Aaron A. Goodarzi

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

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.

γH2AX foci analysis for monitoring DNA double-strand break repair: Strengths, limitations and optimization

DNA double-strand breaks (DSBs) represent an important radiation-induced lesion and impaired DSB repair provides the best available correlation with radiosensitivity. Physical techniques for monitoring DSB repair require high, non-physiological doses and cannot reliably detect subtle defects. One outcome from extensive research into the DNA damage response is the observation that H2AX, a variant form of the histone H2A, undergoes extensive phosphorylation at the DSB, creating γH2AX foci that can be visualised by immunofluorescence. There is a close correlation between γH2AX foci and DSB numbers and between the rate of foci loss and DSB repair, providing a sensitive assay to monitor DSB repair in individual cells using physiological doses. However, γH2AX formation can occur at single-stranded DNA regions which arise during replication or repair and thus does not solely correlate with DSB formation. Here, we present and discuss evidence that following exposure to ionising radiation, γH2AX foci analysis can provide a sensitive monitor of DSB formation and repair and describe techniques to optimise the analysis. We discuss the limitations and benefits of the technique, enabling the procedure to be optimally exploited but not misused.

ATR‐dependent phosphorylation and activation of ATM in response to UV treatment or replication fork stalling

The phosphatidyl inositol 3‐kinase‐like kinases (PIKKs), ataxia‐telangiectasia mutated (ATM) and ATM‐ and Rad3‐related (ATR) regulate parallel damage response signalling pathways. ATM is reported to be activated by DNA double‐strand breaks (DSBs), whereas ATR is recruited to single‐stranded regions of DNA. Although the two pathways were considered to function independently, recent studies have demonstrated that ATM functions upstream of ATR following exposure to ionising radiation (IR) in S/G2. Here, we show that ATM phosphorylation at Ser1981, a characterised autophosphorylation site, is ATR‐dependent and ATM‐independent following replication fork stalling or UV treatment. In contrast to IR‐induced ATM‐S1981 phosphorylation, UV‐induced ATM‐S1981 phosphorylation does not require the Nbs1 C‐terminus or Mre11. ATR‐dependent phosphorylation of ATM activates ATM phosphorylation of Chk2, which has an overlapping function with Chk1 in regulating G2/M checkpoint arrest. Our findings provide insight into the interplay between the PIKK damage response pathways.

DNA‐PK autophosphorylation facilitates Artemis endonuclease activity

The Artemis nuclease is defective in radiosensitive severe combined immunodeficiency patients and is required for the repair of a subset of ionising radiation induced DNA double‐strand breaks (DSBs) in an ATM and DNA‐PK dependent process. Here, we show that Artemis phosphorylation by ATM and DNA‐PK in vitro is primarily attributable to S503, S516 and S645 and demonstrate ATM dependent phosphorylation at serine 645 in vivo. However, analysis of multisite phosphorylation mutants of Artemis demonstrates that Artemis phosphorylation is dispensable for endonuclease activity in vitro and for DSB repair and V(D)J recombination in vivo. Importantly, DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) autophosphorylation at the T2609–T2647 cluster, in the presence of Ku and target DNA, is required for Artemis‐mediated endonuclease activity. Moreover, autophosphorylated DNA‐PKcs stably associates with Ku‐bound DNA with large single‐stranded overhangs until overhang cleavage by Artemis. We propose that autophosphorylation triggers conformational changes in DNA‐PK that enhance Artemis cleavage at single‐strand to double‐strand DNA junctions. These findings demonstrate that DNA‐PK autophosphorylation regulates Artemis access to DNA ends, providing insight into the mechanism of Artemis mediated DNA end processing.

