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MINIREVIEW / MINISYNTHE`SE
Marks to stop the clock: histone modifications and checkpoint regulation in the DNA damage response1 Stephen E. Humpal, David A. Robinson, and Jocelyn E. Krebs
Abstract: DNA damage from endogenous and exogenous sources occurs throughout the cell cycle. In response to this damage, cells have developed a series of biochemical responses that allow them to recover from DNA damage and prevent mutations from being passed on to daughter cells. An important part of the DNA damage response is the ability to halt the progression of the cell cycle, allowing damaged DNA to be repaired. The cell cycle can be halted at semi-discrete times, called checkpoints, which occur at critical stages during the cell cycle. Recent work in our laboratory and by others has shown the importance of post-translational histone modifications in the DNA damage response. While many histone modifications have been identified that appear to facilitate repair per se, there have been surprisingly few links between these modifications and DNA damage checkpoints. Here, we review how modifications to histone H2A serine 129 (HSA129) and histone H3 lysine 79 (H3K79) contribute to the stimulation of the G1/S checkpoint. We also discuss recent findings that conflict with the current model of the way methylated H3K79 interacts with the checkpoint adaptor protein Rad9. Key words: histone modifications, H3K79, H2AS129, checkpoints, DNA damage. Re´sume´ : Les dommages a` l’ADN de sources endoge`nes ou exoge`nes se produisent tout au long du cycle cellulaire. En re´ponse a` ces dommages, les cellules ont de´veloppe´ une se´rie de re´ponses biochimiques qui leur permettent de re´cupe´rer de ces dommages a` l’ADN et qui pre´viennent que les mutations ne soient transmises aux cellules filles. Une partie importante de la re´ponse aux dommages a` l’ADN est la capacite´ d’arreˆter la progression du cycle cellulaire, permettant au dommage a` l’ADN d’eˆtre re´pare´. Le cycle cellulaire peut eˆtre arreˆte´ a` des moments pre´cis appele´s points de controˆle que l’on retrouve a` des e´tapes critiques du cycle cellulaire. Des travaux re´cents re´alise´s dans notre laboratoire et ailleurs ont de´montre´ l’importance des modifications post-traductionnelles des histones en re´ponse aux dommages a` l’ADN. Alors que plusieurs modifications d’histones semblent faciliter intrinse`quement la re´paration, on a fait e´tonnamment peu de liens entre ces modifications et les points de controˆle des dommages a` l’ADN. Nous passons ici en revue les fac¸ons dont les modifications de la se´rine 129 de l’histone H2A (H2AS129) et de la lysine 79 de l’histone H3 (H3K79) contribuent a` la stimulation du point de controˆle G1/S. Nous discutons e´galement des de´couvertes re´centes qui contredisent le mode`le actuel d’interaction de H2K79 et la prote´ine adaptatrice de point de controˆle Rad9. Mots-cle´s : modification d’histones, H3K79, H2AS129, points de controˆle, dommage a` l’ADN. [Traduit par la Re´daction]
Cell cycle The processes of cell division are tightly regulated and subject to many controls that can halt progression due to environmental stress. The family of serine/threonine kinases, known as CDKs (cyclin-dependent kinases), is responsible for cell cycle progression. CDKs are inactive until they associate with cyclins. The budding yeast Saccharomyces cer-
evisiae has only 1 major cell cycle CDK (Cdc28) but a number of different cyclins, the levels of which fluctuate with the cell cycle (recently reviewed in Bloom and Cross 2007). Higher eukaryotes have multiple CDKs, as well as multiple cyclins, that associate in specific combinations to advance the cells through the cell cycle. S. cerevisiae CDK associates with G1-cyclins (Cln1-3) in G1 phase, and this active kinase complex controls progression of the cell cycle by
Received 14 May 2008. Revision received 11 July 2008. Accepted 15 July 2008. Published on the NRC Research Press Web site at bcb.nrc.ca on 11 February 2009. S.E. Humpal, D.A. Robinson, and J.E. Krebs.2 Department of Biological Sciences, University of Alaska–Anchorage, 3211 Providence Drive, Anchorage, AK 99508, USA. 1This
paper is one of a selection of papers published in this Special Issue, entitled 29th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process. 2Corresponding author (e-mail:
[email protected]). Biochem. Cell Biol. 87: 243–253 (2009)
doi:10.1139/O08-109
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activating proteins, such as the Rho-type GTPase Cdc42, which functions in the polarization of the cell and inception of the new bud (Sopko et al. 2007). During all other phases of the cell cycle, CDK function is controlled by the B-type cyclins Clb1-6. Different Clbs promote DNA replication, spindle maturation and segregation, and entry and progression through mitosis (Donaldson 2000; Ku¨ntzel et al. 1996; Mendenhall and Hodge 1998). To ensure that cell cycle activities do not occur prematurely or out of sequence, CDKs and cyclins are specifically regulated. CDK activity is regulated by inhibitory phosphorylation or by association with inhibitory proteins that block ATP-binding sites on the kinases (Mendenhall and Hodge 1998). Cyclins are blocked from associating with CDKs by inhibitory proteins, and while levels of CDKs remain fairly constant, cyclin levels are controlled by rapid degradation via the proteosome (Mendenhall and Hodge 1998). Thus, as the cell progresses through the cell cycle, the levels of different cyclins will fluctuate in a phase-dependent manner. Various feedback mechanisms stimulate the transcription of the subsequent cyclin to advance the cell beyond its current phase. The regulation of the activity of Cdc28/Clb complexes by the inhibitory protein Sic1 illustrates this type of cell cycle control. In response to DNA damage in G1, yeast cells activate Rad53, a checkpoint kinase that causes the downstream suppression of Cln genes, preventing their accumulation. Without the accumulation of Cln1-3, the Sic1 protein, which inhibits the activity of Cdc28/Clb, remains at high levels in the cell, restricting passage from G1 into S phase. Only activation and transcription of the Cln genes can stimulate the phosphorylation of Sic1 and its subsequent ubiquitylation and degradation, releasing the Cdc28/Clb complexes (Harper 2002; Wysocki et al. 2006).
Checkpoints There are a number of different points during the cell cycle where its progression can be paused to recover from a particular stress, or stopped entirely if the damage caused by the stress cannot be overcome, and these points are collectively referred to as checkpoints. The major checkpoints occur at G1/S (Start), during S phase (intra-S and replication checkpoints), and during G2/M. At any point during the cell cycle, various stresses can cause a cell to arrest, including nutrient deprivation, overcrowding, and DNA damage. Checkpoints are usually regulated by a separate set of sensor proteins and signaling kinases that respond to perceived stresses and send signals to halt the cell cycle. In the case of DNA damage, the detection of lesions or breaks in the DNA requires a cell to repair the damage before progressing to the next phase to ensure that genetic information is not altered or lost (Ataian and Krebs 2006; Bartek and Lukas 2007; Branzei and Foiani 2008; Gontijo et al. 2003; Harrison and Haber 2006; Heideker et al. 2007). The detection of DNA damage is achieved by a number of surveillance proteins that respond to distortions in the DNA double helix, the presence of single-stranded DNA, or unprotected exposed ends of DNA. Key members of the phosphatidylinositol-3 kinase-like kinase (PI3KK) family of proteins detect and respond to
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DNA damage, such as double-strand breaks (DSBs), by phosphorylating a wide range of proteins (Cann and Hicks 2007; Lee and Paull 2007). The 2 best characterized of these proteins are Tel1 and Mec1 in S. cerevisiae (ATM and ATR in mammals), which respond to DNA damage by phosphorylating histones (Downs et al. 2000; Du et al. 2006; Fernandez-Capetillo et al. 2004; Foster and Downs 2005; Moore et al. 2007), other kinases, adaptor proteins, chromatin modifying proteins, and even themselves (Clerici et al. 2004; Grenon et al. 2006; Morrison et al. 2007; Paciotti et al. 1998; Sweeney et al. 2005); with selection of their targets dependent on what point in the cell cycle the damage occurs. There are a number of mammalian targets of the ATM/ATR kinases, including the well-known oncoprotein p53, the p53binding protein 53BP1, the BRCA1 oncoprotein, and Smc1. p53 is a transcription factor that initiates transcription of CDK inhibitors to stop cell cycle progression, as well as proapoptotic factors in the presence of a prolonged cell cycle block (Niida and Nakanishi 2006). 53BP1 is related to S. cerevisiae Rad9, and serves in the same capacity as an adaptor protein by stimulating the phosphorylation of the checkpoint effector protein Chk2 (Rad53 in S. cerevisiae) (Pellicioli and Foiani 2005; Sweeney et al. 2005). The oncoprotein BRCA1 has direct activity in cell cycle regulation at the G2/M checkpoint (Gudmundsdottir and Ashworth 2006; Kao et al. 2006; Paull et al. 2000). Smc1, part of the cohesin complex involved in chromosome organization and segregation and DSB repair, has a well-characterized homolog of the same name in yeast (Watrin and Peters 2006). All of these targets play a role in responding to DNA damage within the context of the cell cycle, preventing cells from progressing without proper repair.
