Chromatin control in double strand break repair

CHAPTER THREE

Chromatin control in double strand break repair Anastas Gospodinov, Iva Ugrinova* Roumen Tsanev Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria *Corresponding author: e-mail address: [email protected]

Contents 1. DNA repair and chromatin 2. Chromatin control of double strand break repair 2.1 Mechanisms of DSB repair 3. Chromatin in the temporal control of DSB repair 3.1 DNA damage signaling 3.2 Chromatin events leading to DSB repair pathway choice 4. Spatial control of DSB repair 4.1 Control of local chromatin accessibility 4.2 Large scale re-organization of the repair process 4.3 The nucleolus as a hub involved in genome integrity maintenance 5. Concluding remarks Acknowledgments References

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Abstract DNA double strand breaks (DSB) are the most deleterious type of damage inflicted on DNA by various environmental factors and as consequences of normal cellular metabolism. The multistep nature of DSB repair and the need to assemble large protein complexes at repair sites necessitate multiple chromatin changes there. This review focuses on the key findings of how chromatin regulators exert temporal and spatial control on DSB repair. These mechanisms coordinate repair with cell cycle progression, lead to DSB repair pathway choice, provide accessibility of repair machinery to damaged sites and move the lesions to nuclear environments permissive for repair.

1. DNA repair and chromatin Usually, DNA compaction in chromatin is assumed to be an obstacle to the processes of DNA metabolism, including repair. In line with this thinking, early observations of nuclease sensitivity kinetics of newly Advances in Protein Chemistry and Structural Biology, Volume 115 ISSN 1876-1623 https://doi.org/10.1016/bs.apcsb.2018.11.003

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synthesized DNA in UV irradiated cells suggested that reversible changes of chromatin accessibility accompany repair, leading to the early “access, repair, restore” model (Smerdon & Lieberman, 1978). Accumulating data in the last 30 years have shown multiple roles of chromatin in repair processes. Indeed, as both the processes of DNA restoration and those that control chromatin structure have evolved together, histone modifications and ATP dependent chromatin remodeling events seem to be as much an intrinsic part of the repair process as lesion recognition and removal. This review examines on the roles of chromatin changes (via posttranslational histone modifications and chromatin remodeling) necessary for double strand break (DSB) repair.

2. Chromatin control of double strand break repair Double strand breaks (DSBs) are the most dangerous type of damage as they can cause large chromosomal alterations—loss of fragments or rearrangements. Both endogenous and exogenous factors inflict DSBs. Normal processes such as V(D)J recombination, yeast mating type switching and meiosis as well as replication fork collapse lead to DSBs. Exogenous causes of DSBs include ionizing radiation, crosslinking agents that stall DNA polymerases, topoisomerase poisons, etc. It is estimated that in mammalian cells
10 DSBs per cell are formed daily. If left unrepaired DSBs could lead to cell death or deregulated growth and cancer development. As any process, cellular response to DSBs needs to be controlled spatially and temporally. The latter includes mechanisms to halt cell cycle progression (DNA damage checkpoint) and to facilitate the adequate repair pathway choice depending on the cell cycle phase the afflicted cell is in (Fig. 1). The former mechanisms need to provide accessibility to the repair complexes and—as recently is becoming increasingly evident—to alter local chromatin architecture in order to move the lesion in the appropriate surrounding facilitating repair. In the text below, we will use this distinction based on outcome to describe the functional roles of chromatin changes.

2.1 Mechanisms of DSB repair DSBs are repaired via two major pathways—nonhomologous end joining (NHEJ) and homologous recombination (HR) repair, carried out by distinct sets of factors. NHEJ is active in all phases of the cell cycle and it is the predominant form of DSB repair in G1 phase in mammalian cells. Ku70 and Ku80 proteins initiate NHEJ. The heterodimer caps the free DNA ends

Fig. 1 See legend on next page.

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and physically blocks DSB ends resection (Langerak et al., 2011; Shao et al., 2012). Ku proteins recruit DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which phosphorylates itself and its target proteins (Burma & Chen, 2004). Rejoining of the broken ends is mediated by ligase IV (LeesMiller & Meek, 2003), which is associated with a dimer consisting of XRCC4 (Modesti et al., 2003) and XLF (Ahnesorg, Smith, & Jackson, 2006), which is suggested to play a bridging function (Brouwer et al., 2016). A newly discovered factor involved in NHEJ is PAXX, a protein whose tertiary structure resembles both XLF and XRCC4 and which functions by stimulating the activity of ligase IV in the ligation of non-compatible ends (Tadi et al., 2016; Xing et al., 2015). As NHEJ is not guided by a DNA template it is prone to error. Homologous recombination (HR) repair requires a homologous sister chromatid to repair the break and consequently, it is active in late S and G2 phases (Takata et al., 1998), being an error-free mechanism. The process is initiated by the MRN complex, which searches for free DNA ends by one-dimensional facilitated diffusion on nucleosome-coated DNA. MRN is able to remove Ku proteins or other DNA adducts via an Mre11-dependent nucleolytic reaction on occluded DNA ends (Myler et al., 2017). After that, Exo1 and Sgs1 (BLM helicase in mammals) generate extensive tracts of RPA-coated ssDNA (Mimitou & Symington, 2008) in a mechanism conserved from yeast to mammals (Gravel et al., 2008). In the next step of the process, RPA is later replaced by Rad51, forming a nucleoprotein filament capable of searching for, invading, Fig. 1 A simplified scheme of the temporal control of DSB repair. Following induction of a DSB (1), MRN complex binds DSB ends, leading to recruitment and activation of ATM, H2AX phosphorylation, MDC1 binding and damage signal amplification (2). ATM substrate Chk2 and Chk1 (activated in a similar way by ATR) (3) propagate the signal and induce cell cycle arrest via inactivation of Cdc25 phosphatases or via p53 transcriptional induction of CDK inhibitors, thus putting a break on cyclin-dependent kinases and the cell cycle (4). The repair choice is determined by the antagonistic relationship of BRCA1 and 53BP1 (marked in red), both of which bind ubiquitinate histones (5). 53BP1 channels repair to non-homologous end joining (NHEJ) pathway (by inhibiting resection via shieldin binding). NHEJ is initiated by recruitment to the lesion of Ku proteins, which bind and activate DNA-PKcs kinase activity (6). Following limited processing of the ends by the Artemis nuclease, the rejoining reaction is carried out by DNA ligase IV complexed with XRCC4 and XLF (7) to seal the break (8). In S and G2 when a homologous template is available, resection of DSB ends (9) initiates homologous recombination repair. RPA on single-stranded DNA (9) is replaced by Rad51 (10) in an exchange reaction dependent on Rad51 paralogs and Rad52. Rad51 nucleoprotein filament searches for and invades the homologous duplex and following extension of the invading strands and Holiday junction resolution the broken DNA gets restored (11).

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and transferring strands with a homologous duplex. Formation of the Rad51 nucleoprotein filament is enhanced by interactions with Rad51 paralogs (Paques & Haber, 1999; Rodrigue et al., 2006; Thompson & Schild, 2001) and Rad52 (Benson, Baumann, & West, 1998; New et al., 1998; Shinohara & Ogawa, 1998). Invasion into the duplex donor is ATP dependent (Baumann, Benson, & West, 1996; Stark et al., 2002; Sung, 1994) and it is stimulated by Rad54 (Petukhova, Stratton, & Sung, 1998; Sigurdsson et al., 2002). The invading strand is extended by DNA polymerases and branch migration leads to restoration of the genetic information spanning the break (Paques & Haber, 1999; West, 2003). Initiation of resection of DNA ends establishes the DSB repair pathway choice by promoting HR and preventing NHEJ.

