Ends Of DNA Breaks Research Articles

Fen-1 Facilitates Homologous Recombination by Removing Divergent Sequences at DNA Break Ends

Homologous recombination (HR) requires nuclease activities at multiple steps, but the contribution of individual nucleases to the processing of double-strand DNA ends at different stages of HR has not been clearly defined. We used chicken DT40 cells to investigate the role of flap endonuclease 1 (Fen-1) in HR. FEN-1-deficient cells exhibited a significant decrease in the efficiency of immunoglobulin gene conversion while being proficient in recombination between sister chromatids, suggesting that Fen-1 may play a role in HR between sequences of considerable divergence. To clarify whether sequence divergence at DNA ends is truly the reason for the observed HR defect in FEN-1(-/-) cells we inserted a unique I-SceI restriction site in the genome and tested various donor and recipient HR substrates. We found that the efficiency of HR-mediated DNA repair was indeed greatly diminished when divergent sequences were present at the DNA break site. We conclude that Fen-1 eliminates heterologous sequences at DNA damage site and facilitates DNA repair by HR.

Structure of human sperm DNA and background damage, analysed by in situ enzymatic treatment and digital image analysis.

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a procedure to detect and quantify DNA breaks in situ, on a cell-by-cell basis. A comparison between sperm nuclei versus peripheral blood leukocytes using this method demonstrated that the nucleoids from mature human sperm are 12.7 times more sensitive to alkaline denaturation than those from human peripheral blood leukocytes. To investigate the origin of this alkali sensitivity, different approaches were employed. First, free 3'-OH ends of background DNA breaks were labelled by Klenow polymerase, or by DNA polymerase I following the in situ nick translation assay. Second, the presence of abasic sites, the other recognized DNA lesions that lends to constitutive alkali sensitivity, and DNA breaks with blocked 3' ends, were determined by in situ exonuclease III digestion prior to the polymerase labelling. The results demonstrated that the sperm nucleoid contains approximately 2.5-fold higher density of background DNA breaks with 3'-OH ends, and also approximately 2.8-fold higher density of basal abasic sites and DNA breaks with blocked 3' termini, than leukocytes. These differences only partially explain the significant alkali sensitivity of sperm DNA. However, in situ digestion with mung bean nuclease before DNA break labelling showed that sperm DNA is 9-fold more enriched in segments of ssDNA than DNA from leukocytes. The high frequency of partially denatured regions may result from a greater torsional stress of DNA loops in sperm chromatin due to its higher degree of compaction. Moreover, these short unpaired ssDNA stretches should be included in the category of alkali-labile sites detected by all techniques that measure DNA breaks through an alkaline unwinding step. These results provide new insights into the nature of DNA packaging in sperm nuclei.

Evidence of abundant constitutive alkali-labile sites in human 5 bp classical satellite DNA loci by DBD-FISH

Human blood leukocytes within an agarose matrix were deproteinized and exposed to an alkaline denaturation that generates single-stranded DNA (ssDNA) starting from the ends of spontaneous basal DNA breaks and alkali-labile sites. Since the amount of ssDNA produced within a specific sequence area may be detected by hybridization with a specific probe, we quantified this in situ in different satellite DNA loci (DBD-FISH: DNA Breakage Detection FISH). The DBD-FISH signal, corrected for the respective FISH signals in metaphase, was remarkably strong in the 5 bp classical satellite DNA domains analyzed (D1Z1, D9Z3, DYZ1), intermediate in the classical satellite 1 DNA sequences, and low in the alphoid satellite regions (D1Z5, DXZ1, all centromeres). This result is evidence of a high density of constitutive alkali-labile sites, probably abasic sites, within the 5 bp satellite DNA sequences in human blood leukocytes. The presence and relative abundance of alkali-labile sites could explain the high frequency of spontaneous breakage and rearrangements in pericentromeric heterochromatin of chromosomes 1, 9, and 16, but not in Yqh, when this chromatin is undercondensed through spontaneous or induced demethylation, i.e. ICF syndrome or 5-azacytidine treatment.

Apoptosis in developing anthers and the role of ABA in this process during androgenesis in Hordeum vulgare L.

Intra-nucleosomal cleavage of DNA into fragments of about 200 bp was demonstrated to occur in developing anthers, in which microspores had developed into the mid-late to late uni-nucleate stage in situ, i.e. at the verge of mitosis. The same was observed, but to a much larger extent, if these anthers were pre-treated by a hyper-osmotic shock. Pretreatment of anthers before the actual culture of microspores was required for optimal androgenesis of microspores. The use of the TUNEL reaction, which specifically labels 3' ends of DNA breaks, after intra-nucleosomal cleavage of DNA, revealed that DNA fragmentation mainly occurred in the loculus wall cells, tapetum cells and filament cells. TUNEL staining was absent or infrequently observed in the microspores of developing anthers in situ. Electron microscopy studies showed condensed chromatin in nuclei of loculus wall cells in the developing anthers. These observations at the chromatin and DNA level are known characteristics of programmed cell death, also known as apoptosis. Features of apoptosis were infrequently found in microspores from freshly isolated mature anthers. However, most tapetum cells had disappeared in these anthers and the remaining cell structures showed loss of cellular content. The viability of microspores in pre-treated anthers was comparable to those in freshly isolated anthers and almost four times higher than in anthers from control experiments. This observation was correlated with three to four times less microspores showing TUNEL staining and a two times higher level of ABA in the anther plus medium samples than in controls. Addition of ABA to the controls enhanced the viability and lowered the occurrence of apoptosis linked characteristics in the microspores. These data suggest that pre-treatment is effective in stimulating androgenesis because it leads to an increase in ABA levels which protects microspores from dying by apoptosis.

