Review
Patching Broken DNA: Nucleosome Dynamics and the Repair of DNA Breaks

https://doi.org/10.1016/j.jmb.2015.11.021Get rights and content

Highlights

  • Repair of DNA breaks involves transient accumulation of repressive complexes.

  • Subsequent H2A.Z exchange by NuA4 promotes rapid release of repressors.

  • Shift to open chromatin at breaks requires H4 acetylation by Tip60.

  • Transition from repressive to open, acetylated chromatin required for DNA repair.

  • Dynamic changes in nucleosome organization required to prepare damaged DNA for repair.

Abstract

The ability of cells to detect and repair DNA double-strand breaks (DSBs) is dependent on reorganization of the surrounding chromatin structure by chromatin remodeling complexes. These complexes promote access to the site of DNA damage, facilitate processing of the damaged DNA and, importantly, are essential to repackage the repaired DNA. Here, we will review the chromatin remodeling steps that occur immediately after DSB production and that prepare the damaged chromatin template for processing by the DSB repair machinery. DSBs promote rapid accumulation of repressive complexes, including HP1, the NuRD complex, H2A.Z and histone methyltransferases at the DSB. This shift to a repressive chromatin organization may be important to inhibit local transcription and limit mobility of the break and to maintain the DNA ends in close contact. Subsequently, the repressive chromatin is rapidly dismantled through a mechanism involving dynamic exchange of the histone variant H2A.Z. H2A.Z removal at DSBs alters the acidic patch on the nucleosome surface, promoting acetylation of the H4 tail (by the NuA4-Tip60 complex) and shifting the chromatin to a more open structure. Further, H2A.Z removal promotes chromatin ubiquitination and recruitment of additional DSB repair proteins to the break. Modulation of the nucleosome surface and nucleosome function during DSB repair therefore plays a vital role in processing of DNA breaks. Further, the nucleosome surface may function as a central hub during DSB repair, directing specific patterns of histone modification, recruiting DNA repair proteins and modulating chromatin packing during processing of the damaged DNA template.

Introduction

DNA is constantly exposed to genotoxic agents that can modify the sugar and base residues, create DNA adducts, cross-link the DNA strands or even cleave the phosphate backbone to create single-strand breaks or double-strand breaks (DSBs). To counter these potentially mutagenic events, mammalian cells possess multiple DNA repair pathways that detect and remove modified bases or process and religate DNA strand breaks. DNA repair pathways, referred to collectively as the DNA damage response [1], function as a highly regulated signal transduction pathway that recruit specific DNA repair complexes to damaged DNA. Further, the DNA damage response is intimately linked with the activation of checkpoints at several points in the cell cycle. Checkpoint activation can temporarily block DNA replication and prevent further damage or mutagenesis that may arise when attempting replication on a damaged DNA template. In addition, the complexity of the chromatin architecture at the site of DNA damage also plays a critical role in regulating DNA repair. Chromatin organization was originally proposed to present a “barrier” to repair so that remodeling was required to gain access to the site, followed by repair and restoration of the original chromatin structure (the “access–repair–restore” model [2], [3]). However, it is now clear that chromatin organization, chromatin remodeling complexes and histone modifications are active players in DNA repair [4], [5], [6] and that dynamic changes in nucleosome organization are critical for efficient repair of DNA damage. In this review, we will focus on how nucleosome dynamics, coupled with changes in histone acetylation, work together to create open, flexible chromatin domains that are required for the processing and repair of DNA DSBs.

DSBs are lethal events that, if unrepaired, lead to chromosomal loss, translocations, genome instability and eventually cancer. DSBs can arise from many events, including collapse of replication forks or exposure to free radicals or ionizing radiation. Many anticancer therapies, including radiation therapy and chemotherapy, specifically kill cancer cells by creating DSBs. Further, many tumors contain mutations in key DSB repair proteins, such as brca1 or p53. Consequently, there is significant clinical effort devoted to unraveling the mechanism of DSB repair in normal and tumor cell lines and developing inhibitors of DNA repair that can target defective DNA repair in tumor cells.

