Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks

Abstract

The repair of DNA double-strand breaks (DSBs) is facilitated by the phosphorylation of H2AX, which organizes DNA damage signaling and chromatin remodeling complexes in the vicinity of the lesion (Pilch, D.R., O.A. Sedelnikova, C. Redon, A. Celeste, A. Nussenzweig, and W.M. Bonner. 2003. Biochem. Cell Biol. 81:123–129; Morrison, A.J., and X. Shen. 2005. Cell Cycle. 4:568–571; van Attikum, H., and S.M. Gasser. 2005. Nat. Rev. Mol. Cell. Biol. 6:757–765). The disruption of DNA integrity induces an alteration of chromatin architecture that has been proposed to activate the DNA damage transducing kinase ataxia telangiectasia mutated (ATM; Bakkenist, C.J., and M.B. Kastan. 2003. Nature. 421:499–506). However, little is known about the physical properties of damaged chromatin. In this study, we use a photoactivatable version of GFP-tagged histone H2B to examine the mobility and structure of chromatin containing DSBs in living cells. We find that chromatin containing DSBs exhibits limited mobility but undergoes an energy-dependent local expansion immediately after DNA damage. The localized expansion observed in real time corresponds to a 30–40% reduction in the density of chromatin fibers in the vicinity of DSBs, as measured by energy-filtering transmission electron microscopy. The observed opening of chromatin occurs independently of H2AX and ATM. We propose that localized adenosine triphosphate–dependent decondensation of chromatin at DSBs establishes an accessible subnuclear environment that facilitates DNA damage signaling and repair.

Perinuclear tethers license telomeric DSBs for a broad kinesin- and NPC-dependent DNA repair process

“…These processes may be conserved, as broken DSB ends can exhibit limited or pronounced mobility in mammals [63][64][65][66] . Thus, it will be important to determine whether motor complexes contribute to DSB repair in different organisms including human.…”

Nuclear organization in genome stability: SUMO connections

“…It has been shown that DNA damage checkpoint and repair proteins relocalize from a diffuse distribution to distinct subnuclear foci in a temporally regulated manner upon DNA damage, in both yeast and mammalian cells [31][32][33][34]. However, it is not clear if double-strand breaks (DSBs) are recruited to scaffolds for repair, or if repair factors assemble at sites of damage.…”

“…However, it is not clear if double-strand breaks (DSBs) are recruited to scaffolds for repair, or if repair factors assemble at sites of damage. Indeed, sites of damage arising from a targeted endonuclease [31] or γ-irradiation [32] are relatively immobile in the mammalian nucleus, suggesting that most DSB repair by non-homologous end joining, does not involve relocalization of damage to sites like PML-NBs. It remains possible, however, that specific pathways of DNA repair require specific nuclear subcompartments, since the efficiency of certain types of repair varies with the chromosomal context in which the damage occurs [35].…”

Chromatin dynamics coupled to DNA repair

“…Chromatin relaxation in the vicinity of DSB has been observed in yeast 41 and mammalian cells. 42 Thus, it was shown that a local expansion of damaged chromatin occurs immediately after DSB, independently of ATM activation and H2AX phosphorylation but dependent on processes that hydrolyze ATP. 42 Moreover, it is speculated that local relaxation at the DSB site might also be a signal for ATM activation since chromatin decondensation induced by relaxing agents, such as chloroquine, and inhibitors of deacetylases can trigger ATM activation.…”