The oncogene heregulin induces apoptosis in breast epithelial cells and tumors

The products of a growing number of genes have been shown to display seemingly contradictory functions; namely, the induction of tumorigenesis and the induction of apoptosis. Heregulin's involvement in oncogenesis occurs through its interactions with members of the EGF receptor tyrosine kinase family. Recently one isoform of heregulin, beta2b, was isolated in an in vitro screen for dominant, apoptosis-inducing genes in kidney epithelial cells. Here we show that heregulin is also capable of mediating apoptosis in human and murine mammary tumor cell lines and murine tumors. Furthermore, through transfection of the human breast cancer cell line MCF-7 with the truncated transmembrane/cytoplasmic segment of the heregulin gene, we show that the intracellular region of the heregulin precursor is sufficient for induction of apoptosis. Through the use of DNA fragmentation assays we also show that apoptosis occurs in cell lines established from heregulin-induced mammary gland tumors. TdT addition of digoxigenin labeled nucleotides to 3' OH ends of DNA breaks recapitulated these results in the actual tumors. Finally, over-expression of heregulin is shown to lead to the down-regulation of Bcl-2, an inhibitor of apoptosis. Conversely, the transfection of Bcl-2 into MCF-7 cells inhibits heregulin-mediated programmed cell death.

DNA fragmentation in normal development of the human central nervous system: a morphological study during corticogenesis

Refinement of the cell number by programmed cell death is a major morphogenetic mechanism of the developing central nervous system (CNS) in vertebrates including mammals, which determines to a significant degree its mature cytoarchitecture. We have examined the topography and the extent of cell death in different regions of the human CNS prenatally (11 fetuses), and in the early post-natal weeks (three newborns). Attention was focused on the wall of the telencephalon during a relatively short time period (12th-23rd week of gestation), corresponding to the time of major proliferation in the ventricular zone and to the peak of neuronal migration; both these mechanisms are crucial for corticogenesis. The TUNEL method was used, allowing the recognition of cell death because of its ability to label blunt ends of double-stranded DNA breaks. Morphological features of nuclei at different stages of apoptosis were identified, providing better evidence of the extent of the process than histological stains. Cell labelling was seen in either post-mitotic elements in the ventricular zone, or along the migratory pathways in the intermediate zone and subplate at all prenatal ages examined. No apoptotic nuclei were seen in the cortical plate. These findings suggest that apoptotic cell death drives the selection of cells which are committed to play a role during the early stages of corticogenesis. Lack of evidence of clonally related apoptotic cells also indicates that cell death occurs randomly. Therefore, molecular signals from the surrounding microenvironment seem to be necessary for the apoptotic pathway to be turned on, thus determining the fate of post-mitotic cells.

Interphase and M-phase oral KB carcinoma cells are targetted in staurosporine-induced apoptosis.

The effect of staurosporine, an antimicrobial agent and inhibitor of protein kinase C (PKC) on programmed cell death/apoptosis (PCD) was investigated in the human oral cavity epidermoid carcinoma KB cell line. Staurosporine-treated oral KB carcinoma cells exhibited morphological features characteristic of apoptosis such as (a) cell shrinkage and increased nuclear fluorescence (quantitated by image analysis with laser scanning confocal microscopy), (b) nuclear condensation and fragmentation observed under fluorescence microscopy with propidium-iodide-DNA staining and (c) chromatin condensation seen under transmission electron microscopy. Specific terminal deoxynucleotidyl transferase (TdT)-mediated labeling of 3'OH ends of DNA breaks in staurosporine-treated cells confirmed DNA fragmentation. In addition, we show the concomitant existence of M-phase PCD with interphase PCD in staurosporine-treated KB cells. It would appear that staurosporine induces apoptosis regardless of the cell cycle phase and that mitosis and apoptosis are not necessarily mutually exclusive events.

The effectiveness of restriction endonucleases in cell killing and mutation

The use of restriction endonucleases (RE) to study the importance of DNA break end structures in differential cellular response has proved controversial. The number of DNA cut sites and the accessibility of RE are recognized examples of confounding factors. We have eliminated these factors by comparing the effectiveness of isoschizomers. Additionally, we considered for the first time the tolerance of the enzymes to cellular conditions. Cell killing and mutation were compared to the overall cutting ability of the enzymes in an "intracellular" buffer. We found that the activity of each RE combined with its lifetime, under simulated cellular conditions, were the overriding factors in determining effectiveness.