ARTEMIS Nuclease Facilitates Apoptotic Chromatin Cleavage

Abstract

One hallmark of apoptosis is DNA degradation that first appears as high molecular weight fragments followed by extensive internucleosomal fragmentation. During apoptosis, the DNA-dependent protein kinase (DNA-PK) is activated. DNA-PK is involved in the repair of DNA double-strand breaks (DSB) and its catalytic subunit is associated with the nuclease ARTEMIS. Here, we report that, on initiation of apoptosis in human cells by agents causing DNA DSB or by staurosporine or other agents, ARTEMIS binds to apoptotic chromatin together with DNA-PK and other DSB repair proteins. ARTEMIS recruitment to chromatin showed a time and dose dependency. It required DNA-PK protein kinase activity and was blocked by antagonizing the onset of apoptosis with a pancaspase inhibitor or on overexpression of the antiapoptotic BCL2 protein. In the absence of ARTEMIS, no defect in caspase-3, poly(ADP-ribose) polymerase-1, and XRCC4 cleavage or in H2AX phosphorylation was observed and DNA-PK catalytic subunit was still phosphorylated on S2056 in response to staurosporine. However, DNA fragmentation including high molecular weight fragmentation was delayed in ARTEMIS-deficient cells compared with cells expressing ARTEMIS. In addition, ARTEMIS enhanced the kinetics of MLL gene cleavage at a breakage cluster breakpoint that is frequently translocated in acute or therapy-related leukemias. These results show a facilitating role for ARTEMIS at least in early, site-specific chromosome breakage during apoptosis.

Alternative end-joining pathway(s): Bricolage at DNA breaks

“…C-NHEJ may account for translocation events in some specific situations such as during the recovery from early apoptotic DNA fragmentation [230][231][232], or in the pathogenesis of acute leukemias secondary to cancer treatments based on Topoisomerase-II (TopoII) inhibitors that frequently feature translocations involving the mixed-lineage leukemia (MLL) gene [233,234]. The role of C-NHEJ in chromosomal rearrangements has been more clearly established in the special case of one-ended DSB repair following replication fork collapse [235][236][237][238], but a recent study also suggests a significant role for A-EJ in the repair of one-ended DSBs following hydroxyurea replication fork arrest [83].…”

Cadmium affects viability of bone marrow mesenchymal stem cells through membrane impairment, intracellular calcium elevation and DNA breakage

“…[24] The Comet assay measures DNA damage as the extent of single and double-strand breaks [16] which might be due to the activation of nuclease. [25] The mean percentage of the comet showed a dose-time-dependent increase after treatment with cadmium chloride when compared to control, thus it might be concluded that even lower dose of cadmium chloride after long-term toxicity can induce morphological changes in nuclei and cytoplasm as well as nuclear breakage of the MSCs which can be considered as a risk factor that affects the bone health in industrial or other contaminated areas.…”

“…The cells were plated in appropriate culture dish and allowed to attach for 24 h, then in the presence of the control group, the cells were exposed to 5,15,25,35, and 45 µM of cadmium chloride (Merck Company, Germany). Number of the plated cells and time of exposure were calculated according to the nature of the test and dimension of the culture dish which is mention where ever it is necessary.…”

“…MATERIALS AND METHODS: The rat bone marrow mesenchymal stem cells were grown to confluency in DMEM medium supplemented with 15% fetal bovine serum and penicillin-streptomycin up to third passage. Then the cells were treated with 0,5,15,25, 35, and 45 of CdCl 2 at 12,24, 36, and 48 h, and their viability was investigated using trypan blue staining. In addition, after treatment with selected dose (15 and 45 µM) and time (24 and 48 h) the cell morphology, DNA damage and calcium content of the cells were evaluated.…”

New Insight on Entangled DNA Repair Pathways: Stable Silenced Human Cells for Unraveling the DDR Jigsaw

“…Among the targeted genes and in using our approach we have always obtained at least one vector able to impose long-term shut down greater than 80% as compared with control cells (as evidenced by realtime RT-PCR). Using this technology, more than 160 human genes in different human cell models such as HeLa (Ame et al, 2009, Amine et al, 2009, Aressy et al, 2008, Betous et al, 2009, Biard, 2007, Biard et al, 2005, Biard & Angulo, 2007, Boehler et al, 2011, Bouley et al, 2010, Britton et al, 2009a, Despras et al, 2007, Godon et al, 2008, Le May et al, 2010b, Ousset et al, 2010, Pennarun et al, 2008, Pennarun et al, 2010, Wu et al, 2007, U2OS (Betous et al, 2009) and MRC5-V1 (Bouquet et al, 2011, Britton et al, 2009b, Schmutz et al, 2010 cells have been silenced. Our approach has also been successfully tested in other human tumorderived cell lines, such as RKO (Biard & Angulo, 2007), HCT-116 (Aressy et al, 2008), Caco2 (Coant et al, 2010), SH-SY5Y cells (Schulte et al, 2008), MCF7, MDA-MB 231, K562, UT7 (papers in preparation), and even in mouse NIH-3T3 cells (Meulle et al, 2008).…”