DNA damage response DNA damage can occur at any stage of the cell cycle and can be caused by both endogenous and exogenous sources. The response to damage depends on the cell cycle stage and the type of damage. DNA can be damaged by ultraviolet light (UV), alkylating agents, and errors introduced by DNA polymerases. These types of damage can be repaired by pathways such as nucleotide excision repair and base excision repair. If these repair events do not occur and cell cycle checkpoints remain active, an alternative response of the cell is to replicate across the lesion, using specialized translesion polymerases, such as Pol z. Translesion polymerases are error prone, but do increase the survival of yeast cells by allowing for the completion of DNA replication after exposure to UV and g radiation and alkylating agents. Pol z gene deletion mutants have decreased rates of homologous recombination (HR), indicating that error prone polymerases are also important in other repair pathways (Gan et al. 2008). Other sources of DNA damage result in the formation of DSBs. DSBs can be induced by ionizing radiation (IR), by conversion of single-strand lesions during replication, or experimentally, using radiomimetic drugs or endonucleases. The pathways of nonhomologous end-joining (NHEJ) and HR, the 2 major pathways responsible for repair of DSBs, have been the most extensively studied mechanisms of Published by NRC Research Press
Humpal et al.
DNA repair with respect to histone modifications, so we will focus on these pathways in this review. Signaling of DNA damage involves the recognition of lesions or DSBs and the activation of both cell cycle regulators and repair factors. Cell cycle checkpoint factors, such as Chk2 (Rad53), MDC1, and 53BP1, all regulate the progression of the cell cycle to permit the cell time to repair the DNA, which is essential for maintenance of genomic integrity and cell survival. 53PB1, a proposed homolog to the budding yeast Rad9 protein, is an effector protein that stimulates phosphorylation of other checkpoint regulators, such as Chk2 (Pellicioli and Foiani 2005; Sweeney et al. 2005). Chk2, a homolog of the Rad53 protein, is a kinase that plays a role in establishing checkpoints and halting progression through the cell cycle (Nakamura et al. 2004), while MDC1, which currently has no yeast homolog, plays a similar role and is involved in phosphorylation of targets that control cell cycle progression (Stewart et al. 2003). The recruitment of repair factors, which occurs after the detection of DNA damage, is specific for the type of damage, as well as for the state of the cell cycle at the time the damage is detected (Dasika et al. 1999). When DSBs occur during G1 in haploid cells, the cell can repair the damage through the process of NHEJ, which does not use homologous DNA as a template for the repair process, and can result in deletions or other errors in the DNA sequence following repair (Fig. 1). Yeast usually use NHEJ only at points in the cell cycle where a sister chromatid (or other homologous donor) is not available for HR (Daley et al. 2005; Duda´sova´ et al. 2004; McGinty et al. 2008; Shrivastav et al. 2008). The repair proteins Ku70 and Ku80, which are abundant in the cell, form heterodimers and bind to both ends of the broken DNA. The Ku dimers then recruit the MRX (Mre11-Rad50-Xbs1) complex of proteins to the break, which may assist in bringing the 2 broken ends together, and also participate in the processing of the ends. The Lig4-Lif1 ligase complex then associates and the 2 ends are ligated together. The process of NHEJ is error prone because it does not use a template to make repairs of the DSB and will, therefore, combine together the free ends of any nearby DNA. In instances where multiple DSBs occur, NHEJ can lead to translocations and to loss of genetic information (Haber 2006). DSBs that occur in late S phase or in diploid cells can be repaired by HR (Fig. 1). HR has a much lower chance of causing mutations in the genome than NHEJ, but requires that a sister chromatid or other homologous donor be available for use as a template for repair. Sister chromatids are the preferred template for HR even in diploids, because the use of homologous loci as templates can lead to loss of heterozygocity. There are several recent reviews of HR (for example, Ataian and Krebs 2006; San Filippo et al. 2008), so we will only briefly describe this pathway here. HR is a multistep process that includes recognition of the break, processing of the free ends, searching for homology, invasion of the homologous region, replication of the homologous template, and resolution of the Holliday junction. The MRX complex binds to the ends and participates in the 5’ to 3’ resection, forming single-stranded DNA, which is immediately recognized by the single-stranded DNA binding protein replication protein A (RPA). The presence of single-
245 Fig. 1. Double-strand break (DSB) repair pathways. During G1 and the beginning of S phase, when the cell is in the haploid state, nonhomologous end-joining (NHEJ) is used to repair DSBs (right side of figure). Ku70/80 bind to the break, along with the MRX complex (Mre11, Rad50, and Xbs1), and the ligase Lig4/Lif1 then ligates the 2 blunt ends to repair the DSB (nonblunt ends are processed to blunt ends, if necessary). The homologous recombination (HR) (left side of figure) pathway has increased fidelity over NHEJ, because it uses homologous regions of DNA as a template. HR occurs when cells are in the diploid state during late S phase, G2, and mitosis. The MRX complex first resects the DNA, forming single-stranded ends; replication protein A (RPA) then binds to the single-stranded DNA. Rad52 and Rad55/57 promote the exchange of Rad51 for RPA, and facilitate the search for the homologous donor and subsequent strand invasion by the Rad51 filament. Finally, new DNA synthesis and ligation complete the repair process, and chromatin structure is restored. Nucleosomes are not depicted for simplicity.
stranded DNA associated with RPA constitutes a key signal for checkpoint activation. After RPA is bound, it recruits Rad52, Rad54, and the Rad55/Rad57 heterodimers. This group of proteins replaces RPA with the strand exchange Published by NRC Research Press
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protein Rad51, which is involved in the homology search and subsequent strand exchange, along with Rad57. Strand exchange leads to replication of the homologous template, using the 3’ ends of the nonresected strands as primers for synthesis by DNA polymerase. HR is also an available mechanism for repairing UV damage when homologous sections of DNA are present in diploid cells and during S and G2 (Aboussekhra and Al-Sharif 2005). DSBs that occur during G2 and M phases are, again, ideally repaired by HR, but it is more difficult in this context, owing to the increased level of chromatin compaction and possible separation of sister chromatids, although cohesin proteins holding sister chromatids together increase the likelihood that homology will be found to facilitate HR (Ball and Yokomori 2008). All of the processes described so far take place within the context of the DNA, but of course DNA does not exist in eukaryotes as a naked double helix. The processes of cell cycle control, checkpoint activation, and DNA damage and repair all must be considered in the context of chromatin.
