Histone and Chromatin Dynamics Facilitating DNA repair

Abstract

Our nuclear genomes are complexed with histone proteins to form nucleosomes, the repeating units of chromatin which function to package and limit unscheduled access to the genome. In response to helix-distorting DNA lesions and DNA double-strand breaks, chromatin is disassembled around the DNA lesion to facilitate DNA repair and it is reassembled after repair is complete to reestablish the epigenetic landscape and regulating access to the genome. DNA damage also triggers decondensation of the local chromatin structure, incorporation of histone variants and dramatic transient increases in chromatin mobility to facilitate the homology search during homologous recombination. Here we review the current state of knowledge of these changes in histone and chromatin dynamics in response to DNA damage, the molecular mechanisms mediating these dynamics, as well as their functional contributions to the maintenance of genome integrity to prevent human diseases including cancer.

Introduction

Eukaryotic genomes are packaged into the chromatin nucleoprotein complex within our cells. Chromatin comprises repeating arrays of nucleosomes, where each nucleosome consists of 147 bp of DNA wrapped 1.75 times around the outside of a histone octamer of two molecules each of histones H2A, H2B, H3 and H4 (1) (Fig. 1). Packaging of the genome into chromatin enables not only organization of the genetic material within our nuclei, but also enables regulation of genomic processes including transcription, DNA replication, DNA repair and recombination. The chromatin within each diploid human cell comprises approximately 30 million nucleosomes restricting free access to the genome to these processes, which has a profound influence on our biology. Chromatin-mediated regulation of these key genomic processes occurs in two main ways: 1. Chromatin facilitates recruitment of the cellular protein machinery that mediates genomic processes and 2. chromatin regulates access of this machinery to the DNA template. More specifically, chromatin mediates recruitment of the protein machinery that performs genomic processes via interactions with specific post-translational modifications on the histone proteins (discussed in the accompanying review by X et al). In this way, chromatin helps localize the replication, repair or transcription machinery to the vicinity of where it is needed in the cell. But at the same time, the presence of histones on the DNA limits access to the DNA template by the cellular protein machinery that mediates genomic processes. This provides an opportunity for the cell to utilize additional mechanisms to reveal access of specific segments of the DNA to the protein machinery that mediates genomic processes at the right time and the right place, via removing the histones from that segment of DNA, by the process of chromatin disassembly. By tightly regulating chromatin disassembly, the cell can prevent inappropriate gene expression, replication and repair from occurring, while also facilitating accurate gene expression, replication and DNA repair. Here we review the mechanisms used to disassemble chromatin during DNA repair. It is equally important to replace histones back onto the naked DNA after genomic processes are complete in order to limit unscheduled access to the genome and we will also review how chromatin is reassembled after DNA repair. We will also discuss how the local loss of histones around DNA lesions leads to chromatin decompaction, presumably to facilitate DNA repair, and at least in the case of DNA double-strand breaks, how this histone removal leads to large-scale movements of chromatin segments within the cell to enable accurate homologous recombination.

Fig 1.

Generic overview of stepwise chromatin assembly and disassembly processes to form and dismantle, respectively, nucleosomes. The magnification on the left shows the nucleosome structure determined by Karolin Luger by X-ray crystallography. The zoom out on the right indicates that nucleosomes are the repeating unit of chromatin within the nucleus.

Chromatin assembly and disassembly: the process and the players

From x-ray crystallography studies, we have a clear picture of how the histone components of the nucleosome interact with each other and contact the DNA, all mediated via non-covalent interactions (2–4) (Fig. 1). The core histones fold together as H3/H4 heterodimers and H2A/H2B heterodimers, and within the nucleosome two H3/H4 heterodimers interact to form an H3/H4 heterotetramer. Histones are rich in basic amino acids which promotes their interaction with the negatively charged DNA (5–7). One might assume, just based on the opposite charges of histones and DNA, that nucleosome assembly might be an easy task. However, in vitro, under physiological ionic strength conditions, histones are unable to bind to DNA in ordered structures. Instead they form aggregates (8). Several methods have been utilized to reconstitute nucleosomes from DNA and histones such as using 2 M salt, polyglutamic acid, polyanions and RNA (7, 9, 10). These in vitro data indicate nucleosome reconstitution requires additional negatively charged factors which can slow down the interactions between the histones and DNA, enabling them to occur in a more controlled fashion to achieve the ordered formation of the nucleosome structure. The job for assembling histones and DNA into nucleosomes in vivo has been assigned to a family of proteins called the histone chaperones or as they were termed originally “molecular chaperones” (8). Nucleoplasmin was the first histone chaperone identified to be able bind to histones and prevent them from non-specifically binding to DNA and to assemble the histones into chromatin (11) (Fig 1). There are now over a dozen histone chaperones that have since been identified that have different specificities for H3/H4 heterodimers, H3/H4 heterotetramers, H2A/H2B dimers, or histone variants and that function during different genomic processes (12). This diversity of histone chaperones underscores the importance of preventing histones from binding to other negatively charged molecules, and the cellular imperative for proper chromatin disassembly and the appropriate formation of nucleosomes. The histone chaperones that have been implicated during DNA repair are discussed throughout this review.

