Degradation of chromosomal DNA during apoptosis

Apoptosis is often accompanied by degradation of chromosomal DNA. CAD, caspase-activated DNase, was identified in 1998 as a DNase that is responsible for this process. In the last several years, mice deficient in the CAD system have been generated. Studies with these mice indicated that apoptotic DNA degradation occurs in two different systems. In one, the DNA fragmentation is carried out by CAD in the dying cells and in the other, by lysosomal DNase II after the dying cells are phagocytosed. Several other endonucleases have also been suggested as candidate effectors for the apoptotic degradation of chromosomal DNA. In this review, we will discuss the mechanism and role of DNA degradation during apoptosis.     

Forces during bacteriophage DNA packaging and ejection

The conjunction of insights from structural biology, solution biochemistry, genetics, and single-molecule biophysics has provided a renewed impetus for the construction of quantitative models of biological processes. One area that has been a beneficiary of these experimental techniques is the study of viruses. In this article we describe how the insights obtained from such experiments can be utilized to construct physical models of processes in the viral life cycle. We focus on dsDNA bacteriophages and show that the bending elasticity of DNA and its electrostatics in solution can be combined to determine the forces experienced during packaging and ejection of the viral genome. Furthermore, we quantitatively analyze the effect of fluid viscosity and capsid expansion on the forces experienced during packaging. Finally, we present a model for DNA ejection from bacteriophages based on the hypothesis that the energy stored in the tightly packed genome within the capsid leads to its forceful ejection. The predictions of our model can be tested through experiments in vitro where DNA ejection is inhibited by the application of external osmotic pressure.     

Stable denaturation of chromosomal DNA from Saccharomyces cerevisiae during meiosis.

Partial denaturation of Saccharomyces cerevisiae chromosomal DNA was found to occur spontaneously during meiosis. Short regions of strand separation (300 base pairs long) were seen in DNA molecules prepared for electron microscopy by the aqueous spreading technique. These regions were clustered along the DNA. The time course of their appearance indicated that the denatured regions were present during the periods of premeiotic DNA replication and recombination. A similar pattern of denaturation was also detected in the DNA from vegetatively grown cells of a conditional cdc8 mutant, which is defective in DNA replication.     

Epstein-Barr virus induces fragmentation of chromosomal DNA during lytic infection.

Pulsed-field agarose gel electrophoresis showed that fragmentation of chromosomal DNA in Raji cells was induced by infection with the P3HR-1 strain of Epstein-Barr virus (EBV). S1 nuclease treatment of the agarose plugs containing cells suggested that the majority of DNA fragments did not contain single-strand gaps. Chromosomal DNA fragmentation was inhibited by cycloheximide, indicating that protein synthesis was required for DNA fragmentation. Phosphonoacetic acid, an inhibitor of EBV DNA polymerase, did not inhibit fragmentation of chromosomal DNA. These findings suggest that EBV-specific early proteins participate in fragmentation of chromosomal DNA. Chromosomal DNA of P3HR-1 cells was also fragmented by treatment with n-butyrate plus 12-O-tetradecanoylphorbol-13-acetate (TPA), which induced activation of latent EBV genome following viral replication. In addition, fragmentation of DNA preceded cell death during lytic infection. These results suggest that fragmentation of chromosomal DNA is generally induced during EBV replication and probably contributes to the cytopathic effect of EBV. The role of DNA fragmentation in death of infected cells is discussed in relation to apoptosis.     

The chromosomal distribution of human satellite III DNA during meiosis.

In human meiotic cells DNA satellite II is located in the same sites as in mitotic cells, but in prophase the areas are less condensed. This differs from the situation in Plethodon where the sites of heavy satellite DNA are condensed throughout meiotic prophase (MacGregor and Kezer, 1971). The difference may be ascribed to the fact that Plethodon heavy satellite is pericentrically located, whereas few if any of the human satellite sites are actually at centromeres. A second difference is found in sperm, where the Plethodon satellite is located at a single site in the rear of the nucleus, while the satellite regions in mad do not have a common or constant orientation, suggesting that the respective satellites may well be functionally different.     

