No abstract

Hallmarks of the terminal stages of apoptosis are genomic DNA fragmentation and chromatin condensation. Here, we have studied the mechanism of condensation both in vitro and in vivo. We found that DNA fragmentation per se of isolated nuclei from non-apoptotic cells induced chromatin condensation that closely resembles the morphology seen in apoptotic cells, independent of ATP utilization, at physiological ionic strengths. Interestingly, chromatin condensation was accompanied by release of nuclear actin, and both condensation and actin release could be blocked by reversibly pretreating nuclei with Ca 2؉ , Cu 2؉ , diamide, or low pH, procedures shown to stabilize internal nuclear components. Moreover, specific inhibition of nuclear F-actin depolymerization or promotion of its formation also reduced chromatin condensation. Chromatin condensation could also be inhibited by exposing nuclei to reagents that bind to the DNA minor groove, disrupting native nucleosomal DNA wrapping. In addition, in cultured cells undergoing apoptosis, drugs that inhibit depolymerization of actin or bind to the minor groove also reduced chromatin condensation, but not DNA fragmentation. Therefore, the ability of chromatin fragments with intact nucleosomes to form large clumps of condensed chromatin during apoptosis requires the apparent disassembly of internal nuclear structures that may normally constrain chromosome subdomains in nonapoptotic cells.Characteristics of the terminal stages of apoptosis are genomic DNA fragmentation and chromatin condensation (reviewed in Ref. 1). Although internucleosomal DNA breakdown is often temporally correlated with such chromatin condensation, it is not absolutely required to trigger this event (2-4). Three pathways have been identified that mediate apoptotic chromatin condensation: (i) a caspase-3-independent pathway triggered by mitochondrial apoptosis-inducing factor, which leads to an accompanying large-scale DNA fragmentation without internucleosomal DNA cleavage (5); (ii) a caspase-3-dependent pathway triggered by the protein acinus, which occurs withoutinducinganyDNAfragmentation(6);and(iii)acaspase-3-dependent pathway that leads to internucleosomal DNA cleavage mediated by activated DFF, 1 also termed caspase-activated deoxyribonuclease or caspase-activated nuclease (7-11).Although little is known regarding the mechanisms that lead to chromatin condensation in apoptotic cells, considerable information has appeared in the literature on the components that are linked to chromatin condensation in non-apoptotic cells. In interphase nuclei, heterochromatic regions are often associated with specific chromosomal proteins, post-translational modifications, and methylated DNA (reviewed in Refs. 12-14). In mitotic cells, chromosome condensation requires post-translational modifications and the action of an ATP-dependent complex called "condensin" to introduce positive DNA supercoils into DNA substrates in the presence of topoisomerases (Ref. 15; reviewed in Ref. 16). In summary, many parameters and prot...

To obtain an overall picture of the repair of DNA single and double strand breaks in a defined region of chromatin in vivo, we studied their repair in a ∼170 kb circular minichromosome whose length and topology are analogous to those of the closed loops in genomic chromatin. The rate of repair of single strand breaks in cells irradiated with γ photons was quantitated by determining the sensitivity of the minichromosome DNA to nuclease S1, and that of double strand breaks by assaying the reformation of supercoiled DNA using pulsed field electrophoresis. Reformation of supercoiled DNA, which requires that all single strand breaks have been repaired, was not slowed detectably by the inhibitors of poly(ADP-ribose) polymerase-1 NU1025 or 1,5-IQD. Repair of double strand breaks was slowed by 20–30% when homologous recombination was supressed by KU55933, caffeine, or siRNA-mediated depletion of Rad51 but was completely arrested by the inhibitors of nonhomologous end-joining wortmannin or NU7441, responses interpreted as reflecting competition between these repair pathways similar to that seen in genomic DNA. The reformation of supercoiled DNA was unaffected when topoisomerases I or II, whose participation in repair of strand breaks has been controversial, were inhibited by the catalytic inhibitors ICRF-193 or F11782. Modeling of the kinetics of repair provided rate constants and showed that repair of single strand breaks in minichromosome DNA proceeded independently of repair of double strand breaks. The simplicity of quantitating strand breaks in this minichromosome provides a usefull system for testing the efficiency of new inhibitors of their repair, and since the sequence and structural features of its DNA and its transcription pattern have been studied extensively it offers a good model for examining other aspects of DNA breakage and repair.