Most of our current understanding of chromatin assembly comes from studies following DNA replication, which revealed that histones H3/H4 are first deposited onto naked DNA as a heterotetramer, followed by the addition of two heterodimers of H2A/H2B to complete the nucleosome (13) (Fig. 1). The detailed biochemistry of how histone chaperones assemble histones onto DNA is beyond the scope of this review and is discussed elsewhere (14, 15). Chromatin disassembly is the opposite of chromatin assembly, with the initial removal of H2A/H2B heterodimers by histone chaperones, followed by removal of H3/H4 from the DNA (Fig. 1), and chromatin disassembly is even more poorly understood at the mechanistic level. More often than not, histone chaperones work along with ATP-dependent nucleosome remodelers to disassemble / assemble nucleosomes and arrange them into an ordered chromatin architecture. The assembled histones also need to be positioned properly on the DNA, which is accomplished by the ATP-dependent nucleosome remodelers via nucleosome sliding (16). In various situations, histone chaperones exchange canonical histones for histone variants that have slightly different properties, and play important roles in chromatin packaging, transcription and repair.

The challenge for DNA repair within chromatin

The cell uses specific DNA damage repair pathways, depending on the nature of the DNA lesion. The focus of this review is on histone movement and chromatin movement during nucleotide excision repair (NER) of helix-distorting lesions made by UVC irradiation and the repair of DNA double-strand breaks (DSB), as studies of chromatin dynamics during repair to date have mostly been limited to these repair pathways.

The cell needs to recognize the type of genomic lesion that is buried within the chromatin and repair it efficiently since unrepaired breaks can have detrimental consequences, including genomic instability, mutations and chromosomal abnormalities. Given that the eukaryotic genome is organized into chromatin, to truly learn the mechanism of DNA repair, one must understand how the repair machinery can gain access to DNA lesions within the chromatin. The “access-repair-restore” model of chromatin dynamics during DNA repair was proposed by Michael Smerdon after demonstrating DNA accessibility by nucleases upon UV damage of chromatin (17, 18). This model laid the foundation for the appreciation of local changes in the chromatin structure occurring around DNA damage caused by UVC irradiation. Briefly, “accessing” involves chromatin reorganization to allow the DNA repair factors to access the damaged DNA. Once the repair of the lesion is complete, the chromatin landscape around the repair site needs to be restored. Our understanding of the mechanism of the access-repair-restore model has evolved since it was first proposed, with multiple histone chaperones, histone variants and repair proteins playing their part in the process (19–21). While this model may have been postulated for chromatin dynamics during NER, it is broadly applicable to the situation during DSB repair, and potentially other types of DNA repair also.

Nucleosomes pose a barrier to DNA repair and must be dismantled in order to allow repair proteins to access the damaged DNA (22, 23). There are various types of chromatin alteration events that can occur to allow access to the damaged DNA. These include, histone disassembly, nucleosome sliding, histone post-translational modifications that can influence the state of chromatin compaction, or exchange of canonical histone proteins with histone variants that change the intrinsic stability of the nucleosomes thereby altering the DNA accessibility. The involvement of histone chaperones in the “restoration” or chromatin assembly step of DNA repair is better understood than in the “access” or chromatin disassembly step, but access is likely also mediated by histone chaperones (Fig. 1).

Chromatin disassembly and reassembly in nucleotide excision repair

NER is required for repair of a variety of DNA lesions including bulky DNA adducts introduced by oxidative damage and Ultraviolet type C radiation (UV-C) exposure which can cause distortion in the DNA helix. In brief, during NER the DNA lesion is recognized by either XPC or RNA Polymerase II. Once the lesion is recognized, TFIIH unwinds the DNA helix, followed by incision 5’ of the lesion by an endonuclease and finally gap fill-in synthesis by DNA polymerases (24–26).