Breakdown of chromosomal DNA during apoptosis and phagocytosis

Apoptosis can be triggered by a variety of stimuli including death factors, anti-cancer drugs and factor-deprivation. These apoptotic cells are swiftly phagocytosed by macrophages to prevent the release of noxious or inflammatory materials from dying cells. The molecular analysis of Fas ligand (a death factor)-induced apoptosis indicated that a cascade of proteases (caspases) is activated during this process, which eventually activates a specific DNase (caspase-activated DNase). CAD exists as a complex with its inhibitor (ICAD) in proliferating cells. When the cells are triggered to apoptosis, caspases, in particular caspase 3, in the downstream of the caspase cascade cleave ICAD, which releases CAD to cause DNA degradation in nuclei.     

Redistribution of histones during unfolding of chromosomal DNA

Just in the course of unfolding of chromosomal deoxyribonucleoproteins performed in the absence of magnesium ions all DNA-bound histones may be redistributed. This type of redistribution is completely blocked in already unfolded DNP preparations. If the unfolding-induced histone redistribution is allowed it leads to a significant decrease of the average length of free DNA segments in the unfolded, histone F1-depleted DNP. The implications of these data on the mode of DNA packing in chromatin are discussed.     

Associated chromosomal DNA changes in polyploids.

The 2C and 4C nuclear DNA amounts were estimated in eight diploid species, belonging to three diverse genera (Vicia, Tephrosia, and Phlox) and their corresponding colchitetraploids. In P. drummondii, T. purpurea, and T. oxygona tetraploids the deviation from the expectation was highly significant. The DNA in P. drummondii was further discarded in subsequent (C1, C2) generations, thus attaining an overall reduction of about 25%. The DNA content in the subsequent generations was the same as that of C2. It is concluded that rapid DNA loss in the first and subsequent generations was not only associated with the substantial increase (30-66%) in the seed set, but it also helped in the establishment and stabilization of the tetraploid. The possible relationship between such a nucleotypic change and success of polyploids is discussed. The DNA change from the expected value in the P. drummondii tetraploid was achieved by equal decrement to each chromosome independent of size, i.e., small chromosomes loose the same amount of DNA as the large chromosomes.     

Transitory increase in chromosomal DNA (Fleulgen) during floral differentiation in Rhoeo discolor.

Scanning cytophotometric measurements on 3200 telophase and 1700 interphase nuclei (Feulgen-stained) in vegetative and reproductive buds of Rhoeo discolor revealed a transitory increase in staining intensity in more than half of the cells in early differentiating floral buds. The differences between the vegetative and floral nuclei are significant at the 0.001 level of probability and highly reproducible, independent of the type of hydrolysis used. We suggest that the different Feulgen extinction values reflect different nuclear DNA amounts, because methodical errors can fairly be excluded. The occurrence of an extra DNA (control DNA) of the kind of the floral DNA' detected by Wardell and Skoog (1973) and Wardell (1976) in tobacco is discussed.     

Delayed chromosomal instability induced by DNA damage.

DNA damage induced by ionizing radiation can result in gene mutation, gene amplification, chromosome rearrangements, cellular transformation, and cell death. Although many of these changes may be induced directly by the radiation, there is accumulating evidence for delayed genomic instability following X-ray exposure. We have investigated this phenomenon by studying delayed chromosomal instability in a hamster-human hybrid cell line by means of fluorescence in situ hybridization. We examined populations of metaphase cells several generations after expanding single-cell colonies that had survived 5 or 10 Gy of X rays. Delayed chromosomal instability, manifested as multiple rearrangements of human chromosome 4 in a background of hamster chromosomes, was observed in 29% of colonies surviving 5 Gy and in 62% of colonies surviving 10 Gy. A correlation of delayed chromosomal instability with delayed reproductive cell death, manifested as reduced plating efficiency in surviving clones, suggests a role for chromosome rearrangements in cytotoxicity. There were small differences in chromosome destabilization and plating efficiencies between cells irradiated with 5 or 10 Gy of X rays after a previous exposure to 10 Gy and cells irradiated only once. Cell clones showing delayed chromosomal instability had normal frequencies of sister chromatid exchange formation, indicating that at this cytogenetic endpoint the chromosomal instability was not apparent. The types of chromosomal rearrangements observed suggest that chromosome fusion, followed by bridge breakage and refusion, contributes to the observed delayed chromosomal instability.     

