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Signalling DNA Damage

Written By

Andres Joaquin Lopez-Contreras and Oscar Fernandez-Capetillo

Submitted: April 17th, 2012 Published: September 6th, 2012

DOI: 10.5772/50863

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1. Introduction

1.1. Types and sources of DNA damage

During our lifetime, the genome is constantly being exposed to different types of damage caused either by exogenous sources (radiations and/or genotoxic compound) but also as byproducts of endogenous processes (reactive oxigen species during respiration, stalled forks during replication, eroded telomeres, etc).

From a structural point of view, there are many types of DNA damage including single or double strand breaks, base modifications and losses or base-pair mismatches. The amount of lesions that we face is enormous with estimates suggesting that each of our 1013 cells has to deal with around 10.000 lesions per day [1]. While the majority of these events are properly resolved by specialized mechanisms, a deficient response to DNA damage, and particularly to DSB, harbors a serious threat to human health [2].

DSB can be formed [1] following an exposure to ionizing radiation (X- or γ-rays) or clastogenic drugs; [2] endogenously, during DNA replication, or [3], as a consequence of reactive oxygen species (ROS) generated during oxidative metabolism. In addition, programmed DSB are used as repair intermediates during V(D)J and Class-Switch recombination (CSR) in lymphocytes [3], or during meiotic recombination [4]. Because of this, immunodeficiency and/or sterility problems are frequently associated with DDR-related pathologies.


2. DNA repair of DSB. NHEJ and HR

Mammalian cells are equipped with two mechanisms that repair DSB and prevent dangerous chromosomal rearrangements; [1] Non-homologous end joining (NHEJ), which links together the two ends of broken DNA by direct ligation, and [2] Homologous Recombination (HR), which is only available S and G2 phases when a sister chromatid can be used as a template for the repair reaction (see Figure 1).

Figure 1.

DSBs repair mechanisms. A) NHEJ. DSBs are sensed by the ring-shape heterodimer Ku70/Ku80 which then stabilizes the two DNA ends and recruits DNA-PK. Next, DNA-PK phosphorylates and activates the NHEJ effector complex (ligase IV/XRC44/XLF) that finally religates the broken DNA. B) HR. The ATM kinase is recruited to DSB via an interaction with the MRN (Mre11-Rad50-Nbs1) complex. Once at the break, ATM that becomes activated, phosphorylating multiple substrates. In a reaction that depends in multiple endo and exonuclease activities (including Mre11, Exo1 and CtIP) DSBs are resected forming ssDNA strands. These ssDNA regions attract Rad51 and other associated proteins. The Rad51-coated nucleoprotein filaments then invade the undamaged sister strands forming HJ structures. HR is completed by new DNA synthesis and still to be identified HJ “resolvase” enzymes.

2.1. Non Homologous End Joining (NHEJ)

NHEJ is simpler and faster than HR, and can take place in any cell cycle stage. However, NHEJ can process altered or improper ends is thus more error-prone that HR. Therefore, HR is the preferred repair mechanisms during S/G2 phases. However, due to the preponderance of the G0/G1 stage, NHEJ is key to safeguard genomic integrity in mammalian organisms. Moreover, and probably due to the larger genomes and abundant repetitive sequences which limit HR proficiency, mammalian genomes are also frequently healed by NHEJ in S/G2 [5].

The mechanism by which NHEJ rejoins a DSB is fairly understood. First, DSB are recognized by a heterodimer formed by Ku70 and Ku80 proteins [6]. Ku proteins display a ring-like structure and are able to bind and stabilize the ends of DSB, acting as sensors, and subsequently attracting the transducer kinase, DNA-PK (DNA dependent protein Kinase). DNA-PK is a large Ser/Thr kinase (about 470 Kda), which belongs to the PIKK family (PI3-kinase like family of protein kinases). Other members of this family, ATM (ataxia talangectasia mutated) and ATR (ATM and RAD3-related), are key kinases in the DDR and will be explained in futher detail below. Once DNA-PK is located to the DSB it becomes activated and phosphorylates several substrates involved in the processing and ligation of DNA ends. Significantly, DNA-PK can also auto-phosphorylate in several residues, which seems to be essential for its repair capacity [7].

Finally, the main effectors of NHEJ form a multimeric complex (DNA Ligase IV-XRCC4-XLF), which is recruited to DSB and activated by DNA-PK dependent phosphorylation. This complex is responsible for the actual rejoining of the ends [8]. Ligase IV provides the catalytic activity for the rejoining, while XRCC4 stabilizes and stimulates its activity [9].

Ku70 and Ku 80 (sensors), DNA-PK (transducer) and DNA ligase IV and XRCC4 (effectors) are the core component of NHEJ. But often other accessory components are required for the conversion of altered broken ends. For instance, DSB produced by IR usually requires an end-processing step prior to relegation, such as the one regulated by Artemis, a 5’- 3’ exonuclease, that acquires endonuclease activity when phosphorylated by DNA-PK [10]. This activity of Artemis is important to open closed hairpins in which the two strands of the DSB have linked together. Importantly, such hairpins are normally generated at the DSB generated by RAG nucleases during V(D)J recombination. Note that ATM can also induce the phosphorylation and activation of Artemis [11]. In fact, and in addition to be important for the repair of accidental DSB, NHEJ carries out several specialized functions in lymphocytes during V(D)J and CSR [12, 13]. Accordingly, patients with mutation in Artemis, ATM or DNA-PKcs, as well as in other NHEJ components, often suffer from immune deficiencies as well as from an increased predisposition to cancer [14].

Another context in which difficult-to repair DSB are formed relates to chromatin accessibility. For instance, it is estimated that around 10% of radiation induced DSB are repaired with slow kinetics, this population being enriched adjacent to compacted chromatin (heterochromatin). In this case, ATM and not DNAPK will play an important role in the NHEJ-mediated repair of these difficult-to-repair breaks. In this context, ATM activation is necessary to phosphorylate some substrates like H2AX or KAP1, which would relax chromatin compaction allowing the access of repair proteins to DSB at heterochromatin [11, 15]. Nevertheless, it should be noted that the main cell-type that suffers from ATM deficiency is Purkinje cells which are almost devoid of heterochromatin, so that ATM must play additional roles in euchromatin [16].

2.2. Homologous recombination

The other mechanism that cells use for DSB repair, and the most important for replicating cells, is Homologous Recombination. In contrast to NHEJ, during HR DSB need to be first extensively processed to allow for the search of the homologous undamaged template. First, ends undergo 5’-3´ resection producing two single stranded DNA regions. Rad51-coated ssDNA stretches invade the sister chromatid DNA duplex forming inter-strand structures. How these heteroduplexes are finally resolved is still not fully characterized.

Since HR repairs DSB using the sister chromatid as template, it is restricted to S and G2 phases. Restricting HR in G1 is in fact critical, since if HR takes place in G1 it could lead to loss of heterozigosity (LOH) or chromosomal translocations [17]. To avoid this problem the activity of several HR components is regulated by several S-G2 specific CDKs (cyclin dependent kinases) [18].