Chromatin In eukaryotic cells, the DNA must be packaged to fit into the nucleus and to be properly segregated during mitosis and, thus, associates with histones to form nucleosomes, the basic units of chromatin. Two copies each of 4 types of histones (H2A, H2B, H3, and H4) form the core of 1 nucleosome. A total of 146 base pairs of DNA wrap ~1 3/4 times around the nucleosome, with different lengths of linker DNA between nucleosomes, forming the 10 nm fiber. Each of the core histone proteins has a core globular region, around which the DNA wraps, and a flexible N-terminal tail (H2A has 2 tails, 1 N-terminal, and 1 C-terminal) rich in basic amino acid residues that associate closely with the negative backbone of the DNA (Chodaparambil et al. 2006; Luger 2003, 2006). Even at the lowest level of compaction, the 10 nm fiber, DNA in chromatin is relatively inaccessible. The inner surface of the DNA helix wrapped around the nucleosome is hidden from enzymes and other proteins with sequencespecific recognition sites, and the histone tails themselves cover more of the DNA sequence. Modifications to chromatin structure can allow enzymes involved in DNA metabolism access to the DNA. Chromatin can be modified by the physical modification of DNA–histone associations by ATP-dependent chromatin remodeling enzymes, and by post-translational modification of histone tails by enzymes that covalently attach various chemical groups to modifiable amino acids. Some modifications, such as the acetylation of lysine residues or the phosphorylation of serines and threonines, affect the charge of the histone tails, neutralizing the positive charge in the case of lysine acetylation and adding a negative charge in the case of phosphorylation. Other modifications do not change the charge of the tail, but create novel recognition sites on the tails to promote or prevent binding of other proteins. These types of modifications include mono-, di-, or trimethylation of lysines, mono- or dimethylation of arginines, and ubiquitylation and sumoylation of lysines (Kouzarides 2007; Krebs 2007; Millar and Grunstein 2006; Verdone et al. 2006). Different modifications, and combinations of modi-
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fications in a particular sequence, have been shown to be involved in specific nuclear activities, and these sequences of modifications remain relatively well conserved among eukaryotes (Krebs 2007; Millar and Grunstein 2006; Ruthenburg et al. 2007). For example, methylation of H3K4, which stimulates the acetylation of various other lysines on H3, is linked to transcriptional activation (Wang et al. 2001), while phosphorylation of H2A serine 129 (S129) has been associated with repair of DNA DSBs (Downs et al. 2000; Moore et al. 2007). While histone modifications have been extensively studied in transcription and repair, only 2 modifications have been strongly connected to checkpoint function. In this review, we will focus on the histone modifications that have been implicated in the management of the cell cycle, particularly in DNA damage checkpoints that halt the cell cycle during the process of DNA repair.
Role of histone H2A phosphorylation One of the most extensively characterized repair-specific histone modifications is the phosphorylation of histone H2AS129 (H2AX serine-139 in mammals); the phosphorylated form is often designated as gH2A/gH2AX (for recent reviews, see Altaf et al. 2007; Ataian and Krebs 2006; Chambers and Downs 2007; Escargueil et al. 2008; Karagiannis and El-Osta 2007). H2AS129 is phosphorylated by Mec1 and Tel1 kinases, the yeast homologs of the mammalian ATR and ATM kinases, respectively (Downs et al. 2000; Rogakou et al. 1998). These kinases play a number of very important roles in the DNA damage surveillance and repair systems, which are highly conserved among all eukaryotes. gH2AX foci form in regions extending megabases from the site of damage in response to DNA DSBs and UV irradiation in mammals and ~50 kb around breakpoints in yeast (Shroff et al. 2004; Unal et al. 2004). Mutational analyses of H2AS129 have shown that cells carrying a mutant gene in which S129 has been mutated to an unmodifiable alanine residue (S129A) show sensitivity to DNA DSBs and show an increase in collapsed replication forks (Downs et al. 2000; Moore et al. 2007; Redon et al. 2003, 2006). H2AS129 may also play a role in responding to UV irradiation and oxidative damage, as it is phosphorylated following these treatments, although it is not essential for survival after these types of damage (Moore et al. 2007). The role of gH2A in checkpoint function is controversial, however. In Schizosaccharomyces pombe, lack of gH2A formation results in loss of checkpoint maintenance. Nakamura et al. (2004) showed, in S. pombe, that S129A mutation results in a number of defects, such as loss of IRinduced Crb2 focus formation, and reduced Chk1 and Crb2 phosphorylation. Crb2, the S. pombe homolog of Rad53, is required for the G1 DNA damage checkpoint. They also showed that cells arrested at G2/M (using a temperaturesensitive Cdc25 mutant) show dose-dependent checkpoint effects when irradiated with varying levels of IR and then released into the cell cycle. At low levels of IR, H2AS129A mutants exhibit a normal DNA damage checkpoint, similar to wild-type cells, whereas at higher doses of IR, which require longer cell cycle delays for full DNA repair, S129A mutants exit the checkpoint early, and proceed Published by NRC Research Press
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through the cell cycle, suggesting that gH2A is more important for the maintenance than the establishment of the checkpoint (Nakamura et al. 2004). S. cerevisiae, however, appears able to maintain a checkpoint under similar conditions. Downs et al. (2000) showed that in the presence of methyl methane sulphonate (MMS), a DNA alkylating agent known to induce gH2A, synchronized cultures of wild-type and H2AS129A mutants do not show significantly different rates of progression through the cell cycle, as measured by bud index, whether synchronized with nocodozole or a-factor (Downs et al. 2000). However, another study showed that H2AS129A mutants synchronized at G1 enter into S phase more rapidly after IR treatment than wild-type cells, revealing a slight G1 checkpoint defect, and also show a slight affect on intra-S phase checkpoints in synchronized cells treated with MMS (Javaheri et al. 2006). These researchers saw no difference between the mutant and wild-type cells at G2/M. In addition, both Rad9 phosphorylation and Rad53 kinase activity are reduced in S129A cells. gH2A has been shown to facilitate the recruitment of numerous factors to DSBs, including the INO80 and SWR1 remodeling complexes and the NuA4 acetyltransferase (Downs et al. 2004; Morrison et al. 2004; Van Attikum et al. 2004). NuA4, but not INO80 or SWR1, appears to affect the persistence of the G1 checkpoint (Javaheri et al. 2006). These results suggest that the checkpoint function of gH2A is not dependent on the INO80 or SWR1 remodelers. Others have also observed a G1 checkpoint defect in cells containing an H2A allele in which the codon for S129 is mutated to a stop codon (Hammet et al. 2007). So, in general, S129A cells display a defective (but not completely eliminated) G1 damage checkpoint, resulting in delayed arrest, reduced phosphorylation of the Rad9 checkpoint adaptor (Siede et al. 1993), and decreased phosphorylation of the Rad53 checkpoint effector kinase. The association with Rad9 provides the most notable link between gH2A and checkpoint function (Fig. 2). In S. cerevisiae, Rad9 has been shown to interact with gH2A (Hammet et al. 2007), and the same has been shown for the S. pombe homolog Crb2 (Nakamura et al. 2004). Rad9 contains the highly conserved tandem BRCA1 C-terminal (BRCT) protein domains that have been shown to be indispensable for its recruitment to gH2A. These 2 tandem BRCT domains (BRCT2), when tested against peptides specific for the C-terminal tail of H2A and gH2A, show an affinity for only gH2A. Comparing this with the BRCT2 domains of other proteins, such as mammalian MDC1 and BRCA1, we see that Rad9 is specific for gH2A, while the other BRCT2 proteins do not recognize either H2A or gH2A. This specific recruitment of Rad9 to gH2A has also been associated with its hyperphosphorylation, as well as the hyperphosphorylation of Rad53, the S. cerevisiae homolog to Chk2 (Hammet et al. 2007; Soulier and Lowndes 1999). Rad9 and its homologs in higher eukaryotes will be further discussed in subsequent sections. While the role of gH2A in checkpoint activation is not entirely clear, dephosphorylation of gH2A appears to have an important role in checkpoint recovery (at least for the G2/M checkpoint) after repair is completed. The yeast Pph3 phosphatase, which dephosphorylates Rad53 (O’Neill et al. 2007), is needed to remove the phosphate from S129 as
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well (Keogh et al. 2006). This dephosphorylation occurs on gH2A that has been displaced from chromatin. Similar results have been observed for mammalian gH2AX, where protein phosphatase 2A is required to remove gH2AX foci, although defects in protein phosphatase 2A function may prolong the repair itself rather than affect checkpoint recovery (Chowdhury et al. 2005). gH2A turnover has also been linked to checkpoint adaptation, a phenomenon in which cells eventually re-enter the cell cycle, despite the persistence of unrepaired damage (Papamichos-Chronakis et al. 2006).
