The heterochromatic barrier to DNA double strand break repair: how to get the entry visa

Abstract

Over recent decades, a deep understanding of pathways that repair DNA double strand breaks (DSB) has been gained from biochemical, structural, biophysical and cellular studies. DNA non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the two major DSB repair pathways, and both processes are now well understood. Recent work has demonstrated that the chromatin environment at a DSB significantly impacts upon DSB repair and that, moreover, dramatic modifications arise in the chromatin surrounding a DSB. Chromatin is broadly divided into open, transcriptionally active, euchromatin (EC) and highly compacted, transcriptionally inert, heterochromatin (HC), although these represent extremes of a spectrum. The HC superstructure restricts both DSB repair and damage response signaling. Moreover, DSBs within HC (HC-DSBs) are rapidly relocalized to the EC-HC interface. The damage response protein kinase, ataxia telangiectasia mutated (ATM), is required for HC-DSB repair but is dispensable for the relocalization of HC-DSBs. It has been proposed that ATM signaling enhances HC relaxation in the DSB vicinity and that this is a prerequisite for HC-DSB repair. Hence, ATM is essential for repair of HC-DSBs. Here, we discuss how HC impacts upon the response to DSBs and how ATM overcomes the barrier that HC poses to repair.

Keywords: DNA non-homologous end-joining; ataxia telangiectasia mutated; chromatin; damage response signaling; heterochromatin; homologous recombination.

Figure 1

Differential irradiation induced foci (IRIF) formation between Euchromatin and Heterochromatin. (1) DNA double strand breaks (DSBs) form within either heterochromatin (red) or euchromatin (blue); (2) γH2AX occurs on chromatin at the DSB site (green) enabling the formation of the larger IRIF (yellow star) comprised of proteins such as Mre11, Rad50, NBS1, MDC1, RNF8, RNF168 and 53BP1. In heterochromatin, however, IRIF fail to form to a similar extent (or at all) at the same time point. Rather, the heterochromatic DSB relocates from the heterochromatic core to the peripheral zone bordering on euchromatin; (3) Once relocated to the heterochromatin:euchromatin border, the heterochromatic DSB elicits IRIF formation which expands into the surrounding euchromatic space.

Figure 2

Ataxia telangiectasia mutated (ATM)-dependent heterochromatic DSB repair. (1) DSBs elicit ATM activation and IRIF formation, however repair processes within heterochromatin are inhibited by the compacted nucleosome configuration produced by KAP-1 dependent CHD3 activity; (2) Active ATM phosphorylates KAP-1 at S824, which interferes with the SUMOylation-dependent retention of CHD3 in chromatin; (3) In the absence of CHD3, the chromatin surrounding the DSB site relaxes, allowing (3A) non-homologous end-joining or (3B) if cells are in G2-phase, DNA end resection and homologous recombination mediated repair of the DSB; (4) Once the DSB is rejoined, ATM signaling is deactivated. Heterochromatic nucleosomes re-compact once KAP-1 is dephosphorylated and CHD3 activity is again retained.

Figure 3

The influence of heterochromatin on ATM signaling and cell cycle checkpoint arrest. (A, 1–3) At early times after DSB formation, a significant difference in the magnitude of IRIF formation and consequential ATM signaling is observed based on the relative proximity of the DSB to heterochromatic centers, with the expansion of IRIF forming at DSBs located near or within heterochromatin BEING constrained. (A, 4) Where defective heterochromatin is encountered, as a result of germ-line mutation or siRNA-mediated knockdown of heterochromatic building factors, IRIF expansion is unconstrained and a greater degree of ATM signaling is produced relative to normal. (A, 5) In the absence of 53BP1 or the dense, localized phosphorylation of KAP-1, IRIF penetrance into heterochromatin is severely hindered causing a reduction in the amount of ATM signaling. (B) Cell cycle checkpoint arrest is triggered by a threshold number of DSBs in normal cells, due to a defined amount of ATM signaling being produced per DSB within the cell. The magnitude of ATM signaling per DSB is “set” by the natural balance between euchromatin and heterochromatin. Where defective heterochromatin is present, DSBs within or bordering on heterochromatic centers signal to a greater extent than they would in normal cells, since IRIF expansion and ATM signaling are no longer constrained. Hence, the number of DSBs required to achieve the minimum level of ATM signaling needed to trigger checkpoint arrest is lowered and these cells display hypersensitive checkpoint initiation and prolonged maintenance. Note that the figures display events at early times post irradiation. At later times, IRIF expansion occurs into euchromatin efficiently so that the overall size of the foci from DSBs within euchromatin versus heterochromatin are similar although most of the IRIF occurs within the euchromatin region. At later times, IRIF at DSBs within defective heterochromatin are larger than those within euchromatin suggesting that factors may be recruited to euchromatin to restrict their expansion.