ATM Signaling Facilitates Repair of DNA Double-Strand Breaks Associated with Heterochromatin

Genome Damage and Stability Centre, University of Sussex, East Sussex BN1 9RQ, UK.
Molecular cell (Impact Factor: 14.02). 07/2008; 31(2):167-77. DOI: 10.1016/j.molcel.2008.05.017
Source: PubMed


Ataxia Telangiectasia Mutated (ATM) signaling is essential for the repair of a subset of DNA double-strand breaks (DSBs); however, its precise role is unclear. Here, we show that < or =25% of DSBs require ATM signaling for repair, and this percentage correlates with increased chromatin but not damage complexity. Importantly, we demonstrate that heterochromatic DSBs are generally repaired more slowly than euchromatic DSBs, and ATM signaling is specifically required for DSB repair within heterochromatin. Significantly, knockdown of the transcriptional repressor KAP-1, an ATM substrate, or the heterochromatin-building factors HP1 or HDAC1/2 alleviates the requirement for ATM in DSB repair. We propose that ATM signaling temporarily perturbs heterochromatin via KAP-1, which is critical for DSB repair/processing within otherwise compacted/inflexible chromatin. In support of this, ATM signaling alters KAP-1 affinity for chromatin enriched for heterochromatic factors. These data suggest that the importance of ATM signaling for DSB repair increases as the heterochromatic component of a genome expands.

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    • "Atm −/− MEFs showed elevated unrepaired DSBs at 6 h post ionising radiation, and a persistent fraction of unrepaired DSBs could be detected at 12 days post ionising radiation, which is consistent with previous findings (Fig. 4D; supplementary material Fig. S1C, light grey columns) (Riballo et al., 2001). These have been shown to represent DSBs at heterochromatin regions, which have an essential requirement for ATM for repair (Goodarzi et al., 2008). Strikingly, the rate of DSB repair in ATMi-treated Lig4 Y288C cells was slower than in non-ATMi treated cells but, nonetheless, only a small subset of DSBs remained by 12 days post ionising radiation (Fig. 4D; supplementary material Fig. S1C). "
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    • "Notably, DNA DSBs in heterochromatin relocalize to euchromatic regions during repair (Jakob et al. 2011), and as a consequence heterochromatic regions often appear to lack radiation-induced g-H2AX foci (Cowell et al. 2007; Kim et al. 2007; Goodarzi et al. 2008; Vasireddy et al. 2010; Chiolo et al. 2011; Jakob et al. 2011; Lafon-Hughes et al. 2013). The relocalization of heterochromatin DSBs to the periphery of heterochromatic domains is accompanied by a transient ATM-dependent chromatin relaxation (Ziv et al. 2006; Geuting et al. 2013). "
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    • "siRNA sequences were as follows: SNF2H A, 5-CCGGGCAAAUAGAUUCGAGUAUUUA-3; SNF2H B, 5-CAGGGAAGCUCUUCGUGUUAGUGAA-3; SNF2H mouse, 5-GGGAGGCCUCUGAGAACCUUCCGUU-3; RNF20 A, 5-CCGUG- UCCCAGAUUGUGACUGUUUA-3; RNF20 B, 5-CAGUCACAGUUCU- CCGUCUUGUAUA-3; ACF1 A, 5-CAAGUAUAAAGUGCAACCCAC- UAAA-3; ACF1 B, 5-UCAAGAUCCUCAGGUAUCCACUAAA-3; CHD3 A, 5-GGGCCAUCAUUCGUGAGAAUGAAUU-3; CHD3 B, 5-AGGCA- CAGGUGAAGUUCCAUGUUCU-3; RNF8 A, 5-GGACAAUUAUGGA- CAACAA-3; RNF8 B, 5-UGCGGAGUAUGAAUAUGAA-3; and 53BP1, 5-AGAACGAGGAGACGGUAAUAGUGGG-3. For plasmid expression, adherent cells were transfected with 2 µg plasmid 48 h after siRNA treatment using Metafectene Pro according to methods described in Goodarzi et al. (2008, 2011) and Noon et al. (2010) and, 16–24 h later, were irradiated for analysis. For pEGFP-N1– SNF2H expression constructs (SNF2H GFP ), full human SNF2H cDNA were cloned into the pEGFP-N1 backbone under a cytomegalovirus (CMV) promoter (Takara Bio Inc.; plasmid backbone information found at Addgene). "
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