3. Chromatin in the temporal control of DSB repair 3.1 DNA damage signaling As repair takes time, it is essential to block cell cycle progression and thus prevent events that might aggravate the adverse effects of DNA damage. Mammalian cells possess three DNA damage checkpoints: G1/S, intra-S and G2/M (Sancar et al., 2004) to halt the cell cycle. Checkpoint pathways consist of DNA damage sensors, signal transducers and effectors that prevent the occurrence of a cell cycle event before a certain condition is met. Three kinases of the phosphatidylinositol-3-kinase-like kinase (PIKK) family, DNA-PK, ATR and especially ATM (ataxia-telangiectasia mutated), play key roles in DNA damage signaling. The activation of ATM is associated with the structural changes in chromatin that occur upon DSB generation (Lavin & Kozlov, 2007; Shiloh, 2006). DNA-PK can function redundantly to ATM, but only in the absence of the latter (Stiff et al., 2004). During S-phase, ATR is recruited to RPA-coated single-stranded DNA (ssDNA) formed at sites of blocked replicative polymerases via its interaction partner ATRIP (ATR-interacting protein) (Zou & Elledge, 2003). The activated PIKK kinases propagate the signal by phosphorylating downstream effector kinases: checkpoint kinases 1 and 2 (Chk1 and Chk2). During G1, ATM and Chk2 are required to stabilize p53. This results in the induction of various transcriptional targets, including the Cdk inhibitor p21, which binds and inhibits cyclin–Cdk complexes blocking the cell cycle (Deng et al., 1995; Harper et al., 1993; Kastan et al., 1991). In S-phase, Chk1 and 2 activate the Wee1 kinase (Chow et al., 2003; Watanabe, Broome, & Hunter, 1995), as well as target cell cycle progression via inactivation of Cdc25 phosphatases, which are critical regulators of CDK activity (Donzelli & Draetta,

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2003; Falck et al., 2001). In G2 ATM and Chk2 establish cell cycle arrest after damage (Rainey et al., 2008) but ATR-Chk1-signaling is required for checkpoint maintenance in G2 (Brown & Baltimore, 2003; Shibata et al., 2010). The activation of the main DSB checkpoint kinase ATM critically depends on several chromatin factors. A major regulator of ATM is Tip60 histone acetyltransferase. In response to damage, Tip60 gets phosphorylated and binds to the pre-existing H3K9me3 histone mark (Xu, Xu, & Price, 2012). This triggers Tip60-mediated acetylation of the ATM kinase, promoting DNAdamage-checkpoint activation and cell survival (Kaidi & Jackson, 2013; Sun et al., 2005). The ability of H3K9me3 at DSBs to regulate the activity of Tip60 and the subsequent activation of ATM emphasizes the crucial role played by chromatin architecture in regulating DSB repair (Xu, Xu, et al., 2012). In a similar manner, the nucleosome-binding protein HMGN1 modulates the interaction of ATM with chromatin. HMGN1 depletion reduced the levels of ionizing radiation (IR)-induced ATM autophosphorylation and the activation of several ATM targets. These effects depended on a global damage-induced HMGN1-dependent increase in H3K14 acetylation, suggesting that by regulating the levels of histone modifications, HMGN1 affects ATM activation (Kim et al., 2009). Efficient activation of ATM in response to ionizing radiation was also shown to depend on histone H4K16 acetylation, carried out by the histone acetyltransferase MOF (Gupta et al., 2005). The key chromatin target of PIKK kinases is histone variant H2AX. It is central in coordinating the DNA damage response on chromatin (see below). In addition, phosphorylation of H2AX, along with that of checkpoint kinases themselves (Goodarzi et al., 2004; Shreeram et al., 2006), is involved in temporal regulation of the checkpoint response. Phosphorylated H2AX is targeted by several phosphatases (Chowdhury et al., 2005, 2008; Douglas et al., 2010) as well as chromatin remodeling (Heo et al., 2008) to restore the unphosphorylated H2AX when DNA damage signaling is switched off.

3.2 Chromatin events leading to DSB repair pathway choice The DSB repair pathway choice depends on multiple factors, including the cell cycle phase, local transcription and the types of DNA ends (Aymard et al., 2014; Symington & Gautier, 2011). 50 -30 Resection of DSB ends leads the process toward HR repair, while inhibition of resection favors NHEJ. It has been recognized that an antagonistic relationship between 53BP1 and BRCA1 determines pathway choice (Bouwman et al., 2010;

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Bunting et al., 2010) with 53BP1 (being phosphorylated on multiple sites to interact with RIF1) inhibiting resection (Escribano-Diaz et al., 2013; Zimmermann et al., 2013). Both 53BP1 and Brca1 form foci at the site of damage. High-resolution imaging has been demonstrated that 53BP1 focal enrichment is most prominent in the G0/G1 cell cycle phases and as cells transition through S phase, the recruitment of BRCA1 into the core of IRIF is associated with an exclusion of 53BP1 to the focal periphery, resulting in an overall reduction of 53BP1 occupancy at DNA damage sites (Chapman et al., 2012). RPA foci—indicative of resection—are formed following 53BP1 repositioning (Kakarougkas et al., 2013). This suggests that the major role of BRCA1 is to overcome the barrier against DNA end resection established by 53BP1, while 53BP1 may serve to limit the extent of resection (Shibata, 2017). Recently, several groups reported a 53BP1 effector complex, named shieldin, consisting of C20orf196 (SHLD1), FAM35A (SHLD2), CTC534A2.2 (SHLD3) and REV7. Shieldin complex localizes to DSB sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains analogous to those of RPA1. Loss of shieldin impairs NHEJ and causes hyper-resection. Binding of singlestranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends (Dev et al., 2018; Ghezraoui et al., 2018; Gupta et al., 2018; Mirman et al., 2018; Noordermeer et al., 2018). A complex similar to RPA called CST (CTC1-STN1-TEN1) has been shown to interact with shieldin and to localize with Polα to sites of DNA damage in shieldin-dependent manner. It has been shown that CSTPolα-mediated fill-in counteracts DSB resection (Mirman et al., 2018). Multiple chromatin-associated events are linked with DSB repair pathway choice. Importantly, chromatin recruitment of both 53BP1 and Brca1 is a consequence of a cascade of these. As already mentioned, histone variant H2AX is a key substrate of the DDR kinases and it delineates the chromatin domains changed during the DNA damage response. Phosphorylation of gH2AX on serine 139 by ATM (Burma et al., 2001) or DNA-PK kinases (Stiff et al., 2004; Ward & Chen, 2001) in response to DSBs or ATR in response to replication-stalling lesions (Hanasoge & Ljungman, 2007; Marti et al., 2006) is one of the earliest events following damage. Phosphorylated H2AX spreads to several megabases around the DSB. The spread of its phosphorylation is attributed to a mechanism that involves the MDC1 protein. MDC1 stabilizes the interaction of NBS1 component of MRN with chromatin at the DSB (Lukas et al., 2004), and the MRN complex recruits

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more ATM (Falck, Coates, & Jackson, 2005; You et al., 2005). This induces further H2AX phosphorylation in a positive feed-back loop that marks the region confining the DDR (Kinner et al., 2008; Soria, Polo, & Almouzni, 2012). A similar interaction with TopBP1 anchors the ATR kinase to chromatin at lesions that stall polymerases and favor generation of ssDNA (Wang, Gong, & Chen, 2011). MDC1 protein serves as an interaction platform for other DDR components. Recruitment of the RNF8 (an E3 ubiquitin ligase) by MDC1 (Mailand et al., 2007) initiates a series of ubiquitylation events needed to bring about BRCA1 and 53BP1 recruitment. RNF8 cooperates with UBC13 E2 enzyme (the interaction being facilitated by HERC2; Bekker-Jensen et al., 2010; Plans et al., 2006) to catalyze the formation of K63-linked polyubiquitin chains (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007) initially thought to target H2A histones, but now shown to be mainly of H1 linker histones (Thorslund et al., 2015). This ubiquitylation event serves as a recruitment signal for RNF168, which in turn ubiquitylates H2A-type histones at K13/K15 in a DSB-dependent manner (Doil et al., 2009; Fradet-Turcotte et al., 2013; Mattiroli et al., 2012). The key outcome of the RNF8–RNF168 pathway is the chromatin retention of 53BP1 and BRCA1 at chromatin areas near DSBs. Chromatin retention of 53BP1 requires histone methylation in addition to ubiquitylation. The two methylated lysines associated with the retention of 53BP1 to DSB-flanking chromatin are H3K79me and H4K20me (Huyen et al., 2004; Sanders et al., 2004; Schotta et al., 2008). Both H3K79 methylation and H4K20me2 do not change in response to DNA damage and it has been suggested that chromatin changes in response to damage lead to them getting exposed to mediate 53BP1 binding. However, recent results indicate that H4K20 methylation increases locally upon induction of DSBs and that it is mediated by the MMSET methyltransferase in mammals. Downregulation of MMSET significantly decreases H4K20 methylation and subsequent accumulation of 53BP1 at DSBs (Pei et al., 2011). Chromatin remodeling also appears to be an important factor promoting resection and HR repair (Costelloe et al., 2012; Gospodinov et al., 2011; van Attikum, Fritsch, & Gasser, 2007). The nucleosome remodeler SMARCAD1, which is recruited onto chromatin in a BRCA1/BARDdependent manner, promotes resection (Costelloe et al., 2012). We have found that mammalian INO80 protein associates with chromatin within 10 kb of a defined break site and its knock-down results in deficient HR repair, due to compromised for 50 -30 resection of DSB ends (Gospodinov et al., 2011). Another remodeler of the same family SRCAP has been

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reported to facilitate resection by promoting the chromatin recruitment of CtIP (Dong et al., 2014). Having in mind the recency of the findings regarding the mechanisms governing DSB repair pathway choice, it is likely that many more chromatin interactions controlling the process will soon be found.