Nucleosomes prevent efficient NER on reconstituted chromatin templates from human cell extracts (22). However, nucleosomes exist in a dynamic equilibrium where portions of the nucleosomal DNA spontaneously unwrap from the histones, providing transient access to the DNA. The presence of even a single UV lesion within a biochemically reconstituted mononucleosome enhances this spontaneous unwrapping of the nucleosome which enhances the efficiency of NER (27). In addition to this passive mechanism, other factors also help tip the equilibrium towards chromatin disassembly, including the local presence of DNA repair factors (13). NER efficiency on chromatin is also increased in the presence of hyperacetylated nucleosomes (28), presumably due to the more open chromatin architecture that results from acetylation of histones (29). Histones H3, H4 and H2A are also actively removed locally from the DNA after UV damage via a mechanism that involves the UV damage sensor DDB2 and depends on histone ubiquitylation by the CUL4-DDB-ROC1 complex leading to decompaction of the local chromatin structure (30–32) (Fig. 2). UV exposure of heterochromatin additionally leads to removal of H1 from the DNA in a manner that is dependent on DDB2, promoting decompaction of the heterochromatin containing the UV lesions (33). It is not clear how histone ubiquitylation ultimately lead to histone removal around sites of UV damage, but this allows DNA accessibility for the NER machinery to facilitate NER. The ubiquitylated histones do not appear to be degraded after their removal from around the UV lesions, because parental histones transiently redistribute to the chromatin adjacent to regions of localized UV lesions and DDB2-medicated chromatin opening occurs, without any significant histone degradation (34). It is quite possible that ubiquitylation of histones may help increase histone mobility or disassembly rather than their degradation. A role for histone chaperones in histone disassembly during NER has not yet been documented.

Fig 2.

Model for chromatin dynamics during Nucleotide Excision Repair: Upon UVC damage, DDB2 and associated protein ubiquitinate the histones in the vicinity of the DNA lesion, resulting in removal of parental histones from the chromatin in the vicinity of the DNA lesion, triggering chromatin opening which facilitates access for the DNA repair machinery to the DNA lesion. HIRA assembles H3.3 into the chromatin, followed by CAF-1/ASF1-mediated assembly of newly-synthesized H3.1/H4 and FACT-mediated assembly of H2A/H2B onto the repaired DNA. Parental histones also return to the repaired DNA.

Chromatin dynamics and the role of histone chaperones after UV repair is understood better than how DNA accessibility is achieved to allow repair of UV lesions. Using Xenopus oocytes and human cell extracts, the Almouzni group demonstrated that the histone chaperone chromatin assembly factor 1 (CAF-1), which is specific for newly synthesized canonical histone H3/H4, is required for chromatin assembly after NER (35). Subsequent research from this lab established that CAF-1 interacts with PCNA and another H3/H4 histone chaperone, Anti-silencing function 1 (ASF1) to promote chromatin assembly during NER, where PCNA functions to couple CAF-1 function to the DNA synthesis machinery (36). Meanwhile, ASF1 mediates transfer of histones H3/H4 to CAF-1 for chromatin assembly after NER (37). This chromatin assembly mechanism following UV repair is identical to that used for chromatin reassembly after DNA replication (38). The findings of these biochemical studies were recapitulated in cells where CAF-1 was demonstrated to localize to sites of UV lesions along with PCNA indicating that CAF-1 is directly involved in the local chromatin reassembly (39, 40). Furthermore, CAF-1 was shown to assemble newly synthesized canonical H3 (termed H3.1) into the chromatin at the sites of UV lesions (41). Subsequently, two other histone chaperone complexes, HIRA (Histone Information Regulator A) and FACT (Facilitates Chromatin Transcription) were independently shown to deposit histone H3 variant H3.3/H4 and H2A/H2B, respectively, at sites of UV lesions (42, 43). While HIRA deposits H3.3 into chromatin around UV lesions before NER is complete, this is not required for NER or transcriptional recovery (42, 44). By contrast, CAF-1 deposits new canonical H3/H4 when the NER is complete (Fig. 2) (42). Repair-coupled assembly of new histones by CAF-1, HIRA and DAXX was also observed in pericentromeric heterochromatin after completion of NER (33). After NER repair is complete, the chromatin structure recondenses, likely due to the chromatin reassembly (34) and with the help of chromatin modifiers (33).

New histones have distinct patterns of post-translational modifications as compared to the parental histones that occupied the DNA prior to UV repair. So, is the pre-damage epigenetic landscape restored after repair and if so, how? By fluorescence analyses within cells, while the parental histones were shown to redistribute to the periphery of the UV damaged region during repair, recovery of the parental histones back to the repaired region occurred after repair (34), although the mechanisms at play are unclear. Combining this information with the new histone deposition occurring after NER mediated by CAF-1, it seems that the chromatin includes a mix of new and old histones over the repaired DNA. This may be helpful in restoring the chromatin landscape after repair of UVC induced damage, as the close proximity of the old histones to the new histones may template the incorporation of the parental pattern of histone post-translational modifications onto the new histones. Alternatively, new histones being present on the chromatin over the repaired DNA could be perceived as a “wound” to mark the region as recently damaged.