For teaching purposes, and trying to simplify the complex process that takes place during HR, we will artificially divide it in 4 independent stages. We acknowledge that many of the stepwise events described here are still to be fully validated, but just want to transmit a holistic view of how a DSB can be sensed, signaled and repaired by HR:

Recruitment to the DSB: Foci forming factors

The Mre11-Rad50-Nbs1 (MRN) complex is one of the first in being recruited to DSB [19]. Next, through a direct interaction, MRN recruits ATM which, among other things, phophorylates H2AX (a variant of the canonical histone H2A) at serine 139, forming γH2AX [20]. Direct phospho-binding to γH2AX recruits the adaptor protein MDC1 [21], which through interaction with ATM and Nbs1 forms a positive feedback loop that amplifies the γH2AX signal [22]. In addition, ATM-dependent phosphorylation of MDC1 is recognized by the E3 ubiquitin ligase RNF8, that ubiquitinates histones in the vicinity of the DSB [23, 24]. Another E3 ubiquitin ligase, RNF168 (mutated in the human RIDDLE immunodeficiency syndrome), is also recruited and amplifies the ubquitylation signal, in both cases mediated by UBC13 E2 ligase, eventually recruiting 53BP1 and BRCA1 to DSB-associated foci [25-27]. In the case of BRCA1, RAP80 binds to ubiquinylated substrates at DSB through its UIM (Ubiquitin Interaction Motif) [23]. RAP80 then interacts with Abraxas [28], the deubiquitylating (DUB) enzyme BRCC36 and BRCA1 (breast cancer 1, early onset). BRCA1 is a well known human breast cancer susceptibility gene, responsible for the third part of familiar breast cancer [29]. BRCA1 is a large protein (over 200 kDa) that is constitutively bound to BARD1. Together, BRCA1/BARD1 present E3 ubiquitin ligase activity [30]. Although BRCA1 has been the subject of many studies and it is known to participate in HR, its exact role(s) and whether its ubiquitin ligase activity is critical for its activity is still not well understood. Whereas the road from DSB to BRCA1 is more understood, how 53BP1 is loaded to substrates modified by RNF8/RNF168 remains unknown.

Importantly, it should be noted that all of these events have been inferred from the formation or absence of foci at DSB and, to date, it is not clear to what extent capacity to recruit to foci represents an accurate measurement of the function of the protein [31].

Processing of the end: Resection

As mentioned before, the resection of the two 5’ ends of the DSB is an essential step for HR. Three exonucleases (Mre11, Exo1 and CtIP) and one helicase (BLM) have been involved in resection [32-34]. Importantly, ATM dependent phosphorylation seems to stimulate CtIP-mediated resection, linking DNA damage signaling with the processing of DNA ends [35]. In addition to ATM, ssDNA is quickly coated by RPA (replication protein A) and triggers a parallel pathway resulting in the activation of the kinase ATR [18, 36-38], that will be described below. It remains to be seen to what extent ATM, ATR, and perhaps DNA-PK collaborate in the processing of DSB for HR. One important recent development is the finding that, against all expectations, 53BP1 might limit resection [39]. This would explain how the absence of 53BP1 rescues the cancer and ageing phenotypes of a BRCA1 mutant mouse model, which could be explained by an improved HR through enhanced resection [40].

Sister chromatid invasion

The initiation of recombination requires the loading of the recombinase Rad51, which replaces RPA coating on the ssDNA. Rad51, a protein with DNA-dependent ATPase activity, together with other associated proteins (DSS1, RAD52,RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, RAD54, etc.) form a nucleoprotein filament that mediates strand invasion of the sister chromatid [41]. In mammals, Rad51 loading is mediated by BRAC2 [42]. Once again, BRCA2 is another important breast cancer susceptibility gene suggesting a critical role of HR in the prevention of breast cancer [43]. In contrast to BRCA1, BRCA2 recruitment to DSB is not fully understood, one possible scenario being that BRCA1 could mediate the recruitment of FancD2 and BRCA2 to DSB [44].

The nucleoprotein filament (ssDNA, RAD51 and other HR proteins) searches for an homologous region within the duplex DNA in the sister chromatid and displaces a DNA strand forming a D-loop structure and the 3’ end of the invading strand. This invading end then is extended by un unknown DNA polymerase (in vitro data indicates that DNA pol eta may be implicated [45]). The “X” shaped structure formed as a consequence of this invading process is called a Holliday Junction (HJ) [46]. When the other 3’ end of broken DNA enters the D-loop a double Holliday junction structure is formed. Noteworthy, whereas this model was proposed almost half a century ago, it was only recently that evidence for the double Holliday junction intermediates of repair was found [47]


If HJ formation was remained elusive, the resolution of Holliday junctions is even a bigger mistery, many proteins having been proposed as candidates to be the HJ “resolvase”. Such a protein should have helicase and nuclease activities, like BLM (Bloom syndrome protein), MUS81-EME1 complex or GEN1 [48-51]. All of them have been proposed to be the missing HJ-resolvase at some point. Moreover, recent evidence suggests that another enzyme, SLX4, could be such missing link [52, 53]. Whereas it seems clear that many proteins can deal with HJ-resembling structures in vitro, only clean genetic data will finally resolve this “resolution” problem. We should mention that although the HR classical model consists in the formation of these two Holliday Junctions, there are other alternative models that explain the HR process.

In addition to its role at DSB, HR also has a key role in restarting stalled replication forks (RF). SSBs, interstrand crosslinks or other lesions in the DNA promote stalling of the RF which, if persistent, could lead to breakage that has to be resolved by HR [54]. ATR, another PIKK kinase that is essential for an appropriate progression of RF, may be regulating HR in this case. In fact, ATR and its target kinase Chk1 phosphorylate a number of HR-related proteins such as BRCA1, BLM and Rad51 [55-57].

HR also participates in the maintenance of telomere length, by promoting inter-telomeric HR in the absence of telomerase. This pathway is known as alternative lengthening of telomeres (ALT) [58]. Finally, HR is the repair pathway of the recombination reaction that occurs between homologous chromosomes in meiosis, where DSB are intentionally generated in order to increase genetic variability.

Whereas we here provided a simplified view of DSB repair, it should be noted that other still poorly understood pathways of repair such as micro-homology directed NHEJ, which joins partially resected DSB, are emerging as important genome caretakers.


3. Signaling DSB

We here present a general view of how cells signal DSB, with a particular emphasis on phosphorylation, which is the most known mechanism to date.

3.1. Phosphorylation. The so-called DNA Damage Response (DDR)

The so-called DDR stands for a coordinated signaling response which starts at the DSB and which promotes DNA Repair while it limits the expansion of the damaged cell by apoptotic or cytostatic mechanisms. The canonical DDR begins with the activation of two PIKK, ATM and ATR, upon detection of DSB. The role of DNA-PK, another member of the PIKK family, is mainly to stimulate repair activities locally at the break but without triggering a cellular response. Thus, ATM and ATR are considered the main upstream kinases in the signaling of DNA damage. Whereas they are activated by different types of damage and have some specific substrates and functions, their “response” is frequently interconnected. The activation of any of these kinases starts a phosphorylation cascade leading to cell cycle arrest (Checkpoints). Among others, the tumor suppressor p53 plays a central role in coordinating the apoptotic and checkpoints initiated by DNA damage.

In the last decade the knowledge about the DDR has substantially increased, and besides the classical activation of the PIKK and phosphorylation cascades, other types of signaling such as ubiquitylation or sumoylation have burst [59]. A general overview of this complex pathway will be explained below.

3.1.1. Central players of the DDR: PIKK kinases: ATM, ATR and DNA-PK

The DDR has a well defined hierarchy. Their components have been classified into sensors, transducers, mediators and effectors. ATM and ATR kinases are considered the main DDR transducers.

As mentioned, ATM, ATR and DNA-PK belong to the PIKK (phosphatidylinositol-3-kinase related kinases) family [60]. They are large proteins (more than 300 kDa) with similar structure: a variable number of repeat domains in the N-terminus (HEAT domains), a FAT domain, a catalytic domain homologue to that in PI-3-Kinase (PI3K), a PIKK regulatory domain (PRD) and a FATC domain at the very C-terminus [61]. The PRD domain has been shown to mediate kinase activation, at least for ATR; and HEAT, FAT and FATC domains may be involved in the specific interactions of these proteins with their substrates and modulators [62].

ATM, ATR and DNA-PK specifically phosphorylate serine and threonine residues frequently followed by a glutamine. Upon activation, they are able to phosphorylate hundreds of common substrates regulating many cellular functions [63]. Noteworthy, some of them are preferred by one specific kinase, which is likely due to mediators. For instance, whereas all three can phosphorylate H2AX, Chk1 is only phosphorylated by ATR and this is explained by the need of Claspin in mediating the ATR/Chk1 interaction.