4. Spatial control of DSB repair 4.1 Control of local chromatin accessibility Access of repair factors to DNA lesions is an obvious requirement for repair to occur. There are two ways to modify chromatin structure in order to modulate accessibility—via the posttranslational modifications (PTMs) of histones and by ATP-dependent chromatin remodeling (Fig. 2, accessibility). Histone acetylation is a key modulator of chromatin accessibility. Historically, it was the first to be recognized as a way to provide accessibility for nucleotide excision repair, when it was found that total protein acetylation increases in response to UV irradiation (Ramanathan & Smerdon, 1986) and that repair DNA synthesis is enhanced in hyperacetylated nucleosomes (Ramanathan & Smerdon, 1989). Histone acetylation is essential for efficient repair of DSBs as well. Acetylation of the positively charged lysine and arginine residues of N-terminal histone tails weakens their interaction with the negatively charged DNA backbone. Early yeast studies showed that a deletion of histone H3 N-terminus impairs DSB repair. The enzyme that catalyzes acetylation of N-tail lysines of histone H3—GCN5—was found to be recruited to DSBs and GCN5 deletion mutants lost viability following induction of a single DSB (Tamburini & Tyler, 2005). Similarly, mutation of any of the four N-terminal lysines of histone H4 that could be acetylated abolished repair of both DSB and UV lesions and the defect was linked to the NuA4 acetylase complex (Bird et al., 2002). The mammalian homolog of yeast NuA4, TRRAP-Tip60 complex is required for DSB repair, as well. Ectopic expression of Tip60, lacking acetyltransferase activity, led to defects in DSB and apoptosis (Ikura et al., 2000). In a similar way, depletion of TRRAP-Tip60 subunits TRRAP, as well as RUVBL1 and 2, resulted in HR repair defects that could be overcome by forced chromatin relaxation (Gospodinov, Tsaneva, & Anachkova, 2009; Murr et al., 2006), indicating that the complex controls chromatin accessibility. It was found that Tip60 is recruited to sites of DSB in a TRRAP-dependent manner (Murr et al., 2006) and that hyperacetylation of histone H4 is required for the loading of a subset of repair proteins, including 53BP1, Rad51 and BRCA1 (Murr et al., 2006).

Fig. 2 Major processes in the spatial control of DSB repair. After DSB formation chromatin (1) in the vicinity of the lesion rapidly expands (2), a process dependent on ATP and therefore likely carried out by chromatin remodeler(s). A major determinant of chromatin accessibility is histone acetylation, which changes with time during repair (3) due to the action of various chromatin regulators, including histone deacetylases 1 and 2 and TRRAP-Tip60 acetyltransferase complex. Multiple other chromatin factors modulate chromatin accessibility during specific steps of repair (4). While in yeast damaged locus is highly mobile, in higher eukaryotes it appears to be much more stable. Still, available data suggest that due to increased mobility of the damaged locus, heterochromatic DSBs (5) move outside of the heterochromatin domain (6), and in Drosophila gets translocated to the nuclear periphery, likely to get to a repair-permissive environment (7). In a similar manner, lesions in the nucleolus (8) move to the outside of the organelle (9).

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Histone acetylation by p300 and CBR stimulates NHEJ. Ablation of the acetyltransferases sensitized cells to ionizing radiation and DSB-inducing drug etoposide and suppressed acetylation of lysine 18 within histone H3, and lysines 5, 8, 12, and 16 within histone H4 at the DSBs. This led suppressed recruitment of Ku proteins (Ogiwara et al., 2011) to the DSB sites. Both HR and NHEJ are stimulated by yet another histone acetyltransferase— MOF (Sharma et al., 2010). Its activity increases H4K16 acetylation in response to DSBs (Li et al., 2010; Miller et al., 2010). Much like the effect of TRRAP/Tip60, deletion of MOF impairs recruitment of BRCA1 and 53BP1 (Li et al., 2010; Sharma et al., 2010). In addition to local changes, a recent study showed that H3K14 is acetylated globally following induction of DSBs in an HMGN1-dependent manner. This mark is needed for ATM binding to damaged chromatin suggesting that H3K14 acetylation participates in the regulation of ATM activation (Kim et al., 2009). The acetylation level of chromatin around DSBs, however, both increases and decreases during the course of DSB repair, suggesting that the relationship between acetylation of histones and repair seems to be more complex than a model in which more acetylation equals better accessibility equals more efficient repair. Tamburini and Tyler first reported that the acetylation status of histone H3 and H4 lysines vary during homologous recombination repair and that histone deacetylases (HDACs) are recruited to a site-specific DSB during the course of HR repair (Tamburini & Tyler, 2005). Mammalian HDAC1 and HDAC2 get recruited to DSBs, deacetylate H3K56 and H4K16 and their depletion suppresses NHEJ (Miller et al., 2010). The authors suggested that chromatin compaction may be required to keep Ku proteins concentrated at the DSB ends and prevent them from sliding away (which they do on naked DNA) (Miller et al., 2010). Another way to modulate local accessibility is via ATP-dependent chromatin remodeling. ATP-dependent chromatin remodelers function by weakening the histone-DNA interactions using ATP hydrolysis and slide or evict individual nucleosomes or catalyzes histone variant exchange. In an elegant study in living cells, Kruhlak et al. found that seconds after break introduction chromatin undergoes an energy-dependent local expansion. The change corresponded to a 30–40% reduction in the density of chromatin fibers in the vicinity of the DSB (Kruhlak et al., 2006). The authors proposed a model (Kruhlak, Celeste, & Nussenzweig, 2006) in which the DSB induction causes chromatin decondensation by ATP dependent chromatin remodeling proteins. This facilitates the subsequent recruitment of DNA

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damage checkpoint and repair proteins. In line with this thinking a number of ATP-dependent chromatin remodelers have been shown to participate in DSB repair, mostly at its early stages. SWR1 remodeler participates in yeast NHEJ by facilitating recruitment of the Ku proteins (van Attikum et al., 2007). Early events in mammalian NHEJ depend on SWI/SNF chromatin remodeling. The BRM1 subunit of the SWI/SNF complex is required for efficient NHEJ by facilitating Ku70 recruitment (Ogiwara et al., 2011). Knock-down of this protein or the BRG1 subunit of SWI/SNF impairs H2AX phosphorylation and sensitizes cells to DSB-inducing agents (Lee et al., 2010). Both yeast and mammalian INO80 complexes are recruited to DSBs and strains deficient in their components are less effective in the initial 50 -30 resection at DSB ends (Gospodinov et al., 2011; van Attikum et al., 2004). The H2AZ exchange at DSBs by p400 ATPase has been found to shift chromatin to an open conformation facilitating acetylation and ubiquitination and Brca1 loading (Xu et al., 2012).

