poor in terminally differentiated organs like heart, kidney and liver. Strikingly, NHEJ junctions from these tissues also showed extensive deletions and insertions. Hence, for the first time, we show that despite mode of joining being generally comparable, efficiency of NHEJ varies among primary tissues of mammals.

A novel DNA intercalator, 8-methoxy pyrimido[4′,5′:4,5]thieno (2,3-b)quinoline-4(3H)-one induces apoptosis in cancer cells, inhibits the tumor progression and enhances lifespan in mice with tumor†‡

Polycyclic aromatic molecules such as ellipticine intercalate into double‐stranded DNA and interfere with physiological functions. In the present study, we evaluate the chemotherapeutic potential of MPTQ on animal models and its mode of action. In order to test the antitumor activity, monohydrochloride of MPTQ was orally administered in mice bearing tumor. Results showed a significant inhibition of tumor growth compared to that of untreated controls. More importantly, mean lifespan of tumor bearing animals treated with MPTQ was significantly higher as compared to that of untreated tumor bearing mice suggesting that the treatment affected viability of cancerous cells, but not of normal cells. Consistent with this, we find that administration of MPTQ to normal mice did not cause any major side effects as observed upon hematological and serum profiling. We also found that MPTQ induces cytotoxicity in cancer cell lines, by activating apoptosis both by intrinsic and extrinsic pathways. Thus, MPTQ could be used as a potential cancer therapeutic agent.

Time-Dependent Predominance of Nonhomologous DNA End-Joining Pathways during Embryonic Development in Mice

Repair of DNA double-strand breaks (DSBs) is crucial for maintaining genomic integrity during the successful development of a fertilized egg into a whole organism. To date, the mechanism of DSB repair in postimplantation embryos has been largely unknown. In the present study, using a cell-free repair system derived from the different embryonic stages of mice, we find that canonical nonhomologous end joining (NHEJ), one of the major DSB repair pathways in mammals, is predominant at 14.5 day of embryonic development. Interestingly, all four 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 evidences for canonical NHEJ. Strikingly, in contrast to the above, we observed noncanonical end joining accompanied by DSB resection, dependent on microhomology and ligase III in 18.5-day embryos. Interestingly, we observed an elevated expression of CtIP, MRE11, and NBS1 at this stage, suggesting that it could act as a switch between classical end joining and microhomology-mediated end joining at later stages of embryonic development. Thus, our results establish for the first time the existence of both canonical and alternative NHEJ pathways during the postimplantation stages of mammalian embryonic development.

Nonhomologous DNA End Joining in Cell-Free Extracts

Among various DNA damages, double-strand breaks (DSBs) are considered as most deleterious, as they may lead to chromosomal rearrangements and cancer when unrepaired. Nonhomologous DNA end joining (NHEJ) is one of the major DSB repair pathways in higher organisms. A large number of studies on NHEJ are based on in vitro systems using cell-free extracts. In this paper, we summarize the studies on NHEJ performed by various groups in different cell-free repair systems.

Novel BCL2 inhibitor, Disarib Induces Apoptosis by Disruption of BCL2-BAK Interaction.

ABSTRACT Apoptosis is a highly regulated pathway of programmed cell death relying on the fine balance between pro and antiapoptotic binding partners. Overexpression of the antiapoptotic protein BCL2 in several cancers makes it an ideal target for chemotherapy, with minimum side effects. In one of our previous studies, we designed, synthesized and characterized Disarib, a BCL2‐specific small molecule inhibitor. Interestingly, Disarib showed a novel mode of BCL2 inhibition, by predominantly binding to its BH1 domain, as compared to the BH3‐specific action of other known BCL2 inhibitors. Here, we investigate the mechanism by which Disarib induces cell death, upon binding to BCL2. We find that Disarib specifically disrupted the BCL2‐BAK interaction, but not that of BCL2‐BAX or other members of the proapoptotic family such as PUMA and BIM, in vitro. Biochemical and biophysical studies demonstrate Disarib‐induced inhibition of BCL2‐BAK interaction with a Ki of 12.76 nM. Genetic knockout cells of BAK/BAX and double knockout (DKO) cells confirmed a BAK‐specific action of Disarib, thereby facilitating apoptosis. Importantly, intracellular FRET in BAK/BAX single and double knockout cells demonstrated BCL2‐BAK disruption, and activation of intrinsic pathway of apoptosis upon Disarib treatment. Thus, we report a unique mechanism of action of a BCL2 inhibitor, Disarib, by specifically targeting the interaction of BCL2‐BAK, while sparing that of other proapoptotic binding partners.

Nonhomologous DNA End Joining

Maintaining genomic integrity is imperative for the survival of an organism. Among different DNA damages, double-strand breaks (DSBs) are considered as most deleterious since they can lead to cell death if left unrepaired or chromosomal rearrangements when mis-repaired, leading to cancer. Nonhomologous DNA end joining (NHEJ) and homologous recombination (HR) are the major DSB repair pathways in higher eukaryotes. HR being a precise mechanism uses extensive homology, while NHEJ is error prone as it utilizes no or limited homology. NHEJ is a quick fix mechanism and operates throughout the cell cycle. During NHEJ, KU protein heterodimer is recruited to DNA ends followed by DNA-PKcs in association with ARTEMIS, which processes DSBs. Pol μ and/or λ fills these ends, when required, followed by ligation using XLF–XRCC4–DNA Ligase IV complex. Another repair pathway known as alternative NHEJ (A-NHEJ) or backup NHEJ repairs DSBs when the key proteins responsible for classical NHEJ are absent or defective. The choice of DSB repair pathway between NHEJ and HR depends on cell cycle, end resection, and DSB end structure, which further activate various DNA damage sensors. Recently it has been shown that deregulation of DSB repair poses a threat to genomic integrity and hence can be used as a therapeutic target in the treatment of cancer.