Despite their similarities, these kinases play different roles in the DDR. In brief, DNA-PK is activated in response to DSB and promotes their repair by NHEJ. However, if DSB are located in heterochromatin or they are too numerous, ATM activates additional repair mechanism (e.g. HR) and starts checkpoints signaling. In addition, ATR is activated by ssDNA, generated when DSB persist and are ressected into ssDNA, or when a replication fork is collapsed, promoting a strong checkpoint signaling. Besides, whereas ATM and DNA-PK are active through all the cell cycle, ATR activity is confined only to S and G2 phases [64]. One possible scenario is that the activity of the three kinases follows a stepwise activation if a DSB persist. This model is explained below.


a. DNA-PK. Stimulation of DNA Repair

The first step in this model will be the immediate activation of DNA-PK at a DSB. Previously in this chapter, we described how DNA-PK is activated and promotes NHEJ. In summary, the dimer Ku70/Ku80 binds and stabilizes the DSB ends, it recruits DNA-PK that activates the NHEJ effectors, LigaseIV-XRCC4-XLF. Thus, DNA-PK will provide a fast repair mechanism for easy-to-repair breaks, without the activation of checkpoint activities. It must be emphasized again, that DNA-PK has a very limited role in checkpoint signaling. However in the absence of ATM, DNA-PK may stay longer at the DSB and contribute to checkpoint functions [65].


b. ATM. DSB sensing and ATM Activation

If the DSB persist, one plausible scenario is that ATM could phosphorylate and displace DNA-PK from the DSB, so that repair and checkpoint activities are implemented. ATM is the most studied and likely best known DDR kinase. One reason for this is that its absence is responsible for the human Ataxia-Telangiectasia (A-T) hereditary disorder [66]. A-T is a severe autosomal recessive disease characterized by early onset progressive cerebellar ataxia, oculocutaneous telangectasia, immunodeficiency and lymphoid tumours [67]. Immunodeficiency and predisposition to lymphoid tumours is explained by the role of ATM during T and B lymphocyte development, where DSB are generated as by-products of immune rearrangements. Besides, cells of these patients present numerous breaks, what explains their increase predisposition also to other cancer types.

Whereas it seems clear that the main role of ATM is related to the DDR [68], the two symptoms that named the disease, ataxia and telangiectasia, are still not explained by the role of ATM in relation to DNA damage, suggesting that ATM may have other functions not related to DSB-signaling. Moreover, even though we have significant understanding of the effects of ATM activation, how this is accomplished is still a matter of debate.

As described above, ATM activation is triggered by the recruitment of the MRN complex (sensor) in response to DSB (Figure 2). MRN complex recruits ATM and collaborates for its activation [69]. However, the precise mechanism by which ATM becomes activated is still a matter of controversy [62]. Some authors claimed that, in basal conditions, ATM is an inactive homodimer which undergoes dissociation and activation in response to DSB, due to its autophosphorylation [70]. Importantly, it has later been shown that ATM autophosphorylation is not necessary for its activation in vivo [71, 72].

Figure 2.

Mechanisms of ATM and ATR activation. A) ATM activation. DSBs (red asterisk) are sensed by the MRN (Mre11-Rad50-Nbs1) complex which attracts ATM and probably contributes to its activation. It is not well established how ATM is converted from its inactive to active status, one possibility being that it changes from an inactive dimer to an active monomeric form. B) ATR activation. The input for ATR activation is ssDNA, which can derive from resected DSB (left), or from stalled replication forks (right). RPA coated ssDNA loads the ATRIP-ATR complex and the 9-1-1 complex, which brings together ATR and its allosteric activator TopBP1.

Nbs1, part of the MRN complex, could contribute to ATM phosphorylation and activation [73], and several other proteins, such as the histone acetyl transferase Tip60 [74], have been reported to be involved in ATM activation. In vitro, ATM can be activated in the absence of MRN members. However, the MRN complex amplifies ATM activity and collaborates for an optimal ATM accumulation surrounding the DSB [75]. Noteworthy, defects in two members of the MRN complex cause A-T related syndromes: Nijmegen breakage syndrome (NBS1 mutants) and A-T-like disorder (MRE11 mutants) [76, 77].

ATM has been claimed to be activated by other stimuli rather than DNA damage, like chloroquine or osmotic shock [70]. However, whether those stimuli do not generate damage, particularly during replication, remains to be seen. There are some evidences of a cytoplasmic fraction of ATM that might be helpful to explain part of the symptoms of A-T disease not related to the DDR like the early neurodegeneration. Of note, ATM in neuronal cells has been found predominantly cytosolic [78].


c. ATR. Activation by ssDNA

The last (in fact, its activation occurs later than ATM) main PIKK kinase in the signaling of DSB is ATR. As noted before, despite their homology, ATR responds to different types of stresses than ATM does, and although these two kinases share several substrates, ATR regulates different processes. ATR has an essential role during replication, sensing alterations in fork progression and activating cellular checkpoints if necessary [79, 80].

In contrast to ATM, ATR is essential at the organism and the cellular level [81]. Consequently there is not any human disease lacking ATR. However, patients of a very rare ATR-related disease known as the Seckel syndrome are alive with very low amounts of ATR [82].

The input for ATR is not the DSB itself, but rather unusual large strands of ssDNA that can be generated in several circumstances [64]:

RS (replication stress) is a not well defined concept that refers to a variety of alterations in the normal progression of the replication fork, caused by lesions encountered in the DNA (SSBs, crosslinks, base adducts), dNTPs deficiency or other problems at the RF. When RS is prolonged or, in the absence of ATR, RF collapse and DSB are generated. Notice that there is no such a thing as RS-free replication, and thus ATR is essential to complete replication even in the absence of exogenous DNA damage.

End resection of DSB. Initially DSB lead to DNA-PK and ATM activation. However if DSB occurs in S or G2 phases and they persist enough, DSB are resected by ATM-stimulated exonucleases generating strands of ssDNA which then activate ATR.

Telomeres. ssDNA can be also generated at uncapped telomeres that have lost its capping function, leading to ATR activation.

NER (nucleotide excision repair). The process of NER can generate patches of ssDNA as repair intermediates, which lead to the activation of ATR, perhaps even in G1 [83].

In contrast to ATM, the molecular mechanism of ATR activation by ssDNA is well established (Figure 2). RPA-coated ssDNA [84] recruits the ATR-ATRIP (ATR interacting protein) complex [85]. At the same time, Rad17 and subsequently the 9-1-1 (Rad9-Rad1-Hus1] complex are also brought to the damage sites by RPA [86]. The 9-1-1 complex, with a PCNA-like clamp conformation, then brings the allosteric activator TopBP1 (topoisomerase-binding protein 1] into close proximity of ATR [87]. The interaction of ATR with TopBP1 is then sufficient to unleash ATR activity [88]. Of note, activation of ATR by TopBP1 even in the absence of DNA breaks is sufficient to promote a robust cellular response including senescence, demonstrating the key role of the DDR in responding to DNA damage [89].

3.1.2. Mediators and DDR amplification. Foci forming factors

Downstream of the PIKK, DDR signaling is amplified by several mechanisms, allowing the response to achieve its final cellular outcomes. On one hand, the DDR, like any other phosphorylation cascade, amplifies its signal in subsequent (enzymatic) step. This involves the participation of mediators, which are proteins acting downstream of the transducer kinases ATM and ATR. Several substrates, regulators, recruiters and others proteins acting as scaffold are considered mediators (e.g. Mdc1, 53BP1, MRN complex, Claspin, BRCA1), and, they modulate the activation of the effectors. Another mean of amplifying the signal, is an intense accumulation of many of their components surrounding the DNA lesions in spots so-called IRIF (Ionizing Radiation Induced Foci).