4.2 Large scale re-organization of the repair process Major nuclear processes such as replication and transcription take place in spatially limited compartments that are thought to increase efficiency by concentrating factors and templates together (Misteli, 2007). Similarly repair of DSB breaks is organized in repair foci that can be easily visualized by immunofluorescence or GFP-tagged repair proteins. Existing results indicate that multiple DSB get recruited to a single repair center. This is evidenced by the observation that if at a low dose of irradiation 2–4 repair foci appear, higher doses do not lead to a proportional increase of foci relative to the number of DNA breaks (Lisby, Mortensen, & Rothstein, 2003; Lisby & Rothstein, 2004). Thus, DSB repair process is both spatially confined and damaged chromatin is able to migrate to these repair compartments. Clearly, a factor in spatial organization of repair are the sequential chromatin changes that accompany it and provide binding interfaces to retain repair factors in the proximity of the lesion (Kobayashi, 2004; Stucki & Jackson, 2006; Toh et al., 2006). Recent studies suggest that phase separation events could help in the formation of membrane-free compartments of the repair foci. It has been shown that poly(ADP-ribose) (PAR) nucleates intracellular phase separation (aka “liquid demixing”) and PAR levels are markedly induced at sites of DNA damage. This triggers rapid, yet transient and fully reversible assembly of various intrinsically disordered proteins at DNA break sites

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(Altmeyer et al., 2015). It has been suggested that one of the roles of histone H2AX phosphorylation, taking megabase-sized domains in mammalian cells, has been to promote phase separation and compartmentalization in repair foci. Since electrostatic interactions drive phases separation events extensive accumulation of negative charges due to H2AX phosphorylation could contribute to compartmentalization (Altmeyer et al., 2015; Marnef & Legube, 2017; Mitrea & Kriwacki, 2016). The issue of formation of a specific repair-permissive subspace in the nucleus is further complicated by the ability of damaged chromatin to move and relocalize to specific nuclear structures. Movement of breaks requires that chromatin is relatively mobile and indeed it has been shown that a tagged domain in the yeast nucleus is capable to travel <0.5 μm in about 10 s (of a total nuclear diameter of about 2 μm) (Gasser, 2002). Direct evidence for long range motion of broken chromosomes has been found by the visualization of two independent DSBs that coalescenced into common repair focus (Lisby et al., 2003). It is now well accepted that in yeast, DSB induction increases both the mobility of the chromatin around the DSB, as well as of undamaged chromatin (Mine-Hattab & Rothstein, 2012; Seeber, Dion, & Gasser, 2013) and this depends on Mec1 (ATR) checkpoint kinase and INO80 remodeler as direct effector (Neumann et al., 2012; Seeber et al., 2013). The mobility of damaged chromatin in higher eukaryotes is however rather controversial. Thus, it has been reported that broken chromosome ends decorated by fluorescently tagged markers are essentially immobile in nuclear space (Soutoglou et al., 2007). Similarly, after introduction of multiple lesions by charged particles, and following fluorescently tagged 53BP, no long-range displacements of damaged chromatin domains were observed ( Jakob et al., 2009). In contrast, analysis of mobility of chromatin domains containing DSBs (using again labeled 53BP1) found them to be substantially more mobile than intact chromatin, and are capable of roaming a more than twofold larger area of the cell nucleus (Krawczyk et al., 2012). A possible source of these discrepancies could the use of different damaging agents likely causing damage in different chromatin contexts (Cho et al., 2014). In favor of increased mobility of DSBs are also reports that have found increased mobility of deprotected telomeres as well as of distant sites on the same chromosome during V(D)J recombination (Difilippantonio et al., 2008; Dimitrova et al., 2008). There are multiple hypotheses of the role of increased mobility of DSB containing chromatin—to promote homology search, to provide clustering of multiple damaged regions into centers facilitating repair or ones that

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sequester the breaks into structures that prevent irregular exchanges and chromosomal rearrangements (Marnef & Legube, 2017). All of these implicitly assume migration to nuclear subspaces that are more conductive to these functions. In yeast, real time imaging and immunoprecipitation showed migration of DSBs to the nuclear periphery, where they associated with the nuclear pore complex components and other proteins such as Mps3 (monopolar spindle) (Nagai et al., 2008; Oza et al., 2009). However, it seems that only persistent DSBs are relocalized within 1–2 h to the nuclear periphery. The relocalization process requires the Rad51 recombinase, DSB resection and the ssDNA binding protein Cdc13 (Kalocsay, Hiller, & Jentsch, 2009; Oza et al., 2009). DSBs that are rapidly repaired and fail to induce robust checkpoint activation do not localize to the nuclear periphery (Kalocsay et al., 2009; Nagai et al., 2008). While nuclear periphery repositioning of DSBs has not been observed in mammalian cells, DSBs in heterochromatic and ribosomal regions that are hard to repair move toward the border of their initial environment (Fig. 2, chromatin mobility). Thus, DSBs in human rDNA induced by either the I-PpoI endonuclease or CRISPR/Cas9 relocalize to nucleolar periphery and undergo HR irrespective of the cell cycle stage (van Sluis & McStay, 2015). In mouse cells it was found that while in G1 pericentric heterochromatin DSBs are positionally stable and recruit Ku80, in S/ G2 they relocate to the periphery of heterochromatin, which requires DSB ends resection (Tsouroula et al., 2016). Work in Drosophila found striking dynamic behaviors of heterochromatic DSBs that are repaired specifically by HR repair. These DSBs associate with the nuclear periphery, moving to nuclear pores or to inner nuclear membrane proteins before recruiting Rad51 and continuing repair (Chiolo et al., 2011). Within minutes, phosphorylated H2Av, TopBP1, and ATRIP foci are assembled in heterochromatin, but subsequent repair steps are temporarily halted (Chiolo et al., 2011; Janssen et al., 2016; Ryu et al., 2015; Ryu, Bonner, & Chiolo, 2016). Resection triggers a global expansion of the heterochromatin domain (a distinct structure in Drosophila cells) and relocalization of DSBs to the nuclear periphery, where repair progresses (Chiolo et al., 2011; Dronamraju & Mason, 2011; Janssen et al., 2016; Ryu et al., 2015). Relocalization is dependent on SUMO ligases dPIAS, Quijote and Cervantes, revealing a double function for SUMOylation in the process: blocking HR in the heterochromatin domain and relocalizing DSBs (Ryu et al., 2016). Foci of late repair proteins form outside of the HP1 domains. Rad51 foci only form at the HP1 domains periphery (where HP1 is absent) only after the relocalization is complete.

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The movement is crucially dependent on checkpoint kinases and it apparently mimics the relocalization of yeast DSBs to the nuclear periphery that is also dependent on robust checkpoint activation. The results also suggest that initial processing and Rad51 mediated recombination are not only temporally but also spatially separated and provide an excellent example of the structural complexities of the DSB repair process (Chiolo et al., 2011). Similar migration has been described in mouse cells in which DSBcontaining chromatin leaves heterochromatic “chromocenters” to complete repair (Chiolo et al., 2013; Jakob et al., 2011; Tsouroula et al., 2016). These data indicate that while chromatin in higher eukaryotes is more constrained compared to yeast, it still needs to move locally to complete repair.

4.3 The nucleolus as a hub involved in genome integrity maintenance The nucleolus is a membrane-less organelle—the most prominent subnuclear structure where rRNA is transcribed, processed and ribosomal assembly takes place. The nucleolus is considered to be one of the most unstable regions in the genome (Kobayashi, 2008), since the exceptionally high transcription rates in the nucleolus facilitate the accumulation of RNA:DNA hybrids (R-loops), which impede replication and promote replication-stress induced DSBs. The presence of tandem arrays of rDNA makes it prone to unscheduled DNA recombination events. Deregulated rRNA synthesis has been associated with disease onset. During interphase, each nucleolus is typically surrounded by heterochromatin, which seems to be important not only for nucleolar structure maintenance, but also for safeguarding genomic stability, via the reduced accessibility to genotoxic byproducts of metabolism, and cellular DNA recombination machinery (Tsekrekou, Stratigi, & Chatzinikolaou, 2017). In mammalian cells, rDNA breaks cause nucleolar caps—structures at the nucleolar periphery that contain damaged DNA, that (van Sluis & McStay, 2015) similar to DSBs in other parts of the genome relocalize outside of heterochromatin (nucleolus) and cluster together (Aten et al., 2004). Induction of DNA breaks via DNA damage response kinases, ATM and DNA-PK, inhibits RNA Pol I to cease transcription, processing and ribosomal assembly (Calkins, Iglehart, & Lazaro, 2013). Numerous nucleolar proteins have been shown to participate in the regulation of various processes including cell cycle arrest, arrest of DNA replication, induction of DNA damage repair and apoptosis in addition to their nucleolar role. The highly abundant protein nucleolin (NCL) is one such