Role of histone H3 methylation Another modification linked to checkpoint function in response to DNA damage is methylation of H3K79, although, like H2AS129, its role has been somewhat controversial. Methylation of H3K79 (H3K79me) is a constitutive process, and appears to occur after nucleosomes are assembled. The level of monomethylated H3K79 in HeLa cells decreases in S phase, possibly due to the dilution of the original histone proteins during DNA replication, reaching the lowest levels during G2, increasing during mitosis, and finally reaching the highest levels in G1 (Feng et al. 2002). Levels of dimethylated H3K79 in S. cerevisiae have been shown to be constant throughout the cell cycle and during DNA damage (Wysocki et al. 2005). Feng et al. (2002) proposed that the decrease in monomethylation during S phase was not likely a result of dilution, as decreases of similarly constitutive modifications are not observed during S phase. One possible explanation for this discrepancy is that many modified histone residues are located on the tails, making them accessible for the quick addition of marks before or after nucleosome assembly. H3K79 is hidden in the nucleosome core, making it difficult for histone-modifying enzymes to make modifications immediately after assembly has occurred (Luger et al. 1997). This inaccessibility would require chromatin remodelers to expose the residue for the histone methyltransferases, explaining why methylation of K79 occurs so slowly following DNA replication (Fig. 2). As described above, when damage occurs in G1, Ku70 and Ku80 bind to the ends of the break, initiating NHEJ. The MRX complex then binds to the break, initiating the processing of DNA ends; in NHEJ, single-strand resection is prevented and, instead, blunt-ended DNA, a substrate for DNA ligase, is ultimately generated. The presence of MRX bound to DNA is a checkpoint activation signal. The kinases Tel1 and Mec1 (with their respective partners Tel2 and Ddc2) associate with the MRX complex, leading to the recruitment of Mec1 and Tel1 to the area of the break, resulting in the phosphorylation of H2AS129 both up- and downstream of the break (Anderson et al. 2008; Takai et al. 2007). The SWR1 and INO80 ATP-dependent chromatin remodelers and the NuA4 acetyltransferase are next recruited to the lesion (Downs et al. 2004; Morrison and Shen 2005; Van Attikum et al. 2007). It has been proposed that the action of these remodelers exposes the constitutive H3K79me, thus allowing the adaptor protein Rad9 to bind. The binding of Rad9 leads to its activation, activation of Rad53 by Mec1 and, thus, activation of the G1/S checkpoint (Fig. 2). Recent findings challenge this proposed model by showing that Published by NRC Research Press
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Fig. 2. During G2 and into mitosis, levels of H3K79 increase as the methyltransferase Dot1 adds the methyl group to the fully assembled nucleosomes, a process that might require a chromatin remodeling enzyme to expose the H3K79. When DNA is damaged by ionizing radiation or another DSB-inducing agent in G1, the NHEJ proteins Ku70 and Ku80 bind to the free DNA ends and recruit the MRX complex (and other factors) to clean up the break to produce blunt ends. The MRX complex recruits Tel1, which phosphorylates histone H2A on serine 129 (H2AS129) in a large region up- and downstream of the break. The Rad9 checkpoint adaptor protein, possibly aided by the chromatin structure remodeling (RSC) chromatin remodeling complex, binds to the phospho-S129 of H2A, as well as to the constitutively methylated lysine 79 (K79) on histone H3. The recruitment of Rad9 to the break is followed by hyperphosphorylation of Rad9 by Mec1, which activates Rad9. Activated Rad9 stimulates the Mec1-mediated phosphorylation of the checkpoint kinase Rad53 (Mec1 also stimulates the trans autophosphorylation of Rad53), which in turn phosphorylates and activates downstream targets and pathways to induce cell cycle arrest.
some of the chromatin remodelers recruited to the area are not involved in exposing H3K79 (Javaheri et al. 2006), and that Rad9 does not actually bind to methylated K79 (Lancelot et al. 2007). Here, we discuss the conflicting results. Feng et al. (2002) first discovered the methyltransferase required for H3K79 methylation using a polyclonal antibody that recognizes only monomethylated H3K79. They identified Disruptor of Telomeric silencing 1 (Dot1, also known as Kmt4) as the enzyme responsible for methylation of H3K79, and found that it was conserved from yeast to humans. Dot1 is not only responsible for monomethylation of H3K79, but also dimethylation and trimethylation; these higher methylation states depend on H2BK123 ubiquityla-
tion (K120 in humans) in a histone cross-talk pathway (Feng et al. 2002; McGinty et al. 2008; Ng et al. 2002; Shahbazian et al. 2005; Van Leeuwen et al. 2002; Wysocki et al. 2005). Further evidence supporting histone dilution as the reason for the decrease in H3K79me during S phase comes from in vitro experiments showing that fully assembled nucleosomes are required for Dot1 to add the methyl group onto H3K79; this requirement shows that the modification must be made after nucleosomes are assembled (Feng et al. 2002). Rad9 and the human homolog 53BP1 have both been proposed to bind to H3K79me, which is supported by pulldown assays using the tandem Tudor domain of 53BP1 (residues Published by NRC Research Press
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1157–1634 and 1480–1626) and Rad9 (750–917) as bait incubated with histones purified from calf thymus. This experiment shows that both Rad9 and 53BP1 preferentially bind to H3, monomethylated on K79, compared with H2A, H2B, and H4 (Huyen et al. 2004). Further support for Rad9 binding to H3K79me comes from a study showing that deletion of DOT1 and mutation of the Rad9 Tudor domain (Y798Q) both had an epistatic defect in the G1/S checkpoint after IR-induced damage (Grenon et al. 2007). Nuclear magnetic resonance titrations of the Rad9 Tudor domain (residues 754–947) with dimethylated H3 (H3K79me2) peptide disputed this model by failing to show binding, even though they were able to detect binding of the Tudor domain of 53BP1 to the peptide. The authors cite differences in the sequence and structure of the Tudor domains as a possible explanation for these differences (Lancelot et al. 2007). These authors also showed that the tandem Tudor domain of Rad9 binds both single- and double-stranded DNA, of no specific sequence, with its unique positively charged region that is absent in 53BP1. So, a possible explanation for the contradictory results is that Rad9 is actually binding to the monomethylated form of K79, which was not used in the Lancelot et al. (2007) study but, as stated above, was shown by Feng et al. (2002) to be constitutive in HeLa cells. The choice of the H3K79me2 peptide was likely based on prior results showing that 53BP1 binds to both the mono- and dimethylated forms of H3K79 but has a greater affinity for the dimethylated form (Huyen et al. 2004). Competition assays by Huyen et al. (2004) confirmed the binding of both monoand dimethylated forms of H3K79 to 53BP1, and also showed that 53BP1 had an affinity for dimethylated H3K27 and R26. Direct support of Rad9 binding was shown using a GSTRad9 pulldown assay, where all calf thymus histones were pulled down, the majority being H3 (Huyen et al. 2004). This assay was performed using purified calf thymus histone proteins, bolstering the claim of direct binding to histones (especially H3), rather than general DNA binding. Binding of Rad9 to calf thymus histones could be due to the monomethylated form of K79, since both forms were found to be present in calf thymus lysate. Taken together, it appears that Rad9 binds to nucleosomes through its Tudor domains, interacting with mono(?)methylated H3K79, as well as both double- and single-stranded DNA, which could further stabilize Rad9 recruitment to chromatin. To verify this model, nuclear magnetic resonance titrations could be performed with Rad9 and a monomethylated peptide. Also, an antibody specific for monomethylated H3K79 could be used to verify constitutive levels of monomethylated H3K79 in yeast. However, a role for monomethylated H3K79 does not explain the checkpoint function of Rad6/Bre1-dependent H2B ubiquitylation, which promotes H3K79 di- and trimethylation but is not essential for monomethylation (Wysocki et al. 2005; Shahbazian et al. 2005). rad6/bre1 mutants (or H2BK123 mutants that cannot be ubiquitylated) result in G1 and intra-S checkpoint defects that are indistinguishable from the defects resulting from DOT1 deletions, and include reduced Rad9 phosphorylation and Rad53 activation. It is possible that there are unaccounted-for differences in the study by Lancelot et al. (2007) that resulted in a failure of the Rad9 fragment to bind peptide under the conditions