The basic mechanism by which cells detect and repair DSBs is well defined (Fig. 1). The MRN (mre11-rad50-nbs1) complex binds directly to the DNA ends at DSBs. The MRN complex combines exonuclease and endonuclease activity (mre11), DNA binding functions (mre11) and ATPase activity (rad50) within a single complex [7]. A key function of MRN is to recruit the ATM kinase to the break site, activating ATM's kinase activity and promoting the ATM-dependent phosphorylation of proteins required for DSB repair, checkpoint activation and apoptotic responses to DNA damage [8]. In particular, ATM can phosphorylate the C-terminus of histone H2AX (termed γH2AX [9]), creating a binding site for the BRCT domain of the mdc1 platform protein [10]. Mdc1 recruits activated ATM, facilitating H2AX phosphorylation by ATM further from the break (Fig. 1). This, in turn, recruits additional mdc1 and active ATM, leading to spreading of phosphorylated H2AX for hundreds of kilobases from the break [4], [8], [9], [11]. Mdc1 also serves to recruit additional DSB repair proteins to the site of damage. These include the ubiquitin ligases RNF8 and RNF168 (Fig. 1), which promote chromatin ubiquitination and ubiquitin-dependent loading of the brca1 repair complex [11]. 53BP1, a key regulator of DSB repair, is also recruited to DSBs through a dual interaction with ubiquitinated H2AK13/K15Ub (created by RNF168) and H4K20me2 [12]. Finally, the NuA4-Tip60 remodeling complex [4], [13] exchanges H2A.Z onto nucleosomes at the break and promotes acetylation of histone H4 (Fig. 1). Further, modulation of nucleosome dynamics by NuA4-Tip60 is important for H2A ubiquitination, H4 acetylation and the loading of brca1 and 53BP1 at the DSB, and will be discussed in more detail in later sections.

Repair of DSBs can occur through two distinct mechanisms. In nonhomologous end-joining (NHEJ), the Ku70/80 DNA binding complex and several scaffold proteins are recruited to the break. Damaged bases may be removed and the DSB is then religated by DNA ligase IV [14]. Because processing during NHEJ can create short insertions/deletions, it is considered a low-fidelity repair mechanism. In contrast, homologous recombination (HR) exploits the presence of sister chromatids in late S-phase and G2, which can provide a template for DSB repair. HR requires production of ssDNA at the break, a process referred to as end-resection. Resection is initiated by the CtIP-MRN complex, which then allows the exo1 and dna2 nucleases to extend this to create 3′-ssDNA overhangs [15], [16]. The ssDNA, bound by the rad51 protein, is then used to locate homologous sequences on the sister chromatid that can serve as a template for repair. HR is therefore considered to be a high-fidelity DNA repair mechanism, although HR may still potentially create mutations [17]. Importantly, while NHEJ can occur throughout the cell cycle, HR is largely restricted to S-phase and G2-M, when adjacent sister chromatids are present to provide the template for the HR machinery.

Processing of the DNA for repair by, for example, HR requires both significant remodeling of the damaged chromatin to allow production of ssDNA and remodeling involved in homology search on the sister chromatid. In contrast, NHEJ relies on direct end-ligation of the damaged DNA and is therefore likely to involve less extensive remodeling. Nevertheless, the detection and repair of DSBs within the complex organization of the chromatin is critically dependent on chromatin remodeling to drive efficient DSB repair. Here, we will discuss the earliest events that occur during the initial detection and processing of the damaged chromatin template. In particular, we will focus on how members of the Ino80 family promote progressive changes in nucleosome packing and structure, leading to increased histone acetylation and ubiquitination and promoting the formation of open chromatin structures that facilitate DSB repair.

Section snippets

DSB repair and acetylated chromatin

Early studies demonstrated that DNA damage led to a decrease in chromatin compaction during DNA repair [18]. DSBs increase both the sensitivity of the chromatin to nuclease digestion [19], [20] and the salt solubility of histones [13]. Other studies, using microscopy approaches, indicate a rapid expansion of chromatin at sites of DNA damage [21] and, importantly, that depletion of histone H1 limits the DNA damage response whereas HDAC inhibitors, which promote more open structures, facilitate

Transitioning from Repressive to Open Chromatin

As discussed in the earlier section, DSB repair requires rapid removal of repressive proteins and a shift to a more open, acetylated structure [4]. This transition from repressive to open chromatin involves several remodeling complexes (discussed in several excellent reviews [3], [5], [45], [56], [57]). For example, RSC contributes to nucleosome sliding and ejection at DSBs [58], [59], while FUN30 can regulate resection of the DSB [60], [61]. Here, we will focus on the key role played by the

H2A.Z Turnover and DSB Repair

In addition to regulating nucleosome dynamics at DSBs, it is now clear that H2A.Z and acetylation of histone H4 are also important for promoting additional histone modifications and for the recruitment of several bromodomain proteins to the H4 tail. Here, we will examine how H2A.Z and NuA4-Tip60 (i) regulate the processing of the damaged DNA ends, (ii) direct further histone modification including ubiquitination and (iii) promote recruitment of proteins to the acetylated H4 tail at DSBs.

Conclusion

The interaction between chromatin organization and the DNA repair machinery is complex. The initial cellular response to a DSB is the rapid recruitment of repressive complexes onto the chromatin. This repressive chromatin environment is then further processed to create a more open structure associated with increased histone acetylation, loading of bromodomain proteins and specific patterns of histone ubiquitination. DNA damage therefore initiates rapid rewriting of the preexisting epigenetic

Acknowledgements

We thank members of the Price laboratory for discussion and comments. This study is supported by the National Institutes of Health grants CA64585, CA93602 and CA177884 to B.D.P.

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