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factor that translocates to the nucleoplasm in response to stress, and participates directly in the repair of numerous different DNA lesions. Nucleolin was initially described as a nucleolar protein participating in ribosome biogenesis (Srivastava & Pollard, 1999). Subsequent studies have shown that nucleolin is a multifunctional protein involved in several cellular processes such as transcription, attachment of genomic DNA to nuclear matrix and decondensation of chromatin (Ginisty et al., 1999; Mongelard & Bouvet, 2007; Storck et al., 2007). NCL is a predominantly nucleolar protein under non-stress conditions, though minor but biologically distinct populations have been observed in the nucleoplasm, cytoplasm and at the cell surface in certain cell types/tissues (Borer et al., 1989; Hovanessian et al., 2000). Nucleolin is a highly abundant 77 kDa phosphoprotein, the most abundant nucleolar protein in mammals and the major silver-binding component of argyrophilic nucleolar organizing regions (AgNORs) (Bugler et al., 1982; Orrick, Olson, & Busch, 1973). NCL is conserved throughout eukaryotes, with homologs observed in plants, animals and yeast (Saccharomyces cerevisiae Nsr1p) (Tajrishi, Tuteja, & Tuteja, 2011). It consists of an N-terminal domain containing highly acidic regions, four RNA-binding domains, and a C-terminal RGG domain that is rich in arginine, glycine, and phenylalanine and was shown to interact with ribosomal proteins (Bouvet et al., 1998; Ginisty et al., 1999). NCL exhibits rapid, reversible and post-translationally— regulated nucleoplasmic translocation in response to exposure to genotoxic stress. NCL is able to act as a histone chaperone through its central acidic tracts, thereby supporting chromatin remodeling by the SWI/SNF and ACF complexes in a manner similar to that of NPM1 (nucleophosmin). The protein interacts specifically with the H2A-H2B histone dimer supporting its eviction from histone octamers, an important process for DNA transcription, replication and repair (Angelov et al., 2006; Kobayashi et al., 2012). The observation that NCL undergoes robust yet reversible nucleoplasmic translocation in response to stimuli such as IR, camptothecin and etoposide treatment—each of which induces DSB formation—suggests that NCL may participate in the cellular response to DSB damage (Daniely, Dimitrova, & Borowiec, 2002; Kobayashi et al., 2012). Indeed, several groups have observed that in IR- or camptothecin-treated cells NCL forms nucleoplasmic foci that colocalize with sites of DSB repair, coinciding temporally with the formation of DSBs (Kobayashi et al., 2012), and returns to the nucleolus upon their clearance (Goldstein et al., 2013; Kobayashi et al., 2012), supporting the conclusion that NCL plays a direct role in the repair and clearance of DSBs. Recent studies have begun to provide detailed insights into the potential role of NCL at DSBs

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in vivo. Initial steps in the cellular response to DSBs involve an early recruitment of the MRE11-NBS1-RAD50 (MRN) complex to the DSB site and activation and recruitment of the ataxia telangiectasia mutated (ATM) kinase resulting in the activation of multiple signaling pathways (Bakkenist & Kastan, 2003; Ciccia & Elledge, 2010). Interestingly, depletion of NCL, either by antibody microinjection or siRNA knockdown, did not inhibit recruitment of early DSB repair factors such as the Mre11-Rad50-Nbs1 (MRN) complex or γH2AX, but did cause a failure to repair DSBs by both HR and NHEJ (Goldstein et al., 2013; Kobayashi et al., 2012). Using a ChIP-based assay at a site-specific, NHEJ-targeted DSB, Goldstein et al. (2013) demonstrated that NCL is recruited to sites of DSB repair through an interaction of the RGG domain of nucleolin with the RAD50 subunit of the MRN complex, and that while this recruitment has no effect on the loading of DSB repair factors such as γH2AX, ATM, MDC1, RNF8 and RNF168, it did coincide with the rapid depletion of H2A-H2B, but not H3-H4, dimers within a few kilobases of the DSB site. Kobayashi and colleagues similarly observed reduced H2A-H2B mobilization from chromatin to the nucleoplasm upon depletion of NCL (Kobayashi et al., 2012). Collectively, these data argue for a model in which NCL recruitment to DSBs via the MRN complex facilitates the proximal nucleosome destabilization by chromatin remodeling complexes, possibly through the actions of MDC1 and its targets RNF8/RNF168, and ultimately facilitates NHEJ- and HR-mediated DSB resolution (Goldstein et al., 2013). Another group has investigated the relationship of nucleolin expression with the radiosensitivity of human nonsmall cell lung cancer (NSCLC) cells (Xu et al., 2015). Interesting the authors found out that the expression of NCL is negatively correlated with the radiosensitivity of NSCLC cell lines through the promotion of radiation-induced apoptosis and adjusting the cell cycle to a more radiosensitive stage together with an increase in quantity of γH2AX foci and decreasing radiation-induced DNA damage repair. Furthermore, it was shown that the expression of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) decreased rapidly after irradiation in NCL knockdown cells. As the activity of DNAPKcs phosphorylation sites at the S2056 and T2609 was found significantly suppressed authors concluded that NCL knockdown can inhibit DNA-PKcs phosphorylation activity at these sites, thus reducing the radiation damage repair and increasing the radiosensitivity of NSCLC cells. In a recently published seminal work of Aleksandrov et al. (2018) authors studied the dynamics of repair proteins at complex DNA damage sites and found that NCL belongs to so called first wave removals—proteins with very short half-times of

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removal from the damage site (between 24.7 and 110.5 s). This group also includes PARP1, mPARP2, and mainly PAR-dependent proteins (XRCC1, FUS, mPARG, ADPRHL2) which is consistent with the role of PARP1 dissociation and PAR hydrolysis by PARG and ADPRHL2 in the removal of these proteins (Gibson & Kraus, 2012). Surprisingly NCL is a member of this group, suggesting that it could be PAR dependent as well. In conclusion remarkably, regulatory roles have been found for NCL in the processes of NHEJ and HR (Scott & Oeffinger, 2016). It is important to stress that the roles described above are mediated by direct contact between NCL and components of the discussed repair machineries; however, NCL may promote numerous other, indirect effects on DNA damage repair (Colombo, Alcalay, & Pelicci, 2011; Tajrishi et al., 2011).

5. Concluding remarks During the last two decades, tremendous experimental efforts of many researchers led to the elucidation of the way chromatin participates in DSB repair. The sequential chromatin changes at the DSB lesions leading to pathway choice and then repair, as well as the interfaces of the DNA damage response to cell cycle regulation, have been largely elucidated. The spatial changes as well as those in locus mobility that put breaks in the environment required for efficient repair have started to be understood. There is no doubt that both will continue to yield exciting science as more and more sophisticated imaging, sequencing and biochemical approaches are being introduced. At the same time the accumulation of basic knowledge will inevitably lead to practical results, as novel approaches targeting steps in the DNA damage response get applied in clinical settings to increase efficiency of chemo- and radiotherapy. This knowledge is also ripe to help in the efficient application of Crispr-Cas9 and similar technologies, both in research and in various life science-related practices to fully employ the potential of this scientific revolution. We think that the chromatin control of DSB repair will continue to be a highly exciting field of biology as basic knowledge continues to grow and begins to trickle into practical technologies.

Acknowledgments Work in the laboratory of A.G. is supported by grant # DN11/17 by the Bulgarian National Science Fund. Work in the laboratory of I.U. is supported by grant # DNTS-France-01/13 by the Hubert Curien program Rila 2016.