Autophosphorylation of ataxia‐telangiectasia mutated is regulated by protein phosphatase 2A

Ionizing radiation induces autophosphorylation of the ataxia‐telangiectasia mutated (ATM) protein kinase on serine 1981; however, the precise mechanisms that regulate ATM activation are not fully understood. Here, we show that the protein phosphatase inhibitor okadaic acid (OA) induces autophosphorylation of ATM on serine 1981 in unirradiated cells at concentrations that inhibit protein phosphatase 2A‐like activity in vitro. OA did not induce γ‐H2AX foci, suggesting that it induces ATM autophosphorylation by inactivation of a protein phosphatase rather than by inducing DNA double‐strand breaks. In support of this, we show that ATM interacts with the scaffolding (A) subunit of protein phosphatase 2A (PP2A), that the scaffolding and catalytic (C) subunits of PP2A interact with ATM in undamaged cells and that immunoprecipitates of ATM from undamaged cells contain PP2A‐like protein phosphatase activity. Moreover, we show that IR induces phosphorylation‐dependent dissociation of PP2A from ATM and loss of the associated protein phosphatase activity. We propose that PP2A plays an important role in the regulation of ATM autophosphorylation and activity in vivo.

The influence of heterochromatin on DNA double strand break repair: Getting the strong, silent type to relax

DNA non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the major DNA double strand break (DSB) pathways in mammalian cells, whilst ataxia telangiectasia mutated (ATM) lies at the core of the DSB signalling response. ATM signalling plays a major role in modifying chromatin structure in the vicinity of the DSB and increasing evidence suggests that this function influences the DSB rejoining process. DSBs have long been known to be repaired with two (or more) component kinetics. The majority (∼85%) of DSBs are repaired with fast kinetics in a predominantly ATM-independent manner. In contrast, ∼15% of radiation-induced DSBs are repaired with markedly slower kinetics via a process that requires ATM and those mediator proteins, such as MDC1 or 53BP1, that accumulate at ionising radiation induced foci (IRIF). DSBs repaired with slow kinetics predominantly localise to the periphery of genomic heterochromatin (HC). Indeed, there is mounting evidence that chromatin complexity and not damage complexity confers slow DSB repair kinetics. ATMs role in HC-DSB repair involves the direct phosphorylation of KAP-1, a key HC formation factor. KAP-1 phosphorylation (pKAP-1) arises in both a pan-nuclear and a focal manner after radiation and ATM-dependent pKAP-1 is essential for DSB repair within HC regions. Mediator proteins such as 53BP1, which are also essential for HC-DSB repair, are expendable for pan-nuclear pKAP-1 whilst being essential for pKAP-1 formation at IRIF. Data suggests that the essential function of the mediator proteins is to promote the retention of activated ATM at DSBs, concentrating the phosphorylation of KAP-1 at HC DSBs. DSBs arising in G2 phase are also repaired with fast and slow kinetics but, in contrast to G0/G1 where they all DSBs are repaired by NHEJ, the slow component of DSB repair in G2 phase represents an HR process involving the Artemis endonuclease. Results suggest that whilst NHEJ repairs the majority of DSBs in G2 phase, Artemis-dependent HR uniquely repairs HC DSBs. Collectively, these recent studies highlight not only how chromatin complexity influences the factors required for DSB repair but also the pathway choice.

Identification of in vitro and in vivo phosphorylation sites in the catalytic subunit of the DNA-dependent protein kinase.

The DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs), such as those caused by ionizing radiation and other DNA-damaging agents. DNA-PK is composed of a large catalytic subunit (DNA-PKcs) and a heterodimer of Ku70 and Ku80 that assemble on the ends of double-stranded DNA to form an active serine/threonine protein kinase complex. Despite in vitro and in vivo evidence to support an essential role for the protein kinase activity of DNA-PK in the repair of DNA DSBs, the physiological targets of DNA-PK have remained elusive. We have previously shown that DNA-PK undergoes autophosphorylation in vitro, and that autophosphorylation correlates with loss of protein kinase activity and dissociation of the DNA-PK complex. Also, treatment of cells with the protein phosphatase inhibitor, okadaic acid, enhances DNA-PKcs phosphorylation and reduces DNA-PK activity in vivo. Here, using solid-phase protein sequencing, MS and phosphospecific antibodies, we have identified seven in vitro autophosphorylation sites in DNA-PKcs. Six of these sites (Thr2609, Ser2612, Thr2620, Ser2624, Thr2638 and Thr2647) are clustered in a region of 38 amino acids in the central region of the protein. Five of these sites (Thr2609, Ser2612, Thr2638, Thr2647 and Ser3205) are conserved between six vertebrate species. Moreover, we show that DNA-PKcs is phosphorylated in vivo at Thr2609, Ser2612, Thr2638 and Thr2647 in okadaic acid-treated human cells. We propose that phosphorylation of these sites may play an important role in DNA-PK function.