Once ATM or DNA-PK are activated, the rapid phosphorylation of the histone H2AX (γH2AX) is key for subsequent events. The γH2AX mark is accumulated in such a large amount in the proximity of DSB that can be visualized in spots (IRIF) by standard immunofluorescence techniques. Many DDR proteins (MRN complex, ATM, 53BP1, BRCA1 and others) form IRIF that co-localize with γH2AX foci. Actually, γH2AX modulates the accumulation of repair and signaling proteins in chromatin regions distal to the DSB. However, this has nothing to do with the recruitment of the proteins to the DSB, and rather by altering chromatin conformation in the vicinity of the break [90]. But what are foci good for? One likely possibility is that the accumulation of DDR proteins surrounding DNA lesions is necessary to build a signaling threshold in conditions of limited damage [91].

Among the different factors, 53BP1 is probably one of the most studied examples of an IRIF forming protein. 53BP1 has a pan-nuclear location which, after exposure to genotoxic agents, is quickly repositioned to IRIF. 53BP1 localization into foci is dependent on several upstream events including H2AX phosphorylation [90], recruitment of MDC1, ubiquitinating activity of RNF8 and RNF168 [23, 25, 92, 93], and methylation of histones H3 and H4 [94, 95]. Regardless of their importance for our understanding of the DDR, the absence of foci does not seem to be essential for a proficient DDR. Thus, mouse models lacking H2AX or MDC1, that abolishes IRIF formation, as well as 53BP1 null mice, are viable, but they exhibit phenotypes related to DDR deficiency like genome instability, cancer predisposition or immunodeficiency [22, 96, 97]. In summary, while not essential, IRIF seem to modulate the amplitude of the signaling of DSB which might be important in conditions of low numbers of DSB.

In contrast to ATM, ATR does not undergo any post-translational modifications which would modify its activity, such as autophosphorylation. Nevertheless, a number of mediators of ATR signaling are indeed regulated through phosphorylation. For instance, the ATR activator TopBP1 is phosphorylated by ATM at resected DSB [98]; and probably also by ATR in response to RS. In addition, Claspin phosphorylation is also required for ATR-dependent phosphorylation of Chk1 in response to DSB [99].

In summary, ATM and ATR signaling are amplified and driven by these and many other mediators. The endpoint of the signaling cascade arrives with the activation of the effectors, which would finally be responsible for the cellular responses.

3.1.3. Effectors and cellular outcomes of the DDR

In addition to promote the repair of DNA lesions, the DDR can orchestrate multiple cellular responses orientated to safeguard genome integrity or, in some cases, to avoid transmission of harmful alterations by activating apoptosis or senescence. In this context, one of the main effectors of the DDR is the transcription factor p53 which provides a late sustained response to DNA damage. p53 up-regulation contributes to the activation of checkpoints, and, if the damage persists, may activate senescence, apoptosis or cell differentiation programmes.

Regardless of this p53-centric view, it is clear that many p53-independent pathways are also stimulated by the DDR. For instance, a single proteomic study identified more than 700 ATM/ATR phosphorylation substrates, pointing out the wide variety of effectors and pathways that are regulated by the DDR [63].

Given that there are multiple reviews elsewhere that focus on these cellular responses, we here only briefly describe the main cellular responses promoted by the DDR: cell cycle checkpoints, senescence, apoptosis and differentiation. Transient cell cycle delay: Checkpoints

The transitions through the different stages of the cell cycle are tightly regulated by the activity of cyclin-dependent kinases (CDKs). CDKs are activated by cyclins and inhibited by CDK inhibitors (CKIs) or inhibitory tyrosine phosphorylations [100]. In brief, a simplified scheme depicts that four CDKs are involved in the regulation of cell cycle; CDK2, CDK4 and CDK6 during interphase, and CDK1 being considered the mitotic CDK. An activated DDR can limit the activity of CDKs and thus prevent cell cycle progression into the next stage (G1/S and the G2/M). In addition, the DDR can also slow down replication (intra-S checkpoint), but this is not a full stop and cells with damage in S-phase progress into G2 stop at the G2/M checkpoint [101]. The way that the DDR gets to the CDKs is through effectors that limit CDK activity which we now briefly summarize.

First, the main PIKK targets that regulate checkpoints are the so-called checkpoint kinases Chk1 and Chk2, direct substrates of ATR and ATM respectively [102, 103]. Checkpoint kinases act by regulating CDK inhibitory effectors such as Cdc25a, Wee1 or p53. Cdc25a is a phosphatase that controls CDK1 and CDK2 activities. In response to DNA damage, ATR-activated Chk1 phosphorylates Cdc25a inducing its degradation [104, 105], and thereby, inhibition of CDK activity [106]. An opposing force for Cdc25a is Wee1, the kinase that counteracts Cdc25a activity. Finally, a major mediator of cellular responses to DNA damage is p53. p53 is rapidly stabilized upon DNA damage by a number of inter-dependent PTMs. ATM, ATR, Chk1 and Chk2 are all able to phosphorylate p53 contributing to its stabilization [107, 108]. Besides its apoptotic targets, p53 has a number of transcriptional targets that contribute to checkpoint function, perhaps the most notorious being the CKI p21cip1 which is an important regulator of the G1/S checkpoint.

Specifically, the G1/S checkpoint avoids the replication of damaged DNA (see Figure 3 for a scheme of checkpoint responses). The transition from G1 to S is directed by an increase in CDK2 activity induced by cyclin E [109]. As such, the G1/S checkpoint is directed to limit this burst in CDK activity. An early and late component checkpoint can be separated. First, the G1/S checkpoint is initiated by degradation of Cdc25a, leading to the accumulation of inhibitory phosphorylations on CDK2 [110, 111]. This is an early response that does not require the synthesis of new proteins. If the damage persists, p53-dependent transcriptional upregulation of p21cip1 would implement a stronger G1/S arrest [112].ATM and Chk2, but not ATR/Chk1 are the main DDR mediators of the G1/S transition.

Figure 3.

Chekpoint regulation by the DDR. ATM and ATR orchestrate a transitory delay of the cell cycle in response to DSBs and ssDNA, respectively. Wherease direct phosphorylation of of cdc25a and wee1 allow a rapid establishment of the G1/S and G2/M checkpoints, p53-dependent regulation contributes to checkpoint maintenance at later timepoints. During S and G2 phases DSBs can be resected leading to the generation of ssDNA, which also activates ATR-signaling.

If damage arises in S phase, the intra-S checkpoint is also mainly regulated by the phosphatase Cdc25a, but now under de control of ATR and Chk1 [102]. In addition, DDR-promoted replication inhibition is in part mediated by Cdt1, which participates in the loading of MCM helicases that facilitate DNA unwinding ahead of the RF [113]. How the DDR promotes Cdt1 degradation and thus replication control is yet uncharacterized.

The last key control point is the G2/M checkpoint, that “checks” genome integrity before proceeding into chromosome segregation. Here, the transition from G2 into M is governed by an increase cyclinB-CDK1 activity. Once again, this checkpoint is also established by Cdc25a phosphorylation, in this case mediated by the ATR-dependent Chk1 kinase. In addition, the kinase Wee1 also contributes to the G2/M checkpoint. In a normal G2/M transition Plk1 (Polo kinase 1) phosphorylates Wee1 inducing its degradation and entry into mitosis. Upon damage ATM and ATR are able to phosphorylate and inhibit Plk1, contributing to an increase of Wee1 and therefore reduced CDK1 activity [100]. In addition to this early checkpoint, p53 also regulates the maintenance or sustained G2/M arrest. In this case, rather than mediated by p21, which has little CDK1 inhibitory capacity, other p53 targets such as GADD45 and 14-3-3 act as effectors [114]. Permanent cell cycle arrest: Senescence

By definition, checkpoints are a transitory state from which cells can escape if repair is accomplished and the signal is turned off. In contrast, cellular senescence is a persistent and irreversible cell cycle arrest. It was firstly observed in cell culture in 1965 [115]. Depending on the load of DNA damage or the cell type, a persistent activation of the DDR might direct cells into senescence [89].