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References Ahnesorg, P., Smith, P., & Jackson, S. P. (2006). XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining. Cell, 124(2), 301–313. Aleksandrov, R., et al. (2018). Protein dynamics in complex DNA lesions. Molecular Cell, 69(6), 1046–1061.e5. Altmeyer, M., et al. (2015). Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose). Nature Communications, 6, 8088. Angelov, D., et al. (2006). Nucleolin is a histone chaperone with FACT-like activity and assists remodeling of nucleosomes. The EMBO Journal, 25(8), 1669–1679. Aten, J. A., et al. (2004). Dynamics of DNA double-strand breaks revealed by clustering of damaged chromosome domains. Science, 303(5654), 92–95. Aymard, F., et al. (2014). Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks. Nature Structural & Molecular Biology, 21(4), 366–374. Bakkenist, C. J., & Kastan, M. B. (2003). DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature, 421(6922), 499–506. Baumann, P., Benson, F. E., & West, S. C. (1996). Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro. Cell, 87(4), 757–766. Bekker-Jensen, S., et al. (2010). HERC2 coordinates ubiquitin-dependent assembly of DNA repair factors on damaged chromosomes. Nature Cell Biology, 12(1), 80–86. sup pp 1–12. Benson, F. E., Baumann, P., & West, S. C. (1998). Synergistic actions of Rad51 and Rad52 in recombination and DNA repair. Nature, 391(6665), 401–404. Bird, A. W., et al. (2002). Acetylation of histone H4 by Esa1 is required for DNA doublestrand break repair. Nature, 419(6905), 411–415. Borer, R. A., et al. (1989). Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell, 56(3), 379–390. Bouvet, P., et al. (1998). Nucleolin interacts with several ribosomal proteins through its RGG domain. The Journal of Biological Chemistry, 273(30), 19025–19029. Bouwman, P., et al. (2010). 53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers. Nature Structural & Molecular Biology, 17(6), 688–695. Brouwer, I., et al. (2016). Sliding sleeves of XRCC4-XLF bridge DNA and connect fragments of broken DNA. Nature, 535(7613), 566–569. Brown, E. J., & Baltimore, D. (2003). Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance. Genes & Development, 17(5), 615–628. Bugler, B., et al. (1982). Detection and localization of a class of proteins immunologically related to a 100-kDa nucleolar protein. European Journal of Biochemistry, 128(2–3), 475–480. Bunting, S. F., et al. (2010). 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell, 141(2), 243–254. Burma, S., & Chen, D. J. (2004). Role of DNA-PK in the cellular response to DNA doublestrand breaks. DNA Repair (Amst), 3(8–9), 909–918. Burma, S., et al. (2001). ATM phosphorylates histone H2AX in response to DNA doublestrand breaks. The Journal of Biological Chemistry, 276(45), 42462–42467. Calkins, A. S., Iglehart, J. D., & Lazaro, J. B. (2013). DNA damage-induced inhibition of rRNA synthesis by DNA-PK and PARP-1. Nucleic Acids Research, 41(15), 7378–7386. Chapman, J. R., et al. (2012). BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair. Journal of Cell Science, 125(Pt. 15), 3529–3534. Chiolo, I., et al. (2011). Double-strand breaks in heterochromatin move outside of a dynamic HP1a domain to complete recombinational repair. Cell, 144(5), 732–744.

88

Anastas Gospodinov and Iva Ugrinova

Chiolo, I., et al. (2013). Nuclear dynamics of radiation-induced foci in euchromatin and heterochromatin. Mutation Research, 750(1–2), 56–66. Cho, N. W., et al. (2014). Interchromosomal homology searches drive directional ALT telomere movement and synapsis. Cell, 159(1), 108–121. Chow, J. P., et al. (2003). Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA integrity checkpoints. The Journal of Biological Chemistry, 278(42), 40815–40828. Chowdhury, D., et al. (2005). Gamma-H2AX dephosphorylation by protein phosphatase 2A facilitates DNA double-strand break repair. Molecular Cell, 20(5), 801–809. Chowdhury, D., et al. (2008). A PP4-phosphatase complex dephosphorylates gamma-H2AX generated during DNA replication. Molecular Cell, 31(1), 33–46. Ciccia, A., & Elledge, S. J. (2010). The DNA damage response: Making it safe to play with knives. Molecular Cell, 40(2), 179–204. Colombo, E., Alcalay, M., & Pelicci, P. G. (2011). Nucleophosmin and its complex network: A possible therapeutic target in hematological diseases. Oncogene, 30(23), 2595–2609. Costelloe, T., et al. (2012). The yeast Fun30 and human SMARCAD1 chromatin remodellers promote DNA end resection. Nature, 489(7417), 581–584. Daniely, Y., Dimitrova, D. D., & Borowiec, J. A. (2002). Stress-dependent nucleolin mobilization mediated by p53-nucleolin complex formation. Molecular and Cellular Biology, 22(16), 6014–6022. Deng, C., et al. (1995). Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell, 82(4), 675–684. Dev, H., et al. (2018). Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells. Nature Cell Biology, 20(8), 954–965. Difilippantonio, S., et al. (2008). 53BP1 facilitates long-range DNA end-joining during V(D) J recombination. Nature, 456(7221), 529–533. Dimitrova, N., et al. (2008). 53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility. Nature, 456(7221), 524–528. Doil, C., et al. (2009). RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins. Cell, 136(3), 435–446. Dong, S., et al. (2014). The human SRCAP chromatin remodeling complex promotes DNA-end resection. Current Biology, 24(18), 2097–2110. Donzelli, M., & Draetta, G. F. (2003). Regulating mammalian checkpoints through Cdc25 inactivation. EMBO Reports, 4(7), 671–677. Douglas, P., et al. (2010). Protein phosphatase 6 interacts with the DNA-dependent protein kinase catalytic subunit and dephosphorylates gamma-H2AX. Molecular and Cellular Biology, 30(6), 1368–1381. Dronamraju, R., & Mason, J. M. (2011). MU2 and HP1a regulate the recognition of double strand breaks in Drosophila melanogaster. PLoS One, 6(9), e25439. Escribano-Diaz, C., et al. (2013). A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice. Molecular Cell, 49(5), 872–883. Falck, J., Coates, J., & Jackson, S. P. (2005). Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage. Nature, 434(7033), 605–611. Falck, J., et al. (2001). The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis. Nature, 410(6830), 842–847. Fradet-Turcotte, A., et al. (2013). 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark. Nature, 499(7456), 50–54. Gasser, S. M. (2002). Visualizing chromatin dynamics in interphase nuclei. Science, 296(5572), 1412–1416. Ghezraoui, H., et al. (2018). 53BP1 cooperation with the REV7-shieldin complex underpins DNA structure-specific NHEJ. Nature, 560(7716), 122–127.

Chromatin control in double strand break repair

89

Gibson, B. A., & Kraus, W. L. (2012). New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs. Nature Reviews. Molecular Cell Biology, 13(7), 411–424. Ginisty, H., et al. (1999). Structure and functions of nucleolin. Journal of Cell Science, 112(Pt. 6), 761–772. Goldstein, M., et al. (2013). Nucleolin mediates nucleosome disruption critical for DNA double-strand break repair. Proceedings of the National Academy of Sciences of the United States of America, 110(42), 16874–16879. Goodarzi, A. A., et al. (2004). Autophosphorylation of ataxia-telangiectasia mutated is regulated by protein phosphatase 2A. The EMBO Journal, 23(22), 4451–4461. Gospodinov, A., Tsaneva, I., & Anachkova, B. (2009). RAD51 foci formation in response to DNA damage is modulated by TIP49. The International Journal of Biochemistry & Cell Biology, 41(4), 925–933. Gospodinov, A., et al. (2011). Mammalian Ino80 mediates double-strand break repair through its role in DNA end strand resection. Molecular and Cellular Biology, 31(23), 4735–4745. Gravel, S., et al. (2008). DNA helicases Sgs1 and BLM promote DNA double-strand break resection. Genes & Development, 22(20), 2767–2772. Gupta, A., et al. (2005). Involvement of human MOF in ATM function. Molecular and Cellular Biology, 25(12), 5292–5305. Gupta, R., et al. (2018). DNA repair network analysis reveals shieldin as a key regulator of NHEJ and PARP inhibitor sensitivity. Cell, 173(4), 972–988.e23. Hanasoge, S., & Ljungman, M. (2007). H2AX phosphorylation after UV irradiation is triggered by DNA repair intermediates and is mediated by the ATR kinase. Carcinogenesis, 28(11), 2298–2304. Harper, J. W., et al. (1993). The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell, 75(4), 805–816. Heo, K., et al. (2008). FACT-mediated exchange of histone variant H2AX regulated by phosphorylation of H2AX and ADP-ribosylation of Spt16. Molecular Cell, 30(1), 86–97. Hovanessian, A. G., et al. (2000). The cell-surface-expressed nucleolin is associated with the actin cytoskeleton. Experimental Cell Research, 261(2), 312–328. Huen, M. S., et al. (2007). RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Cell, 131(5), 901–914. Huyen, Y., et al. (2004). Methylated lysine 79 of histone H3 targets 53BP1 to DNA doublestrand breaks. Nature, 432(7015), 406–411. Ikura, T., et al. (2000). Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis. Cell, 102(4), 463–473. Jakob, B., et al. (2009). Live cell microscopy analysis of radiation-induced DNA doublestrand break motion. Proceedings of the National Academy of Sciences of the United States of America, 106(9), 3172–3177. Jakob, B., et al. (2011). DNA double-strand breaks in heterochromatin elicit fast repair protein recruitment, histone H2AX phosphorylation and relocation to euchromatin. Nucleic Acids Research, 39(15), 6489–6499. Janssen, A., et al. (2016). A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin. Genes & Development, 30(14), 1645–1657. Kaidi, A., & Jackson, S. P. (2013). KAT5 tyrosine phosphorylation couples chromatin sensing to ATM signalling. Nature, 498(7452), 70–74. Kakarougkas, A., et al. (2013). Co-operation of BRCA1 and POH1 relieves the barriers posed by 53BP1 and RAP80 to resection. Nucleic Acids Research, 41(22), 10298–10311. Kalocsay, M., Hiller, N. J., & Jentsch, S. (2009). Chromosome-wide Rad51 spreading and SUMO-H2A.Z-dependent chromosome fixation in response to a persistent DNA double-strand break. Molecular Cell, 33(3), 335–343.