Chromatin disassembly in response to DNA double-strand breaks

DNA double-strand breaks (DSBs) are considered to be the most lethal lesions as they can lead to loss of chromosome arms if unrepaired and to translocations, insertions and deletions if inaccurately repaired (45). The cell repairs DSBs by two major mechanisms, non-homologous end joining (NHEJ) and homologous recombination (HR). Briefly, during NHEJ, DNA breaks are bound by the Ku heterodimer following which the DNA-PKcs kinase is recruited and activated by interaction with Ku. The DNA ends are minimally processed, if at all, and ligated by DNA Ligase IV in complex with XRCC4. NHEJ is considered error-prone since it can introduce insertions or deletions (46). HR, on the other hand, is considered error-free since it uses the sister chromatid or homologous chromosome (in the case of meiosis) as a template for repair. HR involves 5’ to 3’ DNA end resection by the MRN, CtIP and Exo1 nucleases to generate ssDNA overhangs which are bound by the single-strand DNA binding protein replication protein A (RPA). Subsequently, RPA is replaced by Rad51 and the Rad51-ssDNA nucleofilament initiates the homology search to identify the template DNA which will be used to accurately repair the DNA break (47).

There is evidence for the presence of nucleosomes limiting the access of the DNA end resection machinery involved in HR. The movement of the DNA resection machinery is inhibited on reconstituted nucleosomal templates, although the addition of an ATP-dependent nucleosome remodeler allows the resection machinery to overcome the nucleosomal barrier in this biochemical scenario (23). In a similar manner, Scott Keeney’s lab mapped resection endpoints from meiotic DSBs in yeast and found that although rates of Exo1-mediated resection were fast, there was a tendency for resection to end at mapped nucleosome positions, indicating that although there is clearly a mechanism for histone eviction or destabilization during meiotic DSB resection, the nucleosomes can present a hurdle to the resection machinery in vivo (48). The chromatin may present a means for cells to restrict excessive resection in vivo.

Using radiation or chemical poisons to induce DSBs in vivo followed by measuring cell death or DNA repair foci persistence are popular approaches to detect defects in DNA repair in eukaryotes. While, these approaches represent more naturally occurring sources of DSBs, the major caveat is that the breaks generated are quite random and throughout the genome. This makes PCR and sequencing-based analyses a challenge. Alternatively, DSBs have been generated by the introduction of exogenous nucleases which can make site-specific breaks such as I-SceI, I-PpoI, TALE nucleases and AsiSI. The introduction of exogenous site-specific nucleases into mitotic yeast and mammalian cells has enabled the quantitative assessment of histone occupancy on the DNA around DSBs by chromatin-immunoprecipitation (ChIP) analyses (49, 50). While these nucleases provide easier capture of DNA repair kinetics, they are limited by the location of these breaks. For example, of the ~1000 sites that AsiSI has on the human genome, only ~150 can be cut efficiently. Theses exclude sites on heterochromatin as AsiSI cutting efficiency is inhibited by DNA methylation (51). For I-SceI, the target DNA sequences have to be introduced into the genome in addition to introducing the nuclease. The pros and cons of using different tools for DSB induction has been reviewed extensively elsewhere (52, 53).

In yeast, induction of the HO endonuclease creates a DSB that is repaired by HR, and this appears to be accompanied by histone removal, as indicated by the fact that histone H3 is absent from the repaired DNA upon blocking chromatin reassembly after HR (50). This suggests that histones are removed from around a DSB at some stage during HR in yeast. Earlier studies showed that nucleosome eviction and DNA end resection appear to be intrinsically coupled during processing of the HO lesion during HR in yeast (54). A recent elegant spatiotemporal analysis of chromatin changes around an induced HO lesion found the immediate eviction of one nucleosome flanking the break even before DNA end resection, and this was accompanied by repositioning of the two adjacent nucleosomes on each side of the break to locations further away from the break. Subsequently, nucleosomes were disassembled from an area of up to 8kb from the break in a manner dependent on end resection (55). The mechanism whereby histones are removed from the DNA during DNA end resection is yeast is unclear. In mammalian cells, induction of I-PpoI endonuclease generates DSBs within the rDNA which is accompanied by removal of all four core histones from the flanking DNA in cycling cells, demonstrating complete nucleosome disassembly around the DSB (49). The nucleolar factor nucleolin was required for the removal of H2A/H2B from around the I-PpoI sites within the rDNA and depletion of nucleolin also prevented removal of H3/H4, given that H2A/H2B need to be removed to allow H3/H4 removal (Fig. 1) (56). Nucleolin depletion also prevented recruitment of RPA to DSBs within the rDNA, indicating that in this scenario the DSB was normally repaired by HR (56). As such, HR in yeast is accompanied by chromatin disassembly within the regions of DNA that are end resected (Fig. 3).

