It is not yet clear how much disassembly of histone octamers must occur at a DSB for NHEJ proteins to function. In contrast to homologous recombination (HR), where kilobases of DNA are involved and γ–H2AX alterations are important, NHEJ probably requires less than 30 bp of DNA on either side of a break.
If randomly distributed, 80% of DSBs would occur on DNA that is wrapped around histone octamers and 20% would occur internucleosomally. For those breaks within a nucleosome, one study showed that Ku can bind, implying that the duplex DNA can separate from the surface of the nucleosome sufficiently to permit Ku to bind (154
Several studies propose that γ–H2AX is important for NHEJ (16
). Much of the evidence is based on immunolocalization studies where the damage site may contain a mixture of HR and NHEJ events within the 2000 angstrom confocal microscope section thickness. Differences in access within the euchromatic versus the heterochromatic regions are likely, but even early genetic insights concerning this are limited to yeast (156
H2AX is only present, on average, in one of every ten nucleosomes because H2A is the predominant species in histone octamers (16
). Therefore, most DSBs would occur about 5 nucleosomes away (about 1 kb) from the nearest octamer containing an H2AX that is eligible for conversion to γ–H2AX via phosphorylation by ATM or DNA-PKcs at serine 139 of H2AX. Given this substantial distance from the site of the enzymatic repair, it is not clear that such γ–H2AX phosphorylation events are critical for NHEJ.
When DNA-PKcs does phosphorylate H2AX, this increases vulnerability of H2AX to the histone exchange factor called FACT (which consists of a heterodimer of Spt16 and SSRP1). Phosphorylated H2AX (γ-H2AX) is more easily exchanged out of the octamer, thereby leaving only a tetramer of (H3)2(H4)2 at the site, and this is more sterically flexible, thereby perhaps permitting DNA repair factors to carry out their work (157
PARP-1 is able to downregulate the activity of FACT by ADP-ribosylation of the Spt16 subunit of FACT. This may be able to shift the equilibrium of γ-H2AX and H2AX in the nucleosomes. That is, PARP-1 activation at a site of damage might shift the equilibrium toward retention of γ-H2AX in the region, perhaps thereby aiding in recruitment or retention of repair proteins (157
Hence, FACT may initially act proximally at the most immediate nucleosome (or closest one) to exchange γ-H2AX out and leave an (H3)2
tetramer at the site of damage for purposes of flexibility of the DNA. FACT may act more regionally (distally) to favor the retention of γ-H2AX for purposes of integrating the repair process with repair protein recruitment, protein retention, and cell cycle aspects (157