90

Anastas Gospodinov and Iva Ugrinova

Kastan, M. B., et al. (1991). Participation of p53 protein in the cellular response to DNA damage. Cancer Research, 51(23 Pt. 1), 6304–6311. Kim, Y. C., et al. (2009). Activation of ATM depends on chromatin interactions occurring before induction of DNA damage. Nature Cell Biology, 11(1), 92–96. Kinner, A., et al. (2008). Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin. Nucleic Acids Research, 36(17), 5678–5694. Kobayashi, J. (2004). Molecular mechanism of the recruitment of NBS1/hMRE11/ hRAD50 complex to DNA double-strand breaks: NBS1 binds to gamma-H2AX through FHA/BRCT domain. Journal of Radiation Research, 45(4), 473–478. Kobayashi, T. (2008). A new role of the rDNA and nucleolus in the nucleus—rDNA instability maintains genome integrity. BioEssays, 30(3), 267–272. Kobayashi, J., et al. (2012). Nucleolin participates in DNA double-strand break-induced damage response through MDC1-dependent pathway. PLoS One, 7(11), e49245. Kolas, N. K., et al. (2007). Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science, 318(5856), 1637–1640. Krawczyk, P. M., et al. (2012). Chromatin mobility is increased at sites of DNA doublestrand breaks. Journal of Cell Science, 125(Pt. 9), 2127–2133. Kruhlak, M. J., Celeste, A., & Nussenzweig, A. (2006). Spatio-temporal dynamics of chromatin containing DNA breaks. Cell Cycle (Georgetown, Tex.), 5(17), 1910–1912. Kruhlak, M. J., et al. (2006). Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks. The Journal of Cell Biology, 172(6), 823–834. Langerak, P., et al. (2011). Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks. PLoS Genetics, 7(9), e1002271. Lavin, M. F., & Kozlov, S. (2007). ATM activation and DNA damage response. Cell Cycle, 6(8), 931–942. Lee, H. S., et al. (2010). A cooperative activation loop among SWI/SNF, gamma-H2AX and H3 acetylation for DNA double-strand break repair. The EMBO Journal, 29(8), 1434–1445. Lees-Miller, S. P., & Meek, K. (2003). Repair of DNA double strand breaks by nonhomologous end joining. Biochimie, 85(11), 1161–1173. Li, X., et al. (2010). MOF and H4 K16 acetylation play important roles in DNA damage repair by modulating recruitment of DNA damage repair protein Mdc1. Molecular and Cellular Biology, 30(22), 5335–5347. Lisby, M., Mortensen, U. H., & Rothstein, R. (2003). Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre. Nature Cell Biology, 5(6), 572–577. Lisby, M., & Rothstein, R. (2004). DNA damage checkpoint and repair centers. Current Opinion in Cell Biology, 16(3), 328–334. Lukas, C., et al. (2004). Mdc1 couples DNA double-strand break recognition by Nbs1 with its H2AX-dependent chromatin retention. The EMBO Journal, 23(13), 2674–2683. Mailand, N., et al. (2007). RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins. Cell, 131(5), 887–900. Marnef, A., & Legube, G. (2017). Organizing DNA repair in the nucleus: DSBs hit the road. Current Opinion in Cell Biology, 46, 1–8. Marti, T. M., et al. (2006). H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks. Proceedings of the National Academy of Sciences of the United States of America, 103(26), 9891–9896. Mattiroli, F., et al. (2012). RNF168 ubiquitinates K13-15 on H2A/H2AX to drive DNA damage signaling. Cell, 150(6), 1182–1195. Miller, K. M., et al. (2010). Human HDAC1 and HDAC2 function in the DNA-damage response to promote DNA nonhomologous end-joining. Nature Structural & Molecular Biology, 17(9), 1144–1151.

Chromatin control in double strand break repair

91

Mimitou, E. P., & Symington, L. S. (2008). Sae2, Exo1 and Sgs1 collaborate in DNA doublestrand break processing. Nature, 455(7214), 770–774. Mine-Hattab, J., & Rothstein, R. (2012). Increased chromosome mobility facilitates homology search during recombination. Nature Cell Biology, 14(5), 510–517. Mirman, Z., et al. (2018). 53BP1-RIF1-shieldin counteracts DSB resection through CSTand Polalpha-dependent fill-in. Nature, 560(7716), 112–116. Misteli, T. (2007). Beyond the sequence: Cellular organization of genome function. Cell, 128(4), 787–800. Mitrea, D. M., & Kriwacki, R. W. (2016). Phase separation in biology; functional organization of a higher order. Cell Communication and Signaling: CCS, 14, 1. Modesti, M., et al. (2003). Tetramerization and DNA ligase IV interaction of the DNA double-strand break repair protein XRCC4 are mutually exclusive. Journal of Molecular Biology, 334(2), 215–228. Mongelard, F., & Bouvet, P. (2007). Nucleolin: A multiFACeTed protein. Trends in Cell Biology, 17(2), 80–86. Murr, R., et al. (2006). Histone acetylation by TRRAP-TIP60 modulates loading of repair proteins and repair of DNA double-strand breaks. Nature Cell Biology, 8(1), 91–99. Myler, L. R., et al. (2017). Single-molecule imaging reveals how Mre11-Rad50-Nbs1 initiates DNA break repair. Molecular Cell, 67(5), 891–898.e4. Nagai, S., et al. (2008). Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase. Science, 322(5901), 597–602. Neumann, F. R., et al. (2012). Targeted INO80 enhances subnuclear chromatin movement and ectopic homologous recombination. Genes & Development, 26(4), 369–383. New, J. H., et al. (1998). Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A. Nature, 391(6665), 407–410. Noordermeer, S. M., et al. (2018). The shieldin complex mediates 53BP1-dependent DNA repair. Nature, 560(7716), 117–121. Ogiwara, H., et al. (2011). Histone acetylation by CBP and p300 at double-strand break sites facilitates SWI/SNF chromatin remodeling and the recruitment of non-homologous end joining factors. Oncogene, 30(18), 2135–2146. Orrick, L. R., Olson, M. O., & Busch, H. (1973). Comparison of nucleolar proteins of normal rat liver and Novikoff hepatoma ascites cells by two-dimensional polyacrylamide gel electrophoresis. Proceedings of the National Academy of Sciences of the United States of America, 70(5), 1316–1320. Oza, P., et al. (2009). Mechanisms that regulate localization of a DNA double-strand break to the nuclear periphery. Genes & Development, 23(8), 912–927. Paques, F., & Haber, J. E. (1999). Multiple pathways of recombination induced by doublestrand breaks in Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews, 63(2), 349–404. Pei, H., et al. (2011). MMSET regulates histone H4K20 methylation and 53BP1 accumulation at DNA damage sites. Nature, 470(7332), 124–128. Petukhova, G., Stratton, S., & Sung, P. (1998). Catalysis of homologous DNA pairing by yeast Rad51 and Rad54 proteins. Nature, 393(6680), 91–94. Plans, V., et al. (2006). The RING finger protein RNF8 recruits UBC13 for lysine 63-based self polyubiquitylation. Journal of Cellular Biochemistry, 97(3), 572–582. Rainey, M. D., et al. (2008). Chk2 is required for optimal mitotic delay in response to irradiation-induced DNA damage incurred in G2 phase. Oncogene, 27(7), 896–906. Ramanathan, B., & Smerdon, M. J. (1986). Changes in nuclear protein acetylation in u.v.-damaged human cells. Carcinogenesis, 7(7), 1087–1094. Ramanathan, B., & Smerdon, M. J. (1989). Enhanced DNA repair synthesis in hyperacetylated nucleosomes. The Journal of Biological Chemistry, 264(19), 11026–11034.