Besides the presence of DSB, other cellular stresses can also promote senescence. These include telomere shortening [116], oxidative stress [117] and oncogenes [118-121]. Nevertheless, all of these stimuli can activate the DDR, so that DNA damage might be the ultimate cause of the senescent response. First, short or unprotected telomeres resemble and are actually sensed by cell as DSB [122, 123]. Second, reactive oxygen species generated by oxidative stress are an obvious source of DNA damage. Finally, several oncogenes have been shown to induce RS so that oncogene-induced senescence (OIS) would be linked to the DDR [124-126]. Importantly, and as it will be discussed below, OIS has been suggested as an early anti-cancer barrier in vivo, providing a link between the DDR and cancer development [127]. However, it must be pointed out that alternative, DDR-independent pathways of promoting OIS and which operate through the Ink4a/ARF locus have been described [128]. Apoptosis

Apoptosis, or programmed cell death, is other phenomenon activated by the DDR in order to eliminate cells with intolerable amounts of DNA damage. As such, apoptosis has an important role in eliminating damaged cells during aging [129] or in the acute responses genotoxic cancer therapies. In addition, apoptosis is a physiological process essential for normal development.

As in the case of senescence, apoptosis is also chiefly governed by p53. DDR-induced p53 promotes the expression of several pro-apoptotic factors, such as Puma, Noxa and Bax [130-132]. This pro-apoptotic p53 program leads to mitochondrial membrane permeabilzation [133], allowing the exit of cytochrome c to the cytosol, forming the apoptosome, which finally activates effector caspases [134].

Why some cells undergo apoptosis and other senescence in response to DNA damage is still a matter of study [135]. Although most cells are capable of both phenomenons, cell type is indeed determinant to undergo one or another. For instance, whereas DNA damage in fibroblasts and epithelial cells specially promotes senescence, low amounts of DNA breaks in lymphocytes are sufficient to trigger apoptosis. Of note, post-mitotic cells like neurons, have a limited capacity to become senescent or undergo apoptosis which might be due to their particular cell cycle status. Other cellular pathways stimulated by the DDR

Given the large amount of PIKK phospho-targets and p53 transcriptional targets it is not surprising that the DDR exerts its function in many cellular processes by means that remain poorly understood. For instance, high doses of DNA damage can induce the differentiation of melanocyte stem cells (SC), resulting in their depletion and thereby promoting aging phenotypes on the skin [136]. Similar results linking the DDR and differentiation have been found in neurons [137]. Related to this, p53 has been shown to regulate the polarity of mammary cancer stem cell divisions [138]. p53 loss promotes symmetric and promiscuous cancer stem cell divisions, which contribute to the expansion of premalignant pools. It is therefore tempting to speculate that one of the means by which DNA damage leads to stem cell exhaustion is by promoting p53-dependent asymmetric cell divisions in SC.

Finally, another process that might be modulated by DNA damage is autophagy, which functions to promote cell survival through the degradation of damaged organelles and molecules. Interestingly, one of the key regulators of autophagy is mTOR (mammalian target of Rapamycin), another member of the PIKK family. Whereas the mechanism remains to be solved, recent studies suggest a connection between the DDR and mTOR that could be the responsible for DNA damage induced autophagy [139, 140]. To what extent these additional effects of the DDR impact on the physiological consequences of a deficient DDR, remains to be seen.

3.1.4. Physiological consequences of the DDR

DDR deficiencies cause important physiological consequences. As commented on this chapter, DSB are physiologically generated during B and T lymphocyte maturation and during meiotic recombination, and many DDR-related proteins are required for normal functioning of these processes. In addition, the DDR has an essential role in facing the stochastic DNA damage that our cells acquire through our lifetimes. In this context, it is not surprising that deficiencies in DDR components cause human diseases associated to immune deficiencies, sterility, premature aging and cancer predisposition. Alterations during embryonic development are also frequent, suggesting that the high division rates occurring at this stage might be prone to accumulate DNA damage, particularly RS.

DDR-mutant mouse models recapitulate many of the phenotypes found in DDR-associated human syndromes. Some of the most relevant DDR-related diseases are shown in table 1. Most of them are of recessive nature, with some exceptions such as variants of Li-Fraumeni, which can be caused by dominant mutations in just one allele of p53.

DiseaseMutated genClinical feature
Ataxia Telangiectasia ATMCerebellar ataxia, telangiectasia, inmunodeficiency, lymphoid tumours.
Nijmegen breakage syndromeNbs1Growth retardation, microcephaly, immunodeficiency, lymphoid tumours.
A-T like disorderMre11Cerebellar ataxia, mild predisposition to tumours.
Seckel SyndromeATRProgeria, microcephaly and other developmental defects.
Li-Fraumenip53Early development of cancer (breast cancer, sarcomas, brain tumours, leukemias, etc.)
Riddle syndromeRNF168Inmunodeficiency, dysmorphic features, mental retardation.
Werner syndromeRECQL2 (WRN)Progeria and age-associated disorders
Fanconi Anemia13 different FA genesBone marrow failure, predisposition to leukemias and solid tumours.

Table 1.

Several human genetic syndromes related to DDR components. The DDR and cancer: Protector and target

The relationship between the DDR and cancer is of particular interest from multiple points of view. First, the DDR is critical to prevent the accumulation of spontaneous pro-cancerous mutations and overall genomic instability. This is why most DDR-related human diseases are prone to cancer development. In addition, the DDR is particularly relevant in preventing chromosome rearrangements during lymphoid maturation which makes lymphomas one of the most frequent cancer in Genomic Instability Syndromes. Note that in some cases mutations in only one allele convey a dramatic increase in cancer predisposition. This is the case for BRCA1 and BRCA2 mutations carriers, whose life-time breast cancer risk raise up to 80%.

Second, the DDR is activated by oncogenes in early stages of tumorigenesis [124, 141]. These studies proposed a model where the activation of certain oncogenes could generate RS which, by activating the DDR, would limit cancer development by promoting cell senescence [142, 143]. In fact, convincing evidence exists to show that certain oncogenes indeed are able to generate DNA damage trough promoting abnormal replication [124, 125]. Note that this type of damage, known as RS, is sensed and signaled mainly by ATR rather than ATM [83], raising the relevance of ATR-signaling in the oncogene-activated DDR model of cancer progression.

Finally, many of the current anti-cancer therapies (including radiotherapy) operate by generating high loads of DNA damage that activate the DDR towards apoptosis. In this regard, there is increasing interest in the development of new anti-cancer strategies that take advantage of our knowledge of the DDR to specifically target tumor cells. The idea behind these new strategies is to exploit synthetic lethal interactions that will only occur in cancer cells. For instance, one of the most promising approaches in this regard is the use of PARP inhibitors for the treatment of BRCA1/2 deficient breast cancers. These inhibitors block a ssDNA repair pathway which is mostly dispensable for normal cells, but essential for cells deficient in HR, such as BRCA1/2 mutant cells [144]. Other examples of synthetic lethality could be the use of Chk1 inhibitors and ATR inhibitors, to treat p53 deficient tumors [145-148]. The role of the DDR in ageing

Ageing is intuitively associated with the natural degeneration of our tissues, which would derive from the accumulation of some “toxic” factor. Studies mostly performed in the last decade have identified DNA damage as this deleterious factor that is associated to the onset of ageing [149, 150]. For instance, aged tissues or stem cells show evidences of an activated DDR [151, 152]. Moreover, most DDR-related genetic diseases suffer from premature ageing, which is likely due to a faster accumulation of intolerable amounts of DNA damage.

The most accepted theory is that DNA damage, when accumulated in SC, activates a DDR that limits their regenerative capacity and thus promotes ageing [151, 153]. To what extent DNA damage is the natural cause of actual ageing in humans, and which types of DNA damage (RS, eroded telomeres, ROS…) are most important in this process, remains to be understood. In what regards to RS, the faster nature of embryonic cell divisions might make this stage particularly susceptible to this type of damage. In fact, recent studies in ATR hypomorphic mice revealed that an intra-uterine exposure to RS can accelerate the later onset ageing [146, 154]. Whereas the exact mechanism of this intrauterine programming of ageing is not fully understood, it raises the question about to what extent our adult well-being can be already conditioned by the stresses to which we were exposed in utero.

