Evidence that DNA fragmentation in apoptosis is initiated and propagated by single-strand breaks

Abstract

Apoptosis is characterised by the degradation of DNA into a specific pattern of high and low molecular weight fragments seen on agarose gels as a distribution of sizes between 50 ± 300 kb and sometimes, but not always, a ladder of smaller oligonucleosomal fragments. Using a 2D pulsed fieldconventional agarose gel electrophoresis technique, where the second dimension is run under either normal or denaturing conditions, we show that single-strand breaks are introduced into DNA at the initial stages of fragmentation. Using singlestrand specific nuclease probes we further show that the complete fragmentation pattern, including release of small oligonucleosomal fragments can also be generated by a single-strand endonuclease. Three classes of sites where single-strand breaks accumulate were identified. The initial breaks produce a distribution of fragment sizes (50 kb to 41 Mb) similar to those generated by Topoisomerase II inhibitors suggesting that cleavage may commence at sites of attachment of DNA to the nuclear matrix. A second class of rare sites is also cut further reducing the size distribution of the fragments to 50-300 kb. Thirdly, single-strand breaks accumulate at the linker region between nucleosomes eventually causing double-strand scissions which release oligonucleosomes. These observations further define the properties of the endonuclease responsible for DNA fragmentation in apoptosis.

Mapping the initial DNA breaks in apoptotic Jurkat cells using ligation-mediated PCR

“…34 There have been a number of reports in the literature suggesting that initiation of apoptotic DNA cleavage occurs at matrix attachment regions. 6,7,[35][36][37] The first supporting evidence came from studies that establish that high molecular weight DNA fragments of 50 and 300 kb, produced in early stages of apoptosis, represent structural domains of chromatin, loops and rosettes, respectively. 1,7 It was then suggested that topoisomerase II, located at matrix attachment sites, is responsible for this cleavage since inhibition of its activity generated the same pattern of DNA fragments.…”

“…6,7,[35][36][37] The first supporting evidence came from studies that establish that high molecular weight DNA fragments of 50 and 300 kb, produced in early stages of apoptosis, represent structural domains of chromatin, loops and rosettes, respectively. 1,7 It was then suggested that topoisomerase II, located at matrix attachment sites, is responsible for this cleavage since inhibition of its activity generated the same pattern of DNA fragments. 6,38 However, others have shown that the activation of endogenous and several exogenous nucleases also results in DNA digestion in the proximity of MARs and produces similar sets of high molecular weight 50 and 300 kb fragments as those generated by topoisomerase II inhibitors.…”

“…This procedure reveals the presence of ss breaks in cleaved DNA fragments. 7,28 Indeed, the denaturation treatment resulted in the liberation of a smear of ss fragments of all sizes, not only from the apoptotic DNA fragments but also from the high molecular weight DNA, which remained in the well after the PFGE separation (Figure 1b, indicated by arrows). There was no significant number of ss DNA breaks in the control sample.…”

“…1,2 The large DNA fragments are thought to arise from the cleavage of DNA domains at matrix attachment regions (MARs) and may result from the accumulation of single-strand (ss) breaks introduced in the vulnerable DNA regions in contact with the nuclear matrix. [3][4][5][6][7] Many chromatinassociated proteins, such as DNA topoisomerase II, PARP, NuMA, lamin B, SAF-A and SATB1, have been shown to undergo site-specific proteolysis during apoptosis. [8][9][10][11][12][13] This process can be temporally linked to the initiation of DNA cleavage, suggesting that the latter may be a consequence of the loss of a protective protein coating.…”

Regulation of DNaseY activity by actinin-α4 during apoptosis

“…This results in the generation of single-strand breaks (ssb) and, subsequently, double-strand (ds) DNA fragmentation. [15][16][17] Recent reports imply that DNaseY is activated by an increase of intracellular Ca 2 þ , which results in early DNA breaks and activation of PARP-1. Poly-ADP-ribosylation of DNaseY by PARP-1 inhibits its endonuclease activity.…”