Fig 3.

Chromatin disassembly and assembly during DSB repair in mammalian cells. During NHEJ the DSB triggers ATM and INO80-dependent disassembly of ~8 nucleosomes around the break, to allow NHEJ. After NHEJ is complete, HIRA, CAF-1 and ASF1 assemble histones onto repaired DNA. During HR, DNA-PK phosphorylates ASF1 which increases its interaction with CAF-1 and histones. ASF1, CAF-1 and the histone interaction in turn are required for recruitment of the RAD51 loader MMS22L/TONSL to the DSB, resulting in the replacement of RPA with RAD51 on ssDNA. TONSL recruitment is via binding to newly-synthesized histones bearing H4K20me0. After Rad51 loading, H3.3 incorporation promotes extended repair synthesis and sister exchange. By analogy to the situation in yeast, it is likely that CAF-1 and ASF1 also assemble new H3/H4 after DNA repair but this has not yet been shown in mammalian cells. Exactly when chromatin compaction occurs during / after DNA repair is unclear.

NHEJ involves minimal, if any, DNA end resection, so whether NHEJ requires histone removal from around the DSB was more of an open question. Induction of I-PpoI sites within the rDNA of mammalian cells arrested in G1 phase, to enrich for DSBs undergoing NHEJ, was accompanied by local removal of H2A/H2B but not H3/H4 around the DSB, in a manner dependent on the nucleolar factor nucleolin (56). This disassembly of H2A/H2B was required for recruitment of the NHEJ factor, XRCC4 (56). The rDNA is packaged into heterochromatin and presumably this role for nucleolin in chromatin disassembly during NHEJ is unique to the nucleolus. However, examination of several DSBs induced by I-PpoI within euchromatin demonstrated that during NHEJ, approximately 8 nucleosomes are disassembled at the site of DSBs in mammalian cells, as indicated by approximately 1500bp of DNA becoming histone H3 depleted around the DSB without detectable end resection and in a manner independent of MRN (57). This chromatin disassembly during NHEJ was largely dependent on activation of the ATM DNA damage checkpoint kinase and on the INO80 ATP-dependent nucleosome remodeler (57). The disassembly of histones from around DSBs during NHEJ appears to be essential for DNA repair, because inactivation of INO80 prevented DNA repair (57). As such, chromatin is disassembled during NHEJ to enable recruitment of the NHEJ machinery to the DNA breaks (Fig. 3).

Chromatin decondensation and histone exchange during DSB repair

Reminiscent of what happens after UV damage, DSB induction also causes a local decondensation of the chromatin structure, as detected by imaging of fluorescently labeled histones following localized microirradiation of mammalian cells (58) (Fig. 3). In the case of NHEJ, this chromatin decondensation is promoted by PARP and the ATP-dependent chromatin remodeler CHD2 (59). Exactly how CHD2 is changing the chromatin structure to decondense the chromatin is not fully understood, nor is it known whether histone removal from the DNA is involved in this chromatin decompaction during NHEJ. During HR, the chromatin decompaction around a DSB in mammalian cells is very transient and is followed by localized recompaction of chromatin, dependent on the incorporation of the histone variant macroH2A1 by the histone chaperones APLF and FACT and histone H3 lysine 9 methylation, and this local chromatin compaction is needed for the DNA damage response, DNA end resection and HR repair (60–63). It is not clear yet how histone removal from DNA around DSBs in mammalian cells correlates with chromatin decompaction after DNA damage or whether histone removal from sites of DSB damage is required for local chromatin decompaction in response to DSBs in mammalian cells, as is the case during NER. The way to address this would be to test whether chromatin decompaction depends on Nucleolin or INO80 in mammalian cells. Susan Gasser’s lab demonstrated that INO80-dependent proteasomal degradation of around 30% of budding yeast histones occurs after global DSB induction and is essential for chromatin decompaction and efficient repair by HR in yeast (Fig 4) (64). Although is it not quite clear exactly how INO80 is involved in ubiquitination and degradation of histones in yeast, given the role of INO80 in removing histones from around DSBs in mammalian cells (57), it is likely that INO80 in yeast also removes the histones from around DNA lesions in response to DSBs, which are then subsequently degraded.

Fig 4.

