Mini-reviewThe influence of heterochromatin on DNA double strand break repair: Getting the strong, silent type to relax
Introduction
Although DNA double strand breaks (DSBs) arise less frequently than DNA single strand breaks (SSBs) and many base alterations, they are, perhaps, the most critical DNA lesion since failure to repair a DSB has a high probability of causing cell death and, as importantly, erroneous DSB repair can lead to chromosomal rearrangements, a causal event in the aetiology of carcinogenesis. Consequently, understanding the factors that influence the efficacy and fidelity of DSB repair is important for assessing risks associated with exposure to DSB inducing agents, including an evaluation of the impact on overall human health as well as cancer avoidance [1]. Furthermore, now that we have at least a basic mechanistic understanding of most DNA repair processes, attention is shifting towards understanding how these pathways operate in vivo in the context of chromatin structure. To accommodate the enormous quantity of genetic material, which measures several metres when fully relaxed, into the dimensions of a nucleus, eukaryotes have evolved mechanisms to tightly compact their DNA. Genomic regions with specialist function or regions which are not undergoing active transcription are often further compressed into even more tightly compacted structures, termed heterochromatin (HC). There is a growing awareness that higher order chromatin architecture exerts just as profound an influence on DNA repair as it does on nuclear processes such as transcription and replication. It is becoming increasingly clear that a range of chromatin remodelling mechanisms function to facilitate these DNA transactions in their cellular context [2], [3]. Such processes involve not only mechanisms that restore chromatin structure when DNA is damaged but also mechanisms that transiently or locally modify chromatin structure to promote DNA repair.
Given the high degree of compaction of HC DNA and its efficacy at blocking transcription, it is perhaps not surprising that it has been shown to act as a barrier for some DNA repair processes. Intriguingly, it appears that in mammals ataxia telangiectasia mutated (ATM) signalling plays a critical role in relieving the constraints on DSB repair posed by the highly compacted HC. In contrast, Tel1, the yeast homologue of ATM, has a less significant role in DSB repair, raising the possibility that ATM signalling has, at least in part, evolved to help overcome the increased chromatin compaction arising in more complex eukaryotic genomes [4]. Here, we review how HC impacts upon DSB repair, focusing on the role of ATM and its damage response mediator proteins in overcoming the constraints posed by the HC superstructure. Further, we discuss how higher order chromatin structure influences DSB repair pathway choice in mammalian cells.
Section snippets
DSB repair and DNA damage response signalling
DNA non-homologous end-joining (NHEJ) represents the major DSB repair process in mammalian cells and functions throughout the cell cycle [5]. This process has been reviewed in detail previously and will only be outlined here. In brief, the Ku70/80 heterodimer (Ku) is a basket shaped structure with a central pore, whose structure confers an avid ability to bind double stranded DNA ends [6]. The central pore allows Ku to thread onto double stranded DNA ends and a ratchet mechanism facilitates its
The slow, ATM-dependent component of DSB repair
A range of studies involving the physical estimation of DNA integrity have shown that DSBs are repaired with at least two kinetically distinct components; approximately 85% of DSBs are repaired with fast kinetics whilst 15–20% are repaired more slowly [30], [31]. More recent approaches to monitor DSB repair have exploited our current understanding of the damage response protein assembly process [29], [32]. IRIF enumeration exploits 53BP1 recruitment or γH2AX foci as a marker of the presence of
The heterochromatic superstructure
The term heterochromatin refers to chromatin that is different (hetero-) from so-called “true” (eu-) chromatin. Indeed, HC is usually spatially segregated from the bulk chromatin in eukaryotic nuclei and is functionally distinct [36]. HC is highly condensed and generally comprises between 10 and 25% of total chromatin, depending on age, cell-type and species (reviewed in Refs. [37], [38]. There are two general classes of HC: constitutive and facultative. Broadly defined, constitutive HC refers
KAP-1 and the heterochromatic barrier to DSB repair
With the exception of brief periods during DNA replication and mitosis, the general integrity of HC remains unperturbed under ordinary circumstances. However, following DSB induction, an elaborate series of events is set into motion within HC to implement dynamic and localised changes necessary for DSB repair. Failure to implement these changes may result in stalled DSB repair within HC regions (Fig. 2E). In recent years, two HC foundation factors have been shown to be robust targets of the DNA
The various roles of 53BP1 in response to DSBs
Recent findings have suggested that 53BP1 exerts functions in response to DSBs which are partly distinct, but also overlapping, with its role(s) in the ATM-to-53BP1 signalling hierarchy. Indeed, it was demonstrated that 53BP1 function enhances the spatial mobility of “uncapped” telomeric ends (generated by TRF2 depletion) within the nucleoplasm, enabling them to ‘locate’ partners for rejoining with increased frequency [62]. Likewise, DSB rejoining during V(D)J recombination has also been
DSB repair events downstream of KAP-1 phosphorylation and chromatin relaxation