Rheological properties of chromosomal and plasmid DNA during alkaline lysis reaction

The experimental apparatus for the simultaneous L-lactic acid fermentation by Rhizopus oryzae immobilized in calcium alginate beads and product separation process was set up in which a three-phase fluidized-bed bioreactor was used as a fermentor and an external electrodialyzer as a separator, and a pump was applied to recycle the fermentation broth between the bioreactor and the separator. The L-lactic acid produced in the fermentor was separated in the separator, product inhibition was alleviated without any addition of alkali or alkali salts and the product purification process could be simplified. The specific productivity and the yield in electrodialysis fermentation (ED-F) process operated in continuous feeding mode were almost the same as that in CaCO₃-buffered fermentation process. A mathematical model of L-lactic acid production in ED-F process was also suggested, in which the model equations for the bioreactor and the electrodialyzer were combined to describe the simultaneous fermentation and product separation. The model predictions were in good agreement with the experimental data.     

Electrophoretic analysis of Histoplasma capsulatum chromosomal DNA.

Seven chromosome-sized DNA molecules in the Downs strain of Histoplasma capsulatum were resolved by using chromosome-specific DNA probes in blot hybridizations of contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE) agarose gels. The sizes of the chromosomal DNA bands extended from that of the largest Saccharomyces cerevisiae chromosome to beyond that of the Schizosaccharomyces pombe chromosomes. Under our experimental conditions, the order of the five largest DNA bands was inverted in the FIGE gel relative to the CHEF gel, demonstrating a characteristic of FIGE whereby large DNA molecules may have greater rather than lesser mobility with increasing size. Comparison of the Downs strain with other H. capsulatum strains by CHEF and FIGE analysis revealed considerable variability in band mobility. The resolution of seven chromosome-sized DNA molecules in the Downs strain provides a minimum estimate of the chromosome number.     

Subunits of chromosomal DNA

A study of chromosomal DNA from Chinese hamster cells and chick fibroblasts by electron microscopy after partial denaturation revealed small regions which melted at 50° and could be stabilized by reaction with formaldehyde. The melted regions remained open so that their length and distribution along the DNA strands could be measured. The measurements indicated regularly spaced sites with low melting points at 0.4–0.5 micron intervals in most of the DNA. The length of the melted regions varied from those just visible to some as long as 0.4–0.5 microns, which probably represents the entire region between two successive sites with low melting points. A computer analysis of the spacings indicated a high probability of melted sites occurring every 2 microns along the DNA strands. Both of these spacings correspond to functional subunits of the DNA which can be isolated under appropriate metabolic conditions.     

Radiation effects on DNA synthesis in a defined chromosomal replicon.

It has recently been shown that the tumor suppressor p53 mediates a signal transduction pathway that responds to DNA damage by arresting cells in the late G1 period of the cell cycle. However, the operation of this pathway alone cannot explain the 50% reduction in the rate of DNA synthesis that occurs within 30 min of irradiation of an asynchronous cell population. We are using the amplified dihydrofolate reductase (DHFR) domain in the methotrexate-resistant CHO cell line, CHOC 400, as a model replicon in which to study this acute radiation effect. We first show that the CHOC 400 cell line retains the classical acute-phase response but does not display the late G1 arrest that characterizes the p53-mediated checkpoint. Using a two-dimensional gel replicon-mapping method, we then show that when asynchronous cultures are irradiated with 900 cGy, initiation in the DHFR locus is completely inhibited within 30 min and does not resume for 3 to 4 h. Since initiation in this locus occurs throughout the first 2 h of the S period, this result implies the existence of a p53-independent S-phase damage-sensing pathway that functions at the level of individual origins. Results obtained with the replication inhibitor mimosine define a position near the G1/S boundary beyond which cells are unable to prevent initiation at early-firing origins in response to irradiation. This is the first direct demonstration at a defined chromosomal origin that radiation quantitatively down-regulates initiation.     

The periodic loss of metabolically unstable DNA during replication of chromosomal DNA of CHO cells

The fate of ³H-thymidine incorporated into newly synthesized DNA of CHO cells was analyzed by either the estimation of the incorporated radioactivity per cell or sedimentation in alkaline sucrose gradient. Under conditions in which DNA synthesis proceeded continuously, of incorporated radioactivity was periodically lost and regained during a 90 min chase, corresponding to a cyclic change in the sedimentation profiles. When DNA synthesis was inhibited by hydroxyurea no cyclic change of the incorporated radioactivity was observed. The cyclic changes were regarded as the result of an actual metabolic change in³H-labelled DNA probaly joining to one of the newly formed sister strands of DNA and the loss of radioactivity seems to require active continued DNA synthesis.     