Histone degradation and chromatin movement during HR in yeast. In response to DSBs, histones are ubiquitinated by ubiquitin ligases and histones are evicted from the DNA in a manner dependent on the INO80 complex. These evicted histones are degraded by the proteasome. The resulting loss of histones causes chromatin decompaction which assists in a faster homology search during HR.

In addition to incorporation of the histone variant macroH2A1 to promote HR repair mentioned above, other histone variants have a role in promoting DSB repair (reviewed comprehensively in (65)). CHD2 is required for histone variant H3.3 incorporation around DSBs, which is required for NHEJ repair, but is not required for the chromatin decondensation that occurs after DSB induction (Fig. 3) (59). This is reminiscent of the early incorporation of H3.3 around sites of UV lesions during NER although that was not required for DNA repair (42). The histone chaperone involved in this H3.3 incorporation prior to NHEJ has not been identified. Exactly how H3.3 promotes NHEJ is unclear, but nucleosomes including H3.3 and the histone H2A variant H2AZ are known to be highly dynamic (66), potentially promoting access for the DNA repair machinery. Noteworthy, H2A.Z has been shown to be rapidly exchanged and removed by the Tip60-p400 ATP-dependent nucleosome remodeler and the Anp32e histone chaperone respectively which is important for early chromatin remodeling events during DSB repair (67, 68). H3.3 is also incorporated in mammalian cells arrested in G2 phase of the cell cycle during HR after the Rad51-dependent homology search, in this case the H3.3 assembly is by the ATP-dependent nucleosome remodeler ATRX working together with the histone chaperone death domain associated protein (DAXX) and PCNA, which leads to extended repair synthesis and sister chromatid exchange (69, 70). In yeast, INO80 and Fun30 have been attributed to remove histones H3 and H2A.Z from around a DSB and additionally both INO80 and Fun30 promote end resection by both Sgs1-Dna2 and Exo1 pathways for the latter. This indicates that histone eviction facilitates DNA end resection (71–74). As such, histone chaperone mediated incorporation, and sometimes also subsequent removal, of histone variants into the chromatin around DSBs plays a key role in DSB repair.

Similar to the requirement for exchange of histone variants into the chromatin flanking DSBs to promote their repair, it also appears that canonical H3/H4 incorporation occurs during DSB repair to promote repair. Specifically, ASF1 and CAF-1-mediated assembly of new H3/H4 was required for Rad51 loading by MMS22L-TONSL complex during HR in human cells (75). MMS22L-TONSL has been shown to load Rad51 on to ssDNA during replication fork stalling and during HR (76–78) and is recruited to the chromatin by unmodified histone H4K20 (H4K20me0) found uniquely in newly synthesized histones (79). This indicates that ASF1 and CAF-1 assemble newly synthesized histones during homologous recombination to allow nucleation of Rad51 on the ssDNA (Fig. 3). These findings indicate that chromatin assembly of canonical H3/H4 doesn’t only occur after DNA repair is complete, but that chromatin assembly of H3/H4 appears to occur during HR and plays a key role in the HR process per se.

While we have discussed chromatin decompaction occurring early in DNA repair and chromatin recompaction occurring later in DNA repair, another possible way to interpret the many chromatin compaction/decompaction events during DSB repair is to consider that changes may occur at multiple levels. For example, while an individual DSB may show increased compaction, the chromatin domain in which it is embedded may display reduced compaction. Large scale vs local changes, and dynamic transition between these states, is a likely outcome during repair.

Chromatin organization and movement play a key role in DSB repair

Chromatin movement within the nucleus is required to find the correct homology donor during HR. The mobility of the chromatin in yeast can be induced in response to even a single DSB and this chromatin mobility is not limited to the break site but also occurs at ectopic sites (80–82). Chromatin movement of the DSB and the ectopic sites presumably assist the cells during the homology search, but is still the rate limiting step during HR in yeast (83). Additionally, cells that are deficient in local break site mobility can still perform strand invasion, as long as ectopic chromatin mobility is maintained (81). In an elegant series of studies in budding yeast (80, 81, 84, 85), the Gasser lab showed that INO80-dependent histone degradation is required for chromatin decompaction and efficient repair by HR by promoting chromatin unfolding, chromatin relocation within the nucleus, and enhanced mobility during the homology search. This dependence on INO80 indicates that chromatin disassembly not only provides DNA access for the DNA repair machinery, but also plays a profound role in enabling increased local motion changes at damage sites and increased nuclear exploration of both the damaged and undamaged loci during HR.

