Elsevier

DNA Repair

Volume 9, Issue 12, 10 December 2010, Pages 1273-1282
DNA Repair

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

https://doi.org/10.1016/j.dnarep.2010.09.013Get rights and content

Abstract

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. ATM's 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.

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

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      The ATM gene encodes a serine/threonine protein kinase that activates DNA damage checkpoint signaling in response to DSBs (Figure 1D) [24,25] and phosphorylates a variety of target proteins ranging from DNA repair and chromatin remodeling factors to cell cycle and cell death regulators [26]. For example, ATM regulates the unfolding of heterochromatin to allow accessibility of the DNA repair factors to the DSB lesion, for which it activates the chromatin compaction regulator Kruppel-associated box (KRAP)-associated protein-1 (KAP1) [27]. Helicases are essential for most functions of the DNA as the double helix needs to be unwound when it is replicated or transcribed and also during most DNA repair and recombination processes.

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