KAP-1 phosphorylation regulates CHD3 nucleosome remodeling during the DNA double-strand break response

KAP-1 poses a substantial barrier to DNA double-strand break (DSB) repair within heterochromatin that is alleviated by ATM-dependent KAP-1 phosphorylation (pKAP-1). Here we address the mechanistic consequences of pKAP-1 that promote heterochromatic DSB repair and chromatin relaxation. KAP-1 function involves autoSUMOylation and recruitment of nucleosome deacetylation, methylation and remodeling activities. Although heterochromatin acetylation or methylation changes were not detected, radiation-induced pKAP-1 dispersed the nucleosome remodeler CHD3 from DSBs and triggered concomitant chromatin relaxation; pKAP-1 loss reversed these effects. Depletion or inactivation of CHD3, or ablation of its interaction with KAP-1SUMO1, bypassed pKAP-1s role in repair. Though KAP-1 SUMOylation was unaffected after irradiation, CHD3 dissociated from KAP-1SUMO1 in a pKAP-1–dependent manner. We demonstrate that KAP-1Ser824 phosphorylation generates a motif that directly perturbs interactions between CHD3′s SUMO-interacting motif and SUMO1, dispersing CHD3 from heterochromatin DSBs and enabling repair.

The impact of heterochromatin on DSB repair

DNA NHEJ (non-homologous end-joining) is the major DNA DSB (double-strand break) repair pathway in mammalian cells. Although NHEJ-defective cell lines show marked DSB-repair defects, cells defective in ATM (ataxia telangiectasia mutated) repair most DSBs normally. Thus NHEJ functions independently of ATM signalling. However, approximately 15% of radiation-induced DSBs are repaired with slow kinetics and require ATM and the nuclease Artemis. DSBs persisting in the presence of an ATM inhibitor, ATMi, localize to heterochromatin, suggesting that ATM is required for repairing DSBs arising within or close to heterochromatin. Consistent with this, we show that siRNA (small interfering RNA) of key heterochromatic proteins, including KAP-1 [KRAB (Krüppel-associated box) domain-associated protein 1], HP1 (heterochromatin protein 1) and HDAC (histone deacetylase) 1/2, relieves the requirement for ATM for DSB repair. Furthermore, ATMi addition to cell lines with genetic alterations that have an impact on heterochromatin, including Suv39H1/2 (suppressor of variegation 3-9 homologue 1/2)-knockout, ICFa (immunodeficiency, centromeric region instability, facial anomalies syndrome type a) and Hutchinson-Guilford progeria cell lines, fails to have an impact on DSB repair. KAP-1 is a highly dose-dependent, transient and ATM-specific substrate, and mutation of the ATM phosphorylation site on KAP-1 influences DSB repair. Collectively, the findings show that ATM functions to overcome the barrier to DSB repair posed by heterochromatin. However, even in the presence of ATM, gamma-H2AX (phosphorylated histone H2AX) foci form on the periphery rather than within heterochromatic centres. Finally, we show that KAP-1s association with heterochromatin is diminished as cells progress through mitosis. We propose that KAP-1 is a critical heterochromatic factor that undergoes specific modifications to promote DSB repair and mitotic progression in a manner that allows localized and transient chromatin relaxation, but precludes significant dismantling of the heterochromatic superstructure.

The Repair and Signaling Responses to DNA Double-Strand Breaks

A DNA double-strand break (DSB) has long been recognized as a severe cellular lesion, potentially representing an initiating event for carcinogenesis or cell death. The evolution of DSB repair pathways as well as additional processes, such as cell cycle checkpoint arrest, to minimize the cellular impact of DSB formation was, therefore, not surprising. However, the depth and complexity of the DNA damage responses being revealed by current studies were unexpected. Perhaps the most surprising finding to emerge is the dramatic changes to chromatin architecture that arise in the DSB vicinity. In this review, we overview the cellular response to DSBs focusing on DNA repair pathways and the interface between them. We consider additional events which impact upon these DSB repair pathways, including regulated arrest of cell cycle progression and chromatin architecture alterations. Finally, we discuss the impact of defects in these processes to human disease.