DSBs have the tendency to cluster within the cell, meaning that there must be a mechanism to move chromatin segments containing DSBs within the nucleus to achieve this clustering (86) (Fig 5). This DSB clustering is ATM dependent (87) and tends to occur in G1 phase of the cell cycle (86) and preferentially at DSBs within active genes in G1 phase (88). It is possible that these DSBs cluster to prevent repair by HR in the absence of the sister chromatid, potentially by decreasing the motility of the DNA ends. This is also reminiscent of the fact that the DNA end protection factor 53BP1 (which prevents DNA end resection) localizes to mega base domains of chromatin only in G1 phase at DSBs that are normally repaired by HR in S and G2 phase (89), suggesting that 53BP1 function is to specifically block HR in G1 at DNA locations that are repaired by HR in other cell cycle phases. It would be interesting to determine the relationship between these DSB clusters, these mega base domains of 53BP1 in G1 phase cells and the accumulation of 53BP1 in liquid-liquid phase separation droplets around DSBs (90) to determine if they are one in the same.

Fig 5.

Types of chromatin dynamics during DSB repair in metazoan cells: Clustering of DSBs occurs where DSBs on different chromosomes cluster. 53BP1 accumulates over mega base lengths of DNA along with γH2A.X to form repair foci in the form of liquid droplets acting as a scaffold to recruit other repair factors. Loop extrusion in TADs and spreading of γH2A.X to mega base regions in the TAD occurs during DSB repair. DSBs in repetitive heterochromatin move to the nuclear periphery to facilitate HR with the sister chromatid, away from all the other repeats.

In higher eukaryotes, chromatin is organized into mega base-sized conserved organizational domains known as Topologically Associated Domains (TADs) (91, 92). Recent studies indicate that the TAD organization of chromatin plays a key role in formation of DNA damage response (DDR) foci, which are microscopically visible and characterized by DNA repair proteins such as 53BP1 and specific chromatin modifications including the histone H2A variant H2A.X phosphorylated (termed γH2A.X) predominantly by ATM (Fig. 5). γH2A.X can be formed by phosphorylation of H2A.X present in the vicinity of damaged DNA or it can be deposited by the FACT histone chaperone complex (93) and typically the domain of γH2A.X extends 0.5 to 2 Mb throughout the entire TAD (94). TADs are formed by cohesion-mediated extrusion of chromatin loops, which stalls at TAD boundaries due to interaction with boundary proteins including CTCF. A seminal recent study from Gaelle Legube’s lab showed that DSB induction by the AsiSI endonuclease leads to loop extrusion triggered by cohesin accumulation, which is dependent on ATM, and that ATM phosphorylates H2A.X during loop extrusion to expand the DDR focus throughout the TAD (95) (Fig 5). This work establishes how the chromatin architecture enables γH2A.X spreading throughout an entire TAD to form DDR foci, and suggests a mechanism for creating a specific repair-prone chromatin compartment which may concentrate repair factors to reduce the time taken for end joining and homology searches by constraining the repair events to within the TAD.

DNA repair within heterochromatin, which contains a lot of repetitive sequences, poses unique challenges for the repair machinery to in term of accessing the damage and accurate repair of the lesions. On one hand, heterochromatin can protect the DNA from certain types of lesions (96) while on the other hand once damaged, repair kinetics are quite slow (97). Additionally, for the HR machinery, identifying the correct repeat on the sister chromatid as the homology donor, rather than using other identical repeats at other locations on the genome for repair, which would result in translocations, insertions or deletions. Ingeniously, the cells utilize the dynamic nature of the chromatin to repair different types of lesions. For example, chromatin decompaction by removing histone H1 to allows repair of UV damage (33). Heterochromatin regions repair DSBs by HR by moving the heterochromatic DSB away from all the other DNA repeats within the heterochromatin (98) to the nuclear periphery and only then does Rad51 recruitment and completion of HR occur (Fig 5) (98, 99) and is reviewed comprehensively in (100, 101).

Chromatin reassembly after DSB repair

After DSB repair is complete it is critical to reestablish the chromatin structure over the repaired DNA. Using high resolution MNase-seq approaches in yeast cells arrested in G1, it has been demonstrated that rapid replication-independent chromatin assembly restores the chromatin architecture after completion of NHEJ repair (55). Strikingly, the nucleosomes resumed their originally occupied positions, despite the nucleosomes being repositioned and then disassembled during DNA repair (55). The mechanism of chromatin reassembly after DSB repair is similar to that which occurs after DNA replication and NER, which is not too surprising given that the chromatin assembly is coupled to DNA synthesis in all three processes. After HR in yeast, CAF-1 and ASF1 reassemble H3/H4 onto the repaired DNA (102) (50) (Fig. 3). Budding yeast cells lacking ASF1, and CAF-1 can repair DSBs but are unable to turn off the DNA damage checkpoint seemingly due to the failure to reassemble chromatin after HR, although a histone-independent mechanism for this has also been proposed (50, 103–105). Additionally, acetylation of lysine 56 of H3, mediated by the histone acetyl transferase by RTT109 on all new H3 in yeast, can override the requirement of ASF1 for chromatin assembly after HR and for turning off the DNA damage checkpoint in yeast (50), presumably because this modification increases the affinity of the histones for CAF-1 as they are passed from ASF1 to CAF-1 (106). Another yeast histone chaperone of H3/H4 Vps75, found in a complex with Rtt109, has been shown to be promote efficient NHEJ and HR, but the mechanism is unclear but may also be related to histone acetylation (107).