3.2. Other posttranslational modifications in the DDR

There is little doubt that PIKK-mediated phosphorylation is a major controller of the DDR. Nevertheless, to end this chapter we would want to describe the role that non-phosphorylation based signaling of DNA damage might play in genome protection. In the light of recent discoveries, DNA damage signaling through other PTMs such as acetylation, methylation, ubiquitination and, sumoylation might also play crucial roles for appropriate DNA damage signaling.

3.3.1. Ubiquitination

Ubiquitination is a highly regulated process that promotes covalent modification of specific proteins substrates with the 76-amino acid protein ubiquitin, catalyzed in three sequential steps by E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) enzymatic activities [155]. Classically, ubiquitination (UQ) was described as a mechanism to target proteins for proteasome mediated degradation. However, at present, other functions of UQ such as during protein localization or in mediating protein-protein interactions are known. In fact, phosphorylation signaling (not only by the DDR) is often coupled to substrate ubiquitination, which increases the potential network of protein-protein interaction [156, 157].

UQ presents seven lysines in its surface: K6, K11, K27, K29, K33, K48 and K63. Whereas poly-ubiquitination linked to K48 residues is usually related to proteasomal degradation [158], K63-linked polyubiquitin chains are more related to other regulatory functions [159]. In addition to the conjugating lysine, ubiquitin chains display an ample structural diversity, that has its counterpart in many different ubiquitin interactif motifs (UIM) in other proteins [160]. The role of UQ is counteracted by deubiquitianting enzymes (DUB), which are responsible for the elimination of UQ conjugates.

Several ubiquitination events have been assoctaied to DSB signaling. As previously discussed, PIKK activation is followed by ubiquitination of various substrates. Again, ATM-dependent phosphorylation of H2AX and MDC1 are sufficient to recruit the E3 ubiquitin ligase RNF8. Together with another E3 ligase known as RNF168, and with Ubc13 as the E2 ligase, RNF8 promotes the accumulation of ubiquitinylated substrates in the vicinity of the DSB. Of note, RNF168 loading to foci occurs trough direct recognition of RNF8-deposited UQ chains through its UIM domains [25, 26]. These poly-ubiquitinations are mainly K63-linked and contribute to attract specific UIM-containing proteins and/or to modulate chromatin architecture via histone ubiquitinylation [159]. Recent works have shown that HERC2 (Hect Domain and RLD2) is an important regulator of RNF8 and RNF168-dependent ubiquitinitalion which mediates through promoting the binding to the E2 ligase Ubc13 [161].

This coordinated cascade of UQ modifications generated at DSB is necessary for the formation of BRCA1 and 53BP1 foci. However, once again, the absence of RNF8 does not lead to major BRCA1 phenotypes, which challenges the relevance of IRIF as predictors of functionality. It should be noted also that there is no evidence of direct binding of 53BP1 to ubiquitin chains, and how UQ mediates 53BP1 foci remains a mistery. In addition, 53BP1 can bind methylated histones which may provide an independent way of loading to DSB [94, 95, 162].

In contrast to 53BP1, the relationship of BRCA1 foci with UQ is well characterized. RAP80, a protein containing several UIMs in tandem, binds to ubiquitinated substrates (perhaps histones) [23], and brings Abraxas [28] and BRCA1. Interestingly, RAP80 also interacts with BRCC36, a DUB, which activity is also required for proper DSB repair and signaling [163, 164]. Thus, ordered ubiquitinylation and deubuquitinylation may be needed during the signaling of DNA damage.

The UQ pathway does not end with 53BP1 or BRCA1. BRCA1 itself is an E3 ubiquitin ligase activity resident in the N-terminal RING domain of this large protein. In fact, this is the only enzymatic activity found in BRCA1. While BRCA1 has E3 activity by itself, this activity increases in several orders of magnitude when bound to its constitutive partner BARD1 [30]. To date, the only well characterized BRCA1 substrates are BRCA1 itself and CtIP. CtIP ubiquitination by BRCA1 [165] has been proposed to promote the resection of DSB, leading to HR and a regulation of the G2/M checkpoint through ATR-activity. Hence, in this system UQ would be upstream of the phosphorylation DDR.

To complicate things further, BRCA1 auto-ubiquitination is linked to K6, which is mediated by the E2 enzyme Ubch5c [166]. Ubiquitinated BRCA1 is found at DSB and also in several endogenous spots during S phase. Interestingly, even though 20% of human BRCA1 mutations predisposing to breast and ovarian cancer are found in the RING domain [30], recent data emerging from mouse models suggest that the E3 ligase activity of BRCA1 might be unrelated to genome maintenance and its tumor suppression role [167, 168]. However, other studies [169, 170] indicate the importance of the RING domain in BRCA1 tumor suppressor function. Certainly, further work needs to be done to solve these divergent observations.

Finally, regardless of DSB there is solid evidence that UQ-mediated signaling pathways also contribute to other genome protective pathways. For instance, UQ plays a key role in the Fanconi Anemia pathway. Fanconi Anemia (FA) is a recessive disease characterized by developmental abnormalities, bone marrow failure and cancer predisposition [171]. The disease is caused by mutations in at least 13 genes which mutations compromise the repair of interstrand DNA cross-links. A key event on the activation of the FA pathway is the monoubiquitination of FancD2 and FancI proteins, with is thought to be essential for its localization at chromatin [44, 172, 173]. Again, there is evidence to suggest that FANCD2 ubiquitinylation is downstream of ATR signaling, providing a further example of the constant interaction between the different signaling pathways that are activated by DNA damage [55].

3.3.2. SUMOylation

SUMO stands for Small Ubiquitin-like modifier due to its similarities with UQ. In fact, SUMOylation is a similar process to ubiquitinylation involving E1, E2 and E3 enzymes. Importantly, very few SUMO E3 ligases exist in the mammalian proteome, which limits the search for potential ligases. In addition, SUMO E3 ligases are mostly dispensable and E2 ligases can complete the SUMOylation reaction largely by themselves. In contrast to UQ, three SUMO variants (SUMO1, SUMO2 and SUMO3 exist [174].

A number of SUMO roles with genome maintenance pathways have been discovered. In what regards to the DDR, two recent studies identified a SUMO-related signaling cascade that also coordinates the foci formation of BRCA1 and 53BP1 in response to DSB [175, 176]. At present, PIAS1 (Protein Inhibitor of Activated STAT-1) and PIAS4, are the E3 SUMO ligase enzymes found to participate in the DDR, using SAE1 (SUMO activating enzyme subunit 1) as E1 and UBC9 (Ubiquitin Conjugating Enzyme 9) as E2 enzymes. All these proteins, as well as SUMO1 and SUMO2/3 conjugates are rapidly recruited to DSB, forming IRIF. Whereas the mechanism is still not totally clear, it seems that PIAS1 promotes SUMO2/3 modification of BRCA1, and PIAS4 is mainly involved in SUMO1 modifications of both 53BP1 and BRCA1. Together, these SUMOylations seem to be necessary for RNF8 and RNF168 function, providing another instance of inter-PTM signaling at DSB [175, 176]. A summary of key SUMO and UQ modifications in the DSB response is illustrated in table 2.

E3 LigaseE2 LigaseMain substratesFunction
FancLUnknownFancD2DNA Cross-link repair
DSB resection
RNF8 Ubc13H2A, H2AXDSB signaling
RNF168Ubc13H2A, H2AXDSB signaling
PIAS1Ubc9BRCA1DSB signaling
PIAS4Ubc9BRCA1, 53BP1DSB signaling

Table 2.

Relevant ubiquitinations and SUMOylations in genome maintenance.