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used. H2AS129 phosphorylation is also involved in Rad9 recruitment (see discussion above), and loss of either gH2A or H3K79me results in loss of Rad9 recruitment to a DSB. Therefore, the interactions of the individual Rad9 BRCT and Tudor domains with single modifications in vitro may not adequately reflect the network of interactions critical in vivo. It is easy to imagine a combination of 2 histone recognition events and nonspecific single- or double-stranded DNA binding resulting in robust recruitment of Rad9 to sites of DNA damage. A key element of this model is that Rad9 must be able to gain access to the normally buried K79me. The prevailing model for Rad9 binding to H3K79me is dependent on the residue being exposed for Rad9 binding by the action of remodelers. As stated above, the phosphorylation of H2AS129, which occurs very rapidly at a DSB, results in recruitment of the Arp4 subunits of the chromatin remodeling enzymes SWR1, INO80, and the acetyltransferase NuA4 to the site of DNA damage (Downs et al. 2004; Morrison et al. 2004; Van Attikum et al. 2004). These remodeling enzymes are needed to expose the damaged region of DNA for repair, and were proposed to be the method by which H3K79 is made available for Rad9 binding. However, recent data contradict the idea that these remodelers are required to expose H3K79 to Rad9 (Javaheri et al. 2006). These researchers knocked out critical subunits of the 3 remodeling or modifying enzymes recruited by gH2A (ARP8 of INO80, SWR1 of the SWR complex, and YNG2 of NuA4), and demonstrated that these individual deletions do not result in a G1 checkpoint delay following DNA damage. The Arp4 subunit is required for the recruitment of all 3 chromatin modifiers by gH2A. A temperature-sensitive allele of ARP4, arp4-12, which blocks recruitment of these complexes to a break, even at the permissive temperature, also has no effect on the G1 checkpoint response. As mentioned above, the only effect on checkpoint activity of any of these mutants is a persistence of the G1 checkpoint in NuA4 mutants, suggesting that this complex may normally oppose checkpoint maintenance. The RSC chromatin remodeler plays an essential function in G1, the G1/S transition, G2, and M phase (Campsteijn et al. 2007). RSC also plays a role in DSB repair. RSC interacts with both the Mre11 subunit of the MRX complex and Ku80, which facilitate recruitment of RSC to breaks (Shim et al. 2005). It is possible that RSC is the chromatin remodeler required for exposing H3K79. This model is supported by a recent study (Liang et al. 2007). These researchers showed that after formation of a DSB, RSC is critical for recruitment of Mec1 and Tel1, phosphorylation of H2A, activation of Rad9 and Rad53, and chromatin remodeling. They proposed a model, based on their findings, suggesting that RSC acts upstream of H2A phosphorylation, at the same level as MRX binding, and that RSC is involved in DSB sensing. Although this model does not directly suggest that RSC is involved in exposing H3K79 to Rad9, the data suggest that this interaction should be pursued further. One caveat to the model of RSC exposing H3K79me to Rad9 is the contradictory evidence showing a decrease in the formation of single-stranded DNA around DSBs when RSC is knocked out, and recent data showing that Rad9 binding to H3K79 actually inhibits the resection of DNA after an HO-induced Published by NRC Research Press
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break (Lazzaro et al. 2008). Mediating this effect on the model is the fact that this latter experiment was performed in G2-synchronized cells, a time when HR is the primary form of DSB repair, and when dot1 and rad9 mutant cells do not show any checkpoint defect (Grenon et al. 2007). The role of H3K79me in repair is still unresolved. We believe the most compelling evidence still supports a model such as that illustrated in Fig. 2, in which exposure of a normally buried modification results in recruitment or stabilization of Rad9 binding to chromatin. gH2A binding and DNA binding could independently or additively contribute to Rad9 recruitment, or to its subsequent activity.
Conclusions: Rad9 and beyond Rad9 homologs in higher eukaryotes have been difficult to identify, owing to lack of strict sequence homology. This lack of sequence homology becomes less troublesome when the secondary structures of the conserved protein domains are compared with proteins with similar checkpoint activities in other species. The mammalian checkpoint protein p53-binding protein 1 (53BP1), BRCA1, and MDC1 have been shown to possess BRCT2 domains and tandem Tudor (Tudor2) domains that interact with H3K79me. The BRCT2 domain of 53BP1 is structurally the most similar to Rad9, despite the lack of sequence identity. Yeast lack homologs of BRCA1 or MDC1. While 53BP1, BRCA1, and MDC1 all have roles in cell cycle checkpoints and genome maintenance, 53BP1, like Rad9, specifically associates with gH2A, as well as H3K79me, and also facilitates phosphorylation of Chk2, the mammalian Rad53 homolog (Huyen et al. 2004; Javaheri et al. 2006; Lancelot et al. 2007; Nakamura et al. 2004; Sweeney et al. 2005). Phosphorylation of Chk2, or Rad53, activates the kinase activity of Chk2/Rad53. Chk2/ Rad53 have a number of phosphorylation targets by which they regulate the progression of the cell cycle or activate cell cycle checkpoints. In summary, stimulation of a checkpoint response in the presence of DNA damage during G1 cells is facilitated by 2 critical histone marks. One is the constitutive methylation of H3K79, and the second is the damage-induced phosphorylation of H2AS129. (H3K79 di- and trimethylation is further controlled by a third critical mark, H2BK123 ubiquitylation.) Neither of these marks alone is enough to efficiently recruit Rad9 to chromatin, and without these marks the activating phosphorylation of Rad9 is reduced, affecting the downstream role of Rad9 in checkpoint activation while DNA repair proceeds. The recruitment of Rad9 to these histone modifications connects the DNA damage checkpoint response to the epigenetic changes that occur during DNA repair, and specifically link the checkpoint to modifications that have been shown to be important for the repair process itself, in addition to their checkpoint functions.
Acknowledgements We heartily thank both Dr. Oya Yazgan and Shannon Uffenbeck for their time and effort with this article. Both provided invaluable technical and editorial assistance, and Dr. Yazgan provided considerable assistance with the figures.