92

Anastas Gospodinov and Iva Ugrinova

Rodrigue, A., et al. (2006). Interplay between human DNA repair proteins at a unique double-strand break in vivo. The EMBO Journal, 25(1), 222–231. Ryu, T., Bonner, M. R., & Chiolo, I. (2016). Cervantes and Quijote protect heterochromatin from aberrant recombination and lead the way to the nuclear periphery. Nucleus, 7(5), 485–497. Ryu, T., et al. (2015). Heterochromatic breaks move to the nuclear periphery to continue recombinational repair. Nature Cell Biology, 17(11), 1401–1411. Sancar, A., et al. (2004). Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annual Review of Biochemistry, 73, 39–85. Sanders, S. L., et al. (2004). Methylation of histone H4 lysine 20 controls recruitment of Crb2 to sites of DNA damage. Cell, 119(5), 603–614. Schotta, G., et al. (2008). A chromatin-wide transition to H4K20 monomethylation impairs genome integrity and programmed DNA rearrangements in the mouse. Genes & Development, 22(15), 2048–2061. Scott, D. D., & Oeffinger, M. (2016). Nucleolin and nucleophosmin: Nucleolar proteins with multiple functions in DNA repair. Biochemistry and Cell Biology, 94, 419–432. Seeber, A., Dion, V., & Gasser, S. M. (2013). Checkpoint kinases and the INO80 nucleosome remodeling complex enhance global chromatin mobility in response to DNA damage. Genes & Development, 27(18), 1999–2008. Shao, Z., et al. (2012). Persistently bound Ku at DNA ends attenuates DNA end resection and homologous recombination. DNA Repair (Amst), 11(3), 310–316. Sharma, G. G., et al. (2010). MOF and histone H4 acetylation at lysine 16 are critical for DNA damage response and double-strand break repair. Molecular and Cellular Biology, 30(14), 3582–3595. Shibata, A. (2017). Regulation of repair pathway choice at two-ended DNA double-strand breaks. Mutation Research, 803–805, 51–55. Shibata, A., et al. (2010). Role of ATM and the damage response mediator proteins 53BP1 and MDC1 in the maintenance of G(2)/M checkpoint arrest. Molecular and Cellular Biology, 30(13), 3371–3383. Shiloh, Y. (2006). The ATM-mediated DNA-damage response: Taking shape. Trends in Biochemical Sciences, 31(7), 402–410. Shinohara, A., & Ogawa, T. (1998). Stimulation by Rad52 of yeast Rad51-mediated recombination. Nature, 391(6665), 404–407. Shreeram, S., et al. (2006). Wip1 phosphatase modulates ATM-dependent signaling pathways. Molecular Cell, 23(5), 757–764. Sigurdsson, S., et al. (2002). Homologous DNA pairing by human recombination factors Rad51 and Rad54. The Journal of Biological Chemistry, 277(45), 42790–42794. Smerdon, M. J., & Lieberman, M. W. (1978). Nucleosome rearrangement in human chromatin during UV-induced DNA-repair synthesis. Proceedings of the National Academy of Sciences of the United States of America, 75(9), 4238–4241. Soria, G., Polo, S. E., & Almouzni, G. (2012). Prime, repair, restore: The active role of chromatin in the DNA damage response. Molecular Cell, 46(6), 722–734. Soutoglou, E., et al. (2007). Positional stability of single double-strand breaks in mammalian cells. Nature Cell Biology, 9(6), 675–682. Srivastava, M., & Pollard, H. B. (1999). Molecular dissection of nucleolin’s role in growth and cell proliferation: New insights. The FASEB Journal, 13(14), 1911–1922. Stark, J. M., et al. (2002). ATP hydrolysis by mammalian RAD51 has a key role during homology-directed DNA repair. The Journal of Biological Chemistry, 277(23), 20185–20194. Stiff, T., et al. (2004). ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation. Cancer Research, 64(7), 2390–2396. Storck, S., et al. (2007). Functions of the histone chaperone nucleolin in diseases. Sub-Cellular Biochemistry, 41, 125–144.

Chromatin control in double strand break repair

93

Stucki, M., & Jackson, S. P. (2006). GammaH2AX and MDC1: Anchoring the DNAdamage-response machinery to broken chromosomes. DNA Repair (Amst), 5(5), 534–543. Sun, Y., et al. (2005). A role for the Tip60 histone acetyltransferase in the acetylation and activation of ATM. Proceedings of the National Academy of Sciences of the United States of America, 102(37), 13182–13187. Sung, P. (1994). Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein. Science, 265(5176), 1241–1243. Symington, L. S., & Gautier, J. (2011). Double-strand break end resection and repair pathway choice. Annual Review of Genetics, 45, 247–271. Tadi, S. K., et al. (2016). PAXX is an accessory c-NHEJ factor that associates with Ku70 and has overlapping functions with XLF. Cell Reports, 17(2), 541–555. Tajrishi, M. M., Tuteja, R., & Tuteja, N. (2011). Nucleolin: The most abundant multifunctional phosphoprotein of nucleolus. Communicative & Integrative Biology, 4(3), 267–275. Takata, M., et al. (1998). Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. The EMBO Journal, 17(18), 5497–5508. Tamburini, B. A., & Tyler, J. K. (2005). Localized histone acetylation and deacetylation triggered by the homologous recombination pathway of double-strand DNA repair. Molecular and Cellular Biology, 25(12), 4903–4913. Thompson, L. H., & Schild, D. (2001). Homologous recombinational repair of DNA ensures mammalian chromosome stability. Mutation Research, 477(1–2), 131–153. Thorslund, T., et al. (2015). Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage. Nature, 527(7578), 389–393. Toh, G. W., et al. (2006). Histone H2A phosphorylation and H3 methylation are required for a novel Rad9 DSB repair function following checkpoint activation. DNA Repair (Amst), 5(6), 693–703. Tsekrekou, M., Stratigi, K., & Chatzinikolaou, G. (2017). The nucleolus: In genome maintenance and repair. International Journal of Molecular Sciences, 18(7), E1411. Tsouroula, K., et al. (2016). Temporal and spatial uncoupling of DNA double strand break repair pathways within mammalian heterochromatin. Molecular Cell, 63(2), 293–305. van Attikum, H., Fritsch, O., & Gasser, S. M. (2007). Distinct roles for SWR1 and INO80 chromatin remodeling complexes at chromosomal double-strand breaks. The EMBO Journal, 26(18), 4113–4125. van Attikum, H., et al. (2004). Recruitment of the INO80 complex by H2A phosphorylation links ATP-dependent chromatin remodeling with DNA double-strand break repair. Cell, 119(6), 777–788. van Sluis, M., & McStay, B. (2015). A localized nucleolar DNA damage response facilitates recruitment of the homology-directed repair machinery independent of cell cycle stage. Genes & Development, 29(11), 1151–1163. Wang, J., Gong, Z., & Chen, J. (2011). MDC1 collaborates with TopBP1 in DNA replication checkpoint control. The Journal of Cell Biology, 193(2), 267–273. Ward, I. M., & Chen, J. (2001). Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. The Journal of Biological Chemistry, 276(51), 47759–47762. Watanabe, N., Broome, M., & Hunter, T. (1995). Regulation of the human WEE1Hu CDK tyrosine 15-kinase during the cell cycle. The EMBO Journal, 14(9), 1878–1891. West, S. C. (2003). Molecular views of recombination proteins and their control. Nature Reviews. Molecular Cell Biology, 4(6), 435–445. Xing, M., et al. (2015). Interactome analysis identifies a new paralogue of XRCC4 in nonhomologous end joining DNA repair pathway. Nature Communications, 6, 6233.

94

Anastas Gospodinov and Iva Ugrinova

Xu, Y., Xu, C., & Price, B. D. (2012). Mechanistic links between ATM and histone methylation codes during DNA repair. Progress in Molecular Biology and Translational Science, 110, 263–288. Xu, Y., et al. (2012). Histone H2A.Z controls a critical chromatin remodeling step required for DNA double-strand break repair. Molecular Cell, 48(5), 723–733. Xu, J. Y., et al. (2015). Knocking down nucleolin expression enhances the radiosensitivity of non-small cell lung cancer by influencing DNA-PKcs activity. Asian Pacific Journal of Cancer Prevention, 16(8), 3301–3306. You, Z., et al. (2005). ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1. Molecular and Cellular Biology, 25(13), 5363–5379. Zimmermann, M., et al. (2013). 53BP1 regulates DSB repair using Rif1 to control 50 end resection. Science, 339(6120), 700–704. Zou, L., & Elledge, S. J. (2003). Sensing DNA damage through ATRIP recognition of RPAssDNA complexes. Science, 300(5625), 1542–1548.