Condensin II resolves chromosomal associations to enable anaphase I segregation in Drosophila male meiosis

Several meiotic processes ensure faithful chromosome segregation to create haploid gametes. Errors to any one of these processes can lead to zygotic aneuploidy with the potential for developmental abnormalities. During prophase I of Drosophila male meiosis, each bivalent condenses and becomes sequestered into discrete chromosome territories. Here, we demonstrate that two predicted condensin II subunits, Cap-H2 and Cap-D3, are required to promote territory formation. In mutants of either subunit, territory formation fails and chromatin is dispersed throughout the nucleus. Anaphase I is also abnormal in Cap-H2 mutants as chromatin bridges are found between segregating heterologous and homologous chromosomes. Aneuploid sperm may be generated from these defects as they occur at an elevated frequency and are genotypically consistent with anaphase I segregation defects. We propose that condensin II–mediated prophase I territory formation prevents and/or resolves heterologous chromosomal associations to alleviate their potential interference in anaphase I segregation. Furthermore, condensin II–catalyzed prophase I chromosome condensation may be necessary to resolve associations between paired homologous chromosomes of each bivalent. These persistent chromosome associations likely consist of DNA entanglements, but may be more specific as anaphase I bridging was rescued by mutations in the homolog conjunction factor teflon. We propose that the consequence of condensin II mutations is a failure to resolve heterologous and homologous associations mediated by entangled DNA and/or homolog conjunction factors. Furthermore, persistence of homologous and heterologous interchromosomal associations lead to anaphase I chromatin bridging and the generation of aneuploid gametes.     

DNA damage during mitosis in human cells delays the metaphase/anaphase transition via the spindle-assembly checkpoint

BACKGROUND: DNA damage during mitosis triggers an ATM kinase-mediated cell cycle checkpoint pathway in yeast and fly embryos that delays progression through division. Recent data suggest that this is also true for mammals. Here we used laser microsurgery and inhibitors of topoisomerase IIalpha to break DNA in various mammalian cells after they became committed to mitosis. We then followed the fate of these cells and emphasized the timing of mitotic progression, spindle structure, and chromosome behavior. RESULTS: We find that DNA breaks generated during late prophase do not impede entry into prometaphase. If the damage is minor, cells complete mitosis on time. However, more significant damage substantially delays exit from mitosis in many cell types. In human (HeLa, CFPAC-1, and hTERT-RPE) cells, this delay occurs during metaphase, after the formation of a bipolar spindle and the destruction of cyclin A, and it is not dependent on a functional p53 pathway. Pretreating cells with ATM kinase inhibitors does not abrogate the metaphase delay due to chromosome damage. Immunofluorescence studies reveal that cells blocked in metaphase by chromosome damage contain one or more Mad2-positive kinetochores, and the block is rapidly overridden when the cells are microinjected with a dominant-negative construct of Mad2 (Mad2deltaC). CONCLUSIONS: We conclude that the delay in mitosis induced by DNA damage is not due to an ATM-mediated DNA damage checkpoint pathway. Rather, the damage leads to defects in kinetochore attachment and function that, in turn, maintain the intrinsic Mad-2-based spindle assembly checkpoint.     

Acinetobacter calcoaceticus liberates chromosomal DNA during induction of competence by cell lysis

A transformation assay was used to assay the amount of DNA present in the extracellular medium of a growing culture of Acinetobacter calcoaceticus. It was observed that small amounts of DNA were liberated during the entire exponential growth phase in a batch culture. Release of DNA could be fully accounted for by lysis of cells. Lysis was quantified via simultaneous measurement of -galactosidase activity of cells and supernatant, with a strain that contained a plasmid (pAPA100) with lacZ under control of a constitutive -lactamase promoter. In conclusion, no evidence could be obtained indicating that Acinetobacter calcoaceticus actively excretes DNA, to be used for DNA exchange.     

Phosphorylation of CAP-G is required for its chromosomal DNA localization during mitosis

Condensin is a 5 subunit complex that plays an important role in the structure of chromosomes during mitosis. It is known that phosphorylation of condensin subunits by cdc2/cyclin B at the beginning of mitosis is important for condensin activity, but the sites of these phosphorylation events have not been identified nor has their role in regulating condensin function. Here we identify two threonine residues in the CAP-G subunit of condensin, threonines 308 and 332, that are targets of cdc2/cyclin B phosphorylation. Mutation of these threonines to alanines results in defects in CAP-G localization with chromosomes during mitosis. These results are the first to identify phosphorylation sites within the condensin complex that regulate condensin localization with chromosomal DNA.