Biochem. Cell Biol. Vol. 87, 2009
References Aboussekhra, A., and Al-Sharif, I.S. 2005. Homologous recombination is involved in transcription-coupled repair of UV damage in Saccharomyces cerevisiae. EMBO J. 24: 1999–2010. doi:10. 1038/sj.emboj.7600665. PMID:15902273. Altaf, M., Saksouk, N., and Coˆte´, J. 2007. Histone modifications in response to DNA damage. Mutat. Res. 618: 81–90. PMID:17306843. Anderson, C.M., Korkin, D., Smith, D.L., Makovets, S., Seidel, J.J., Sali, A., and Blackburn, E.H. 2008. Tel2 mediates activation and localization of ATM/Tel1 kinase to a double-strand break. Genes Dev. 22: 854–859. doi:10.1101/gad.1646208. PMID:18334620. Ataian, Y., and Krebs, J.E. 2006. Five repair pathways in one context: chromatin modification during DNA repair. Biochem. Cell Biol. 84: 490–504. doi:10.1139/O06-075. PMID:16936822. Ball, A.R., Jr., and Yokomori, K. 2008. Damage-induced reactivation of cohesin in post-replicative DNA repair. Bioessays, 30: 5– 9. doi:10.1002/bies.20691. PMID:18081005. Bartek, J., and Lukas, J. 2007. DNA damage checkpoints: from initiation to recovery or adaptation. Curr. Opin. Cell Biol. 19: 238–245. doi:10.1016/j.ceb.2007.02.009. PMID:17303408. Bloom, J., and Cross, F.R. 2007. Multiple levels of cyclin specificity in cell-cycle control. Nat. Rev. Mol. Cell Biol. 8: 149–160. doi:10.1038/nrm2105. PMID:17245415. Branzei, D., and Foiani, M. 2008. Regulation of DNA repair throughout the cell cycle. Nat. Rev. Mol. Cell Biol. 9: 297–308. doi:10.1038/nrm2351. PMID:18285803. Campsteijn, C., Wijnands-Collin, A.-M.J., and Logie, C. 2007. Reverse genetic analysis of the yeast rsc chromatin remodeler reveals a role for rsc3 and snf5 homolog 1 in ploidy maintenance. PLoS Genet. 3: e92. doi:10.1371/journal.pgen.0030092. PMID:17542652. Cann, K.L., and Hicks, G.G. 2007. Regulation of the cellular DNA double-strand break response. Biochem. Cell Biol. 85: 663–674. doi:10.1139/O07-135. PMID:18059525. Chambers, A.L., and Downs, J.A. 2007. The contribution of the budding yeast histone h2a c-terminal tail to DNA-damage responses. Biochem. Soc. Trans. 35: 1519–1524. doi:10.1042/ BST0351519. PMID:18031258. Chodaparambil, J.V., Edayathumangalam, R.S., Bao, Y., Park, Y.J., and Luger, K. 2006. Nucleosome structure and function. Ernst Schering Res. Found. Workshop, 57: 29–46. doi:10.1007/3-54037633-X_2. PMID:16568947. Chowdhury, D., Keogh, M.-C., Ishii, H., Peterson, C.L., Buratowski, S., and Lieberman, J. 2005. Gamma-h2ax dephosphorylation by protein phosphatase 2A facilitates DNA doublestrand break repair. Mol. Cell, 20: 801–809. doi:10.1016/j. molcel.2005.10.003. PMID:16310392. Clerici, M., Baldo, V., Mantiero, D., Lottersberger, F., Lucchini, G., and Longhese, M.P. 2004. A tel1/mrx-dependent checkpoint inhibits the metaphase-to-anaphase transition after uv irradiation in the absence of mec1. Mol. Cell. Biol. 24: 10126–10144. doi:10.1128/MCB.24.23.10126-10144.2004. PMID:15542824. Daley, J.M., Palmbos, P.L., Wu, D., and Wilson, T.E. 2005. Nonhomologous end joining in yeast. Annu. Rev. Genet. 39: 431–451. doi:10.1146/annurev.genet.39.073003.113340. PMID:16285867. Dasika, G.K., Lin, S.-C.J., Zhao, S., Sung, P., Tomkinson, A., and Lee, E.Y.-H.P. 1999. DNA damage-induced cell cycle checkpoints and DNA strand break repair in development and tumorigenesis. Oncogene, 18: 7883–7899. doi:10.1038/sj.onc.1203283. PMID:10630641. Donaldson, A.D. 2000. The yeast mitotic cyclin Clb2 cannot subPublished by NRC Research Press
Humpal et al. stitute for S phase cyclins in replication origin firing. EMBO Rep. 1: 507–512. PMID:11263495. Downs, J.A., Lowndes, N.F., and Jackson, S.P. 2000. A role for Saccharomyces cerevisiae histone H2A in DNA repair. Nature, 408: 1001–1004. doi:10.1038/35050000. PMID:11140636. Downs, J.A., Allard, S., Jobin-Robitaille, O., Javaheri, A., Auger, A., Bouchard, N., et al. 2004. Binding of chromatin-modifying activities to phosphorylated histone h2a at DNA damage sites. Mol. Cell, 16: 979–990. doi:10.1016/j.molcel.2004.12.003. PMID:15610740. Du, L.-L., Nakamura, T.M., and Russell, P. 2006. Histone modification-dependent and -independent pathways for recruitment of checkpoint protein Crb2 to double-strand breaks. Genes Dev. 20: 1583–1596. doi:10.1101/gad.1422606. PMID:16778077. Duda´sova´, Z., Duda´s, A., and Chovanec, M. 2004. Non-homologous end-joining factors of Saccharomyces cerevisiae. FEMS Microbiol. Rev. 28: 581–601. doi:10.1016/j.femsre.2004.06.001. PMID:15539075. Escargueil, A.E., Soares, D.G., Salvador, M., Larsen, A.K., and Henriques, J.A.P. 2008. What histone code for DNA repair? Mutat. Res. 658: 259–270. doi:10.1016/j.mrrev.2008.01.004. PMID:18296106. Feng, Q., Wang, H., Ng, H.H., Erdjument-Bromage, H., Tempst, P., Struhl, K., and Zhang, Y. 2002. Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain. Curr. Biol. 12: 1052–1058. doi:10.1016/S0960-9822(02) 00901-6. PMID:12123582. Fernandez-Capetillo, O., Lee, A., Nussenzweig, M., and Nussenzweig, A. 2004. H2ax: The histone guardian of the genome. DNA Repair (Amst.), 3: 959–967. doi:10.1016/j. dnarep.2004.03.024. PMID:15279782. Foster, E.R., and Downs, J.A. 2005. Histone H2A phosphorylation in DNA double-strand break repair. FEBS J. 272: 3231–3240. doi:10.1111/j.1742-4658.2005.04741.x. PMID:15978030. Gan, G.N., Wittschieben, J.P., Wittschieben, B.Ø., and Wood, R.D. 2008. DNA polymerase zeta (pol zeta) in higher eukaryotes. Cell Res. 18: 174–183. doi:10.1038/cr.2007.117. PMID: 18157155. Gontijo, A.M.D.M.C., Green, C.M., and Almouzni, G. 2003. Repairing DNA damage in chromatin. Biochimie, 85: 1133–1147. doi:10.1016/j.biochi.2003.10.018. PMID:14726019. Grenon, M., Magill, C.P., Lowndes, N.F., and Jackson, S.P. 2006. Double-strand breaks trigger MRX- and Mec1-dependent, but Tel1-independent, checkpoint activation. FEM. Yeast Res. 6: 836–847. doi:10.1111/j.1567-1364.2006.00076.x. Grenon, M., Costelloe, T., Jimeno, S., O’Shaughnessy, A., Fitzgerald, J., Zgheib, O., et al. 2007. Docking onto chromatin via the Saccharomyces cerevisiae Rad9 tudor domain. Yeast, 24: 105–119. doi:10.1002/yea.1441. PMID:17243194. Gudmundsdottir, K., and Ashworth, A. 2006. The roles of BRCA and BRCA2 and associated proteins in the maintenance of genomic stability. Oncogene, 25: 5864–5874. doi:10.1038/sj.onc. 1209874. PMID:16998501. Haber, J.E. 2006. Transpositions and translocations induced by sitespecific double-strand breaks in budding yeast. DNA Repair (Amst.), 5: 998–1009. doi:10.1016/j.dnarep.2006.05.025. PMID:16807137. Hammet, A., Magill, C., Heierhorst, J., and Jackson, S.P. 2007. Rad9 BRCT domain interaction with phosphorylated H2AX regulates the G1 checkpoint in budding yeast. EMBO Rep. 8: 851– 857. doi:10.1038/sj.embor.7401036. PMID:17721446. Harper, J.W. 2002. A phosphorylation-driven ubiquitination switch for cell-cycle control. Trends Cell Biol. 12: 104–107. doi:10. 1016/S0962-8924(01)02238-3. PMID:11859016.