It has been difficult to determine which machinery is involved in chromatin reassembly after HR in mammalian cells because ASF1 and CAF-1 play a role in HR per se, as discussed above, so in their absence one cannot ask if they assemble chromatin after HR, because HR does not get completed. Following the completion of NHEJ in mammalian cells, HIRA, CAF-1 and ASF1 are all required for nucleosome reassembly occurring after NHEJ at euchromatic I-PpoI sites (57). This indicates that both the replication-dependent (involving CAF-1) and the replication-independent (involving HIRA) pathways are assembling chromatin after NHEJ in mammalian cells. It was somewhat unusual for CAF-1 to assemble chromatin during HR because PCNA promotes recruitment of CAF-1 to sites of replication, HR and NER which all involve DNA synthesis. NHEJ, by contrast, does not involve DNA synthesis nor has a requirement for PCNA, so other mechanisms must be used to recruit CAF-1 during NHEJ. In agreement, newly synthesized H3.3 and H3.1 have both been observed to be incorporated at microirradiation lines in mammalian cells (59). Once the chromatin has been reassembled, presumably the new histones will need to gain the histone modifications that were present on the parental histones prior to DNA repair, to restore gene expression patterns after DNA repair. While this has not yet been examined directly, a mouse model has been generated where the I-PpoI endonuclease was induced, cutting at over 140 sites numerous times, and the parental gene expression patterns returned in a manner independent of cell division (108). This is consistent with DNA damage not leading to epigenetic dysfunction, suggesting that mechanisms must exist to restore the epigenetic information after DNA repair. Understanding how the parental pattern of histone modifications is reestablished on the new histones deposited after DNA repair is a major unanswered question, and will likely require development of new technologies, which will then allow the mechanism of epigenetic inheritance during DNA repair to be revealed.

Conclusion and perspective

Our understanding of chromatin and histone dynamics during DNA repair greatly advanced due to the development of new technologies. Fluorescent SNAP-tagging of new and old histones has been instrumental for microscopically following histone incorporation into chromatin, their fate and mobility during DNA repair. The development of site-specific inducible endonuclease systems coupled with ChIP has enabled the detection of proteins including histones and histone modifications, at DNA lesions and during DNA repair. The 3C-based techniques have allowed the mapping of chromatin architecture during DNA repair and fluorescent labeling of chromatin regions and DNA breaks has allowed mobility of chromatin regions to be measured. Thanks to these technological developments, it is now apparent that chromatin is locally disassembled to allow access of the DNA repair machinery, and that chromatin is reassembled after DNA repair is complete. It is also clear that incorporation of histone variants and canonical histones in response to DNA damage and during DNA repair is important for DNA repair. We also now know that chromatin segments that carry DSBs move around the nucleus to either cluster together for repair or to find their correct homologous donor sequence to promote accurate repair. Most recently, we are gaining a better understanding of the formation of DDR foci. Our understanding of how chromatin influences genome stability has come a long way in the last three decades. However, many important questions remain unanswered, and new technologies may need to be developed to answer some of these questions. For example: What are the mechanisms by which histone variants promote DNA repair? Is it based on specific post-translational modifications, protein interactions or intrinsic changes to the nucleosome structure? How is epigenetic information reestablished on the new histones over the repaired DNA? How is clustering of DSBs achieved? How exactly does the Rad51 nucleofilament sample DNA sequences packaged within chromatin to find the correct region of homology? How exactly do chromatin segments with the DSB and ectopic chromatin loci move within the nucleus? Do chromatin segments return to their original nuclear location after DSB repair? And if so, how does this happen? The field awaits the answers to these, any many other questions, to reveal the true nature of maintenance of genomic integrity within our cells.

Highlights.

• We discuss how chromatin is disassembled to provide access to the DNA repair machinery

• We discuss how incorporation of canonical and variant histones plays an active role in DNA repair

• We discuss how chromatin structure is reestablished after DNA repair