Regardless of the DDR, other genome maintenance pathways are also controlled by SUMO. One example is the role of a SUMO ligase called Mms21, which is an essential component of a cohesin and condensin related complex formed by Smc5 and Smc6 [177]. Whereas the role of this complex is far from being understood, it seems it might be important to prevent the accumulation of hemicatenates at stalled replication forks [178].

3.3.3. Interplay of PTMs in the DDR

Other post-translational modifications, such as methylation and acetylation, are also involved in the regulatory network of the DDR. A paradigm of multiple and interconnected PTM is found in the regulation of p53 levels, that involves a coordinated network of phosphorylation, acetylation, methylation and ubiquitination [179].

In the absence of DNA damage, p53 levels are kept low due to its ubiquitination by the E3 UQ ligase MDM2 and rapid degradation by the proteasome [180]. In fact, one of the ways by which DNA damage and other stress signals promote p53 stabilization is by PTMs which alters the MDM2/p53 interaction. p53 also suffers DDR-dependent phosphorylations [107] are thought to stabilize the protein and allow its association with the CBP/p300 acetyltransferase complex, promoting p53 acetylation and further stabilization [181]. Besides, p53 activity is limited by Set8/Pr-Set7 mediated methylations [182], which are also upon DNA damage [183].

Another relevant example of the interplay between different PTMs in the DDR is found in histones. Indeed, post-translational modifications of histones regulate many other processes related to chromatin compaction and structure, such as replication and transcription. The best known histone modification upon DNA damage is the already described phosphorylation of H2AX, that it is mediated by ATM, ATR or DNA-PK [20]. We also discussed how RNF8 and RNF168 can ubiquitinate histones in the proximity of DSB [26, 92]. Finally, methylations of histones H3 and H4 could modulates the recruitment of 53BP1 [94, 95, 162]; and several acetylations of histones have been described to play roles in genome maintenance which are still not completely understood [184-186]. How histone modifications enhance DSB repair is a very active area of research and discussion. One possibility is that histone modifications could increase chromatin accessibility, which would therefore facilitate the repair and signaling of the breaks [187].


4. Future perspectives

We have here provided a general overview of how cells signal the presence of DNA damage, and how a proper signaling is necessary to maintain a healthy genome. Still, whereas the amount of PTMs that coordinate the cellular response to DNA damage is already intimidating, it is likely that we are only seeing the tip of the iceberg. Many other targets and even PTM (ADP-rybosylation, Neddylation, N-terminal glycosylation, etc…) will probably be involved in mounting a proper DDR. Without a doubt, the fast development of massive proteomic technologies will soon provide a breathtaking picture of how cells detect, signal and repair DNA breaks; by promoting a myriad of PTMs in almost every molecule involved in the DDR.