251 Harrison, J.C., and Haber, J.E. 2006. Surviving the breakup: the DNA damage checkpoint. Annu. Rev. Genet. 40: 209–235. doi:10.1146/annurev.genet.40.051206.105231. PMID:16805667. Heideker, J., Lis, E.T., and Romesberg, F.E. 2007. Phosphatases, DNA damage checkpoints and checkpoint deactivation. Cell Cycle, 6: 3058–3064. PMID:18075314. Huyen, Y., Zgheib, O., Ditullio, R.A., Gorgoulis, V.G., Zacharatos, P., Petty, T.J., et al. 2004. Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks. Nature, 432: 406– 411. doi:10.1038/nature03114. PMID:15525939. Javaheri, A., Wysocki, R., Jobin-Robitaille, O., Altaf, M., Coˆte´, J., and Kron, S.J. 2006. Yeast G1 DNA damage checkpoint regulation by H2A phosphorylation is independent of chromatin remodeling. Proc. Natl. Acad. Sci. U.S.A. 103: 13771–13776. doi:10. 1073/pnas.0511192103. PMID:16940359. Kao, J., Milano, M.T., Javaheri, A., Garofalo, M.C., Chmura, S.J., Weichselbaum, R.R., and Kron, S.J. 2006. Gamma-H2AX as a therapeutic target for improving the efficacy of radiation therapy. Curr. Cancer Drug Targets, 6: 197–205. doi:10.2174/ 156800906776842957. PMID:16712457. Karagiannis, T.C., and El-Osta, A. 2007. Chromatin modifications and DNA double-strand breaks: the current state of play. Leukemia, 21: 195–200. doi:10.1038/sj.leu.2404478. PMID:17151702. Keogh, M.-C., Kim, J.-A., Downey, M., Fillingham, J., Chowdhury, D., Harrison, J.C., et al. 2006. A phosphatase complex that dephosphorylates gammaH2AX regulates DNA damage checkpoint recovery. Nature, 439: 497–501. doi:10. 1038/nature04384. PMID:16299494. Kouzarides, T. 2007. Chromatin modifications and their function. Cell, 128: 693–705. doi:10.1016/j.cell.2007.02.005. PMID: 17320507. Krebs, J.E. 2007. Moving marks: dynamic histone modifications in yeast. Mol. Biosyst. 3: 590–597. doi:10.1039/b703923a. PMID:17700858. Ku¨ntzel, H., Schulz, A., and Ehbrecht, I.M. 1996. Cell cycle control and initiation of DNA replication in Saccharomyces cerevisiae. Biol. Chem. 377: 481–487. PMID:8922282. Lancelot, N., Charier, G., Couprie, J., Duband-Goulet, I., AlphaBazin, B., Que´meneur, E., et al. 2007. The checkpoint Saccharomyces cerevisiae Rad9 protein contains a tandem tudor domain that recognizes DNA. Nucleic Acids Res. 35: 5898–5912. doi:10.1093/nar/gkm607. PMID:17726056. Lazzaro, F., Sapountzi, V., Granata, M., Pellicioli, A., Vaze, M., Haber, J.E., et al. 2008. Histone methyltransferase Dot1 and Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped telomeres. EMBO J. 27: 1502–1512. PMID:18418382. Lee, J.H., and Paull, T.T. 2007. Activation and regulation of ATM kinase activity in response to DNA double-strand breaks. Oncogene, 26: 7741–7748. doi:10.1038/sj.onc.1210872. PMID: 18066086. Liang, B., Qiu, J., Ratnakumar, K., and Laurent, B.C. 2007. Rsc functions as an early double-strand-break sensor in the cell’s response to DNA damage. Curr. Biol. 17: 1432–1437. doi:10. 1016/j.cub.2007.07.035. PMID:17689960. Luger, K. 2003. Structure and dynamic behavior of nucleosomes. Curr. Opin. Genet. Dev. 13: 127–135. doi:10.1016/S0959437X(03)00026-1. PMID:12672489. Luger, K. 2006. Dynamic nucleosomes. Chromosome Res. 14: 5– 16. doi:10.1007/s10577-005-1026-1. PMID:16506092. Luger, K., Ma¨der, A.W., Richmond, R.K., Sargent, D.F., and Richmond, T.J. 1997. Crystal structure of the nucleosome core particle at 2.8 e´ resolution. Nature, 389: 251–260. doi:10.1038/ 38444. PMID:9305837. McGinty, R.K., Kim, J., Chatterjee, C., Roeder, R.G., and Muir, Published by NRC Research Press
252 T.W. 2008. Chemically ubiquitylated histone H2B stimulates hDot1l-mediated intranucleosomal methylation. Nature, 453: 812–816. doi:10.1038/nature06906. PMID:18449190. Mendenhall, M.D., and Hodge, A.E. 1998. Regulation of Cdc28 cyclin-dependent protein kinase activity during the cell cycle of the yeast Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 62: 1191–1243. PMID:9841670. Millar, C.B., and Grunstein, M. 2006. Genome-wide patterns of histone modifications in yeast. Nat. Rev. Mol. Cell Biol. 7: 657–666. doi:10.1038/nrm1986. PMID:16912715. Moore, J.D., Yazgan, O., Ataian, Y., and Krebs, J.E. 2007. Diverse roles for histone h2a modifications in DNA damage response pathways in yeast. Genetics, 176: 15–25. doi:10.1534/genetics. 106.063792. PMID:17028320. Morrison, A.J., and Shen, X. 2005. DNA repair in the context of chromatin. Cell Cycle, 4: 568–571. PMID:15753656. Morrison, A.J., Highland, J., Krogan, N.J., Arbel-Eden, A., Greenblatt, J.F., Haber, J.E., and Shen, X. 2004. Ino80 and gamma-h2ax interaction links atp-dependent chromatin remodeling to DNA damage repair. Cell, 119: 767–775. doi:10.1016/j. cell.2004.11.037. PMID:15607974. Morrison, A.J., Kim, J.-A., Person, M.D., Highland, J., Xiao, J., Wehr, T.S., et al. 2007. Mec1/tel1 phosphorylation of the ino80 chromatin remodeling complex influences DNA damage checkpoint responses. Cell, 130: 499–511. doi:10.1016/j.cell.2007.06. 010. PMID:17693258. Nakamura, T.M., Du, L.-L., Redon, C., and Russell, P. 2004. Histone H2A phosphorylation controls Crb2 recruitment at DNA breaks, maintains checkpoint arrest, and influences DNA repair in fission yeast. Mol. Cell. Biol. 24: 6215–6230. doi:10.1128/ MCB.24.14.6215-6230.2004. PMID:15226425. Ng, H.H., Xu, R.-M., Zhang, Y., and Struhl, K. 2002. Ubiquitination of histone H2B by Rad6 is required for efficient Dot1-mediated methylation of histone H3 lysine 79. J. Biol. Chem. 277: 34655–34657. doi:10.1074/jbc.C200433200. PMID: 12167634. Niida, H., and Nakanishi, M. 2006. DNA damage checkpoints in mammals. Mutagenesis, 21: 3–9. doi:10.1093/mutage/gei063. PMID:16314342. O’Neill, B.M., Szyjka, S.J., Lis, E.T., Bailey, A.O., Yates, J.R., Aparicio, O.M., and Romesberg, F.E. 2007. Pph3-psy2 is a phosphatase complex required for rad53 dephosphorylation and replication fork restart during recovery from DNA damage. Proc. Natl. Acad. Sci. U.S.A. 104: 9290–9295. doi:10.1073/ pnas.0703252104. PMID:17517611. Paciotti, V., Lucchini, G., Plevani, P., and Longhese, M.P. 1998. Mec1p is essential for phosphorylation of the yeast DNA damage checkpoint protein Ddc1p, which physically interacts with Mec3p. EMBO J. 17: 4199–4209. doi:10.1093/emboj/17.14. 4199. PMID:9670034. Papamichos-Chronakis, M., Krebs, J.E., and Peterson, C.L. 2006. Interplay between Ino80 and Swr1 chromatin remodeling enzymes regulates cell cycle checkpoint adaptation in response to DNA damage. Genes Dev. 20: 2437–2449. doi:10.1101/gad. 1440206. PMID:16951256. Paull, T.T., Rogakou, E.P., Yamazaki, V., Kirchgessner, C.U., Gellert, M., and Bonner, W.M. 2000. A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr. Biol. 10: 886–895. doi:10.1016/S0960-9822(00) 00610-2. PMID:10959836. Pellicioli, A., and Foiani, M. 2005. Signal transduction: how Rad53 kinase is activated. Curr. Biol. 15: R769–R771. doi:10.1016/j. cub.2005.08.057. PMID:16169479. Redon, C., Pilch, D.R., Rogakou, E.P., Orr, A.H., Lowndes, N.F.,