Whilst it is now clear that the slow, ATM/53BP1-dependent component of DSB repair strongly correlates with regions of HC, the ectopic perturbation of HC (by depletion of KAP-1 or expression of the phosphomimetic KAP-1S824D for example) does not significantly enhance the speed of DSB repair in normal cells [35]. This initially surprising result suggested that, despite the fact that HC is rendered amenable to repair by ATM function, the processes able to implement fast repair kinetics within EC
The world in G2 phase
Studies addressing the utilisation of DSB repair pathways during the G2 phase of the mammalian cell cycle have been hampered for many years by technical difficulties. Firstly, the necessity to use extremely high IR doses has limited our ability to quantitatively assess the contribution of NHEJ and HR for DSB repair in G2, a limitation which is particularly relevant in G2 where cells have a greater propensity to undergo apoptosis than in G1 [68]. The utilisation of γH2AX foci analysis as a tool
Future perspective
Examination of the choreography of proteins recruited to DSBs has enabled DSB formation and repair to be examined in vivo with exquisite sensitivity and the defined steps in the processes to be delineated. These studies have provided insight into the interplay between the two major DSB repair pathways, NHEJ and HR. Moreover, they have exposed the significant impact that chromatin structure exerts on the DNA damage response processes. It was not so long ago that the basis underlying the dramatic
Acknowledgements
The ML laboratory is supported by the Deutsche Forschungsgemeinschaft (Lo 677/4-1/2) and the Bundesministerium für Bildung und Forschung via Forschungszentrum Karlsruhe (02S8335, 02S8355) and Forschungszentrum Jülich (03NUK001C). The PAJ laboratory is supported by the Medical Research Council, the Association for International Cancer Research, the Wellcome Research Trust and the Department of Health. We thank members of both laboratories for their contributions towards the work discussed in
References (71)
- et al.
TEL1, an S. cerevisiae homologue of the human gene mutated in ataxia telangiectasia. Is functionally related to the yeast checkpoint gene MEC1
Cell
(1995) - et al.
Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)
J. Recombination Cell
(2002) Studies on mammalian mutants defective in rejoining double-strand breaks in DNA
Mutat. Res.
(1990)- et al.
DNA damage-induced activation of ATM and ATM-dependent signaling pathways
DNA Repair (Amst.)
(2004) - et al.
A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to gamma-H2AX foci
Mol. Cell
(2004) - et al.
MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks
Cell
(2005) - et al.
Regulatory ubiquitylation in response to DNA double-strand breaks
DNA Repair (Amst.)
(2009) - et al.
53BP1 promotes ATM activity through direct interactions with the MRN complex
EMBO J.
(2010) - et al.
A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage
Curr. Biol.
(2000) - et al.
Role of Artemis in DSB repair and guarding chromosomal stability following exposure to ionizing radiation at different stages of cell cycle
Mutat Res.
(2007)
ATM signaling facilitates repair of DNA double-strand breaks associated with heterochromatin
Mol. Cell
Heterochromatin protein 1: don’t judge the book by its cover!
Curr. Opin. Genet. Dev.
Doxorubicin down-regulates Kruppel-associated box domain-associated protein 1 sumoylation that relieves its transcription repression on p21WAF1/CIP1 in breast cancer MCF-7 cells
J. Biol. Chem.
Role for KAP1 serine 824 phosphorylation and sumoylation/desumoylation switch in regulating KAP1-mediated transcriptional repression
J. Biol. Chem.
PHD domain-mediated E3 ligase activity directs intramolecular sumoylation of an adjacent bromodomain required for gene silencing
Mol. Cell
Higher-order chromatin structure in DSB induction, repair and misrepair
Mutat. Res.
Chromatin structure influences the sensitivity of DNA to gamma-radiation
Biochim. Biophys. Acta
53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks
Cell
Distinct roles of chromatin-associated proteins MDC1 and 53BP1 in mammalian double-strand break repair
Mol. Cell
ATM-dependent chromatin changes silence transcription in cis to DNA double-strand breaks
Cell
The impact of a negligent G2/M checkpoint on genomic instability and cancer induction
Nat. Rev. Cancer
Chromatin dynamics and the preservation of genetic information
Nature
Chromatin remodeling finds its place in the DNA double-strand break response
Nucleic Acids Res.
Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining
Biochem. J.
Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair
Nature
trans Autophosphorylation at DNA-dependent protein kinase's two major autophosphorylation site clusters facilitates end processing but not end joining
Mol. Cell. Biol.
DNA-PK autophosphorylation facilitates Artemis endonuclease activity
EMBO J.
Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis
Nat. Rev. Mol. Cell Biol.
Double-strand-break-induced homologous recombination in mammalian cells
Biochem. Soc. Trans.
Inverted repeats fuse to generate acentric and dicentric palindromic chromosomes by a replication template exchange mechanism
Genes Dev.
DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1
Nat. Cell Biol.
Ataxia-telangiectasia: from a rare disorder to a paradigm for cell signalling and cancer
Nat. Rev. Mol. Cell Biol.
Requirement of the MRN complex for ATM activation by DNA damage
EMBO J.
DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation
Nature
ATM and DNA-PK function redundantly to phosphorylate H2AX following exposure to ioninsing radiation
Cancer Res.
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