Biochem. Cell Biol. Vol. 87, 2009 and Bonner, W.M. 2003. Yeast histone 2A serine 129 is essential for the efficient repair of checkpoint-blind DNA damage. EMBO Rep. 4: 678–684. doi:10.1038/sj.embor.embor871. PMID:12792653. Redon, C., Pilch, D.R., and Bonner, W.M. 2006. Genetic analysis of Saccharomyces cerevisiae H2A serine 129 mutant suggests a functional relationship between h2a and the sister-chromatid cohesion partners Csm3-Tof1 for the repair of topoisomerase Iinduced DNA damage. Genetics, 172: 67–76. doi:10.1534/ genetics.105.046128. PMID:16219777. Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S., and Bonner, W.M. 1998. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273: 5858–5868. doi:10.1074/jbc.273.10.5858. PMID:9488723. Ruthenburg, A.J., Li, H., Patel, D.J., and David Allis, C. 2007. Multivalent engagement of chromatin modifications by linked binding modules. Nat. Rev. Mol. Cell Biol. 8: 983–994. doi:10. 1038/nrm2298. PMID:18037899. San Filippo, J., Sung, P., and Klein, H. 2008. Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 77: 229– 257. doi:10.1146/annurev.biochem.77.061306.125255. PMID: 18275380. Shahbazian, M.D., Zhang, K., and Grunstein, M. 2005. Histone H2b ubiquitylation controls processive methylation but not monomethylation by Dot1 and Set1. Mol. Cell, 19: 271–277. doi:10.1016/j.molcel.2005.06.010. PMID:16039595. Shim, E.Y., Ma, J.-L., Oum, J.-H., Yanez, Y., and Lee, S.E. 2005. The yeast chromatin remodeler rsc complex facilitates end joining repair of DNA double-strand breaks. Mol. Cell. Biol. 25: 3934–3944. doi:10.1128/MCB.25.10.3934-3944.2005. PMID: 15870268. Shrivastav, M., De Haro, L.P., and Nickoloff, J.A. 2008. Regulation of DNA double-strand break repair pathway choice. Cell Res. 18: 134–147. doi:10.1038/cr.2007.111. PMID:18157161. Shroff, R., Arbel-Eden, A., Pilch, D., Ira, G., Bonner, W.M., Petrini, J.H., et al. 2004. Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break. Curr. Biol. 14: 1703–1711. doi:10.1016/j.cub.2004.09.047. PMID:15458641. Siede, W., Friedberg, A.S., and Friedberg, E.C. 1993. Rad9dependent g1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 90: 7985–7989. doi:10.1073/pnas.90. 17.7985. PMID:8367452. Sopko, R., Huang, D., Smith, J.C., Figeys, D., and Andrews, B.J. 2007. Activation of the Cdc42p gtpase by cyclin-dependent protein kinases in budding yeast. EMBO J. 26: 4487–4500. doi:10. 1038/sj.emboj.7601847. PMID:17853895. Soulier, J., and Lowndes, N.F. 1999. The BRCT domain of the S. cerevisiae checkpoint protein rad9 mediates a rad9-rad9 interaction after DNA damage. Curr. Biol. 9: 551–554. doi:10.1016/ S0960-9822(99)80242-5. PMID:10339432. Stewart, G.S., Wang, B., Bignell, C.R., Taylor, A.M., and Elledge, S.J. 2003. MDC1 is a mediator of the mammalian DNA damage checkpoint. Nature, 421: 961–966. doi:10.1038/nature01446. PMID:12607005. Sweeney, F.D., Yang, F., Chi, A., Shabanowitz, J., Hunt, D.F., and Durocher, D. 2005. Saccharomyces cerevisiae Rad9 acts as a Mec1 adaptor to allow rad53 activation. Curr. Biol. 15: 1364– 1375. doi:10.1016/j.cub.2005.06.063. PMID:16085488. Takai, H., Wang, R.C., Takai, K.K., Yang, H., and De Lange, T. 2007. Tel2 regulates the stability of pi3k-related protein kinases. Cell, 131: 1248–1259. doi:10.1016/j.cell.2007.10.052. PMID: 18160036. Published by NRC Research Press
Humpal et al. Unal, E., Arbel-Eden, A., Sattler, U., Shroff, R., Lichten, M., Haber, J.E., and Koshland, D. 2004. DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain. Mol. Cell, 16: 991–1002. doi:10. 1016/j.molcel.2004.11.027. PMID:15610741. Van Attikum, H., Fritsch, O., Hohn, B., and Gasser, S.M. 2004. Recruitment of the ino80 complex by h2a phosphorylation links ATP-dependent chromatin remodeling with DNA double-strand break repair. Cell, 119: 777–788. doi:10.1016/j.cell.2004.11. 033. PMID:15607975. Van Attikum, H., Fritsch, O., and Gasser, S.M. 2007. Distinct roles for Swr1 and Ino80 chromatin remodeling complexes at chromosomal double-strand breaks. EMBO J. 26: 4113–4125. doi:10.1038/sj.emboj.7601835. PMID:17762868. Van Leeuwen, F., Gafken, P.R., and Gottschling, D.E. 2002. Dot1p modulates silencing in yeast by methylation of the nucleosome core. Cell, 109: 745–756. doi:10.1016/S0092-8674(02)00759-6. PMID:12086673. Verdone, L., Agricola, E., Caserta, M., and Di Mauro, E. 2006. His-
253 tone acetylation in gene regulation. Brief Funct. Genomic Proteomic. 5: 209–221. doi:10.1093/bfgp/ell028. PMID:16877467. Wang, H., Cao, R., Xia, L., Erdjument-Bromage, H., Borchers, C., Tempst, P., and Zhang, Y. 2001. Purification and functional characterization of a histone H3-lysine 4-specific methyltransferase. Mol. Cell, 8: 1207–1217. doi:10.1016/S1097-2765(01) 00405-1. PMID:11779497. Watrin, E., and Peters, J.-M. 2006. Cohesin and DNA damage repair. Exp. Cell Res. 312: 2687–2693. doi:10.1016/j.yexcr.2006. 06.024. PMID:16876157. Wysocki, R., Javaheri, A., Allard, S., Sha, F., Coˆte´, J., and Kron, S.J. 2005. Role of dot1-dependent histone H3 methylation in G1 and S phase DNA damage checkpoint functions of Rad9. Mol. Cell. Biol. 25: 8430–8443. doi:10.1128/MCB.25.19.84308443.2005. PMID:16166626. Wysocki, R., Javaheri, A., Kristjansdottir, K., Sha, F., and Kron, S.J. 2006. Cdk Pho85 targets cdk inhibitor Sic1 to relieve yeast G1 checkpoint arrest after DNA damage. Nat. Struct. Mol. Biol. 13: 908–914. doi:10.1038/nsmb1139. PMID:16964260.
Published by NRC Research Press