  1. 1. LindahlT.BarnesD. E.Repair of endogenous DNA damage. Cold Spring Harb Symp Quant Biol20006512733
  2. 2. JacksonS. P.BartekJ.The DNA-damage response in human biology and disease. Nature2009Oct 22;461726710718
  3. 3. DudleyD. D.ChaudhuriJ.BassingC. H.AltF. W.Mechanism and control of V(D)J recombination versus class switch recombination: similarities and differences. Adv Immunol20058643112
  4. 4. ZicklerD.KlecknerN.The leptotene-zygotene transition of meiosis. Annu Rev Genet19983261997
  5. 5. MaoZ.BozzellaM.SeluanovA.GorbunovaV.Comparison of nonhomologous end joining and homologous recombination in human cells. DNA Repair (Amst)2008Oct 1;710176571
  6. 6. Doherty AJ, Jackson SP. DNA repair: how Ku makes ends meet.Curr Biol2001Nov 13;11(22):R9204
  7. 7. BurmaS.ChenD. J.Role-Pof. D. N. A.inK.thecellular.responseto. D. N. A.double-strandbreaks. D. N.DNA-PK in the cellular response to DNA double-strand breaks. DNA Repair (Amst)2004Aug-Sep;3(8-9):909-18.
  8. 8. Martin IV, MacNeill SA. ATP-dependent DNA ligases.Genome Biol2002REVIEWS3005.
  9. 9. CalsouP.DelteilC.FritP.DrouetJ.SallesB.Coordinated assembly of Ku and 460subunits of the DNA-dependent protein kinase on DNA ends is necessary for XRCC4-ligase IV recruitment. J Mol Biol2003Feb 7;326(1):93-103.
  10. 10. MaPannickeY.SchwarzU.LieberK.M. R.Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination. Cell2002Mar 22;108678194
  11. 11. RiballoE.KuhneM.RiefN.DohertyA.SmithG. C.MJRecioet.alA.pathwayof.double-strandbreak.rejoiningdependent.uponA. T. X.Mol Cell2004Dec 3;16571524
  12. 12. GuY.JinS.GaoY.WeaverD. T.AltF. W.Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination. Proc Natl Acad Sci U S A1997Jul 22;9415807681
  13. 13. ChaudhuriJ.AltF. W.Class-switchrecombination.interplayof.transcriptionD. N. A.deaminationrepairD. N. A.NatRev.Immunol2004Jul;4754152
  14. 14. MoshousD.PannetierC.ChassevalRd. R.DeistFl. al.PartialT.lymphocyteB.immunodeficiencypredispositionto.lymphomain.patientswith.hypomorphicmutations.inArtemis.J Clin Invest2003Feb;11133817
  15. 15. AAGoodarziNoon. A. T.DeckbarD.ZivY.ShilohY.LobrichM.etal. A. T. M.signalingfacilitates.repairof. D. N. A.double-strandbreaks.associatedwith.heterochromatinMol Cell2008Jul 25;31216777
  16. 16. Fernandez-CapetilloO.NussenzweigA. A. T. M.breaksinto.heterochromatinMol.Cell2008Aug 8;3133034
  17. 17. RichardsonC.JasinM.Frequent chromosomal translocations induced by DNA double-strand breaks. Nature2000Jun 8;4056787697700
  18. 18. JazayeriA.FalckJ.LukasC.BartekJ.SmithG. C.LukasJ.etal. A. T. M.cellcycle-dependent.regulationof. A. T. R.inresponse.toD. N. A.double-strandbreaks.Nat Cell Biol2006Jan;813745
  19. 19. de JagerM.van NoortJ.van GentD. C.DekkerC.KanaarR.WymanC.HumanRad5.Mreisa.flexiblecomplex.thatcan.tetherD. N. A.endsMol Cell2001Nov;85112935
  20. 20. Fernandez-CapetilloO.LeeA.NussenzweigM.NussenzweigA. H. A. X.thehistone.guardianof.thegenome. D. N.DNA Repair (Amst)2004Aug-Sep;3(8-9):959-67.
  21. 21. StuckiM.ClappertonJ. A.MohammadD.YaffeM. B.SmerdonS. J.JacksonS. P. M. D. C.directlybinds.phosphorylatedhistone. H. A. X.toregulate.cellularresponses.toD. N. A.double-strandbreaks.Cell2005Dec 29;1237121326
  22. 22. LouZ.Minter-DykhouseK.FrancoS.GostissaM.MARiveraCeleste. A.etal. M. D. C.maintainsgenomic.stabilityby.participatingin.theamplification.ofA. T.M-dependentD. N. A.damagesignals.Mol Cell2006Jan 20;212187200
  23. 23. MailandN.Bekker-JensenS.FaustrupH.MelanderF.BartekJ.LukasC.etal. R. N. F.ubiquitylateshistones.atD. N. A.double-strandbreaks.promotesassembly.ofrepair.proteinsCell2007Nov 30;1315887900
  24. 24. MASantosHuen.MSJankovicM.ChenH. T.Lopez-ContrerasA. J.KleinI. al.Class switching and meiotic defects in mice lacking the E3 ubiquitin ligase RNF8. J Exp Med May 10;207597381
  25. 25. DoilC.MailandN.Bekker-JensenS.MenardP.LarsenD. H.PepperkokR.etal. R. N.F1bindsamplifiesubiquitin.conjugateson.damagedchromosomes.toallow.accumulationof.repairproteins.Cell2009Feb 6;136343546
  26. 26. StewartG. S.PanierS.TownsendK.Al-HakimA. K.KolasN. K.MillerE. al.TheR. I. D. D. L. E.syndromeprotein.mediatesa.ubiquitin-dependentsignaling.cascadeat.sitesof. D. N. A.damageCell2009Feb 6;136342034
  27. 27. al.Genomicinstability.defectivespermatogenesis.immunodeficiencycancerin. a.mousemodel.ofthe. R. I. D. D. L. E.syndromeP.LoPLoS Genet Apr;7(4):e1001381.
  28. 28. WangB.ElledgeS. J.Ubc1Rnfubiquitin.ligasescontrol.fociformation.ofthe.Rap8AbraxasBrca.Brcccomplexin.responseto. D. N. A.damageProc Natl Acad Sci U S A2007Dec 26;104522075963
  29. 29. Fackenthal JD, Olopade OI.Breast cancer risk associated with BRCA1 and BRCA2 in diverse populations. Nat Rev Cancer2007Dec;71293748
  30. 30. al.TheR. I. N. G.heterodimerB. R. C. A.isB. A. R. D.ubiquitina.ligaseinactivated.bya.breastcancer-derived.mutationJ Biol Chem2001May 4;276181453740
  31. 31. Fernandez-CapetilloO.CelesteA.NussenzweigA.Focusingon.fociH. A. X.therecruitment.ofD. N.A-damageresponse.factorsCell Cycle2003Sep-Oct;254267
  32. 32. PaullT. T.GellertM.The 3’ to 5’ exonuclease activity of Mre 11 facilitates repair of DNA double-strand breaks. Mol Cell1998Jun;1796979
  33. 33. NimonkarA. V.OzsoyA. Z.GenschelJ.ModrichP.KowalczykowskiS. C.Humanexonuclease. .helicaseB. L. M.interactto.resectD. N. A.initiateD. N. A.repairProc Natl Acad Sci U S A2008Nov 4;105441690611
  34. 34. AASartoriLukas. al.HumanCt. I. P.promotesD. N. A.endresection.Nature2007Nov 22;450716950914
  35. 35. YouZ.ShiL. Z.ZhuQ.WuP.ZhangY. al.CtI. P.linksD. N. A.double-strandbreak.sensingto.resectionMol Cell2009Dec 25;36695469
  36. 36. CuadradoM.Martinez-PastorB.MurgaM.ToledoL. I.Gutierrez-MartinezP.LopezE.etal. A. T. M.regulatesA. T. R.chromatinloading.inresponse.toD. N. A.double-strandbreaks.J Exp Med2006Feb 20;2032297303
  37. 37. Adams KE, Medhurst AL, Dart DA, Lakin ND. Recruitment of ATR to sites of ionising radiation-induced DNA damage requires ATM and components of the MRN protein complex.Oncogene2006Jun 29;25283894904
  38. 38. MyersJ. S.CortezD.Rapid activation of ATR by ionizing radiation requires ATM and Mre11. J Biol Chem2006Apr 7;28114934650
  39. 39. BuntingS. F.CallenE.WongN.ChenH. T.PolatoF.GunnA.etal. .inhibitsB. P.homologousrecombination.inBrca1-deficient.cellsby.blockingresection.ofD. N. A.breaksCell Apr 16;141224354
  40. 40. CaoL.XuX.BuntingS. F.LiuJ.WangR. H.CaoL. L.etal. A.selectiverequirement.for.inB. P.thebiological.responseto.genomicinstability.inducedby.Brcadeficiency.Mol Cell2009Aug 28;35453441
  41. 41. BaumannP.BensonF. E.WestS. C.HumanRad.proteinpromotes. A. T.P-dependenthomologous.pairingstrandtransfer.reactionsin.vitroCell1996Nov 15;87475766
  42. 42. TarsounasM.DaviesD.WestS. C. B. R. C.BRCA2-dependent and independent formation of RAD51 nuclear foci. Oncogene2003Feb 27;228111523
  43. 43. WagnerJ. al.Germline mutations in BRCA2: shared genetic susceptibility to breast cancer, early onset leukemia, and Fanconi anemia. Blood2004Apr 15;103832269
  44. 44. Garcia-HigueraI.TaniguchiT.GanesanS.MSMeynTimmers. al.Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. Mol Cell2001Feb;7224962
  45. 45. MJMc IlwraithVaisman. A.LiuY.FanningE.WoodgateR.WestS. C.HumanD. N. A.polymeraseeta.promotesD. N. A.synthesisfrom.strandinvasion.intermediatesof.homologousrecombination.Mol Cell2005Dec 9;20578392
  46. 46. HollidayR.The Induction of Mitotic Recombination by Mitomycin C in Ustilago and Saccharomyces. Genetics1964Sep;5032335
  47. 47. BzymekM.ThayerN. H.OhS. D.KlecknerN.HunterN.Double Holliday junctions are intermediates of DNA break repair. Nature Apr 8;464729093741
  48. 48. WuL.HicksonI. D.The Bloom’s syndrome helicase suppresses crossing over during homologous recombination. Nature2003Dec 18;42669688704
  49. 49. ChenX. B.MelchionnaR.DenisC. M.GaillardP. H.BlasinaA.Van de al.Human Mus81-associated endonuclease cleaves Holliday junctions in vitro. Mol Cell2001Nov;85111727
  50. 50. ConstantinouA.ChenX. B.Mc GowanC. H.WestS. C.Holliday junction resolution in human cells: two junction endonucleases with distinct substrate specificities. EMBO J2002Oct 15;2120557785
  51. 51. Svendsen JM, Harper JW. GEN1/Yen1 and the SLX4 complex: Solutions to the problem of Holliday junction resolution.Genes Dev Mar 15;24652136
  52. 52. FekairiS.ScaglioneS.ChahwanC.TaylorE. al.HumanS. L. X.isa.Hollidayjunction.resolvasesubunit.thatbinds.multipleD. N. 10;13817889
  53. 53. SvendsenJ. M.SmogorzewskaA.MESowaO’Connell. B. C.GygiS. P.ElledgeS. al.MammalianB. T. B.D1assemblesS. L. X.Hollidaya.junctionresolvase.isrequired.forD. N. A.repairCell2009Jul 10;13816377
  54. 54. HelledayT.Homologous recombination in cancer development, treatment and development of drug resistance. Carcinogenesis Mar 29.
  55. 55. AndreassenP. R.D’AndreaA. D.TaniguchiT. A. T. R.couplesF. A. N. C. D.monoubiquitinationto.theD. N.A-damageresponse.Genes Dev2004 Aug 15;18195816195863
  56. 56. TibbettsR. S.CortezD.BrumbaughK. M.ScullyR.LivingstonD.ElledgeS. al.Functional interactions between BRCA1 and the checkpoint kinase ATR during genotoxic stress. Genes Dev2000Dec 1;142329893002
  57. 57. DaviesS. L.NorthP. S.DartA.LakinN. D.HicksonI. D.Phosphorylation of the Bloom’s syndrome helicase and its role in recovery from S-phase arrest. Mol Cell Biol2004Feb;243127991
  58. 58. Henson JD, Neumann AA, Yeager TR, Reddel RR.Alternative lengthening of telomeres in mammalian cells. Oncogene2002Jan 21;214598610
  59. 59. Harper JW, Elledge SJ. The DNA damage response: ten years after. MolCell2007Dec 14;28573945
  60. 60. ShilohY. A. T. M.relatedprotein.kinasessafeguarding.genomeintegrity.Nat Rev Cancer2003Mar;3315568
  61. 61. CALovejoyCortez. D.Common mechanisms of PIKK regulation. DNA Repair (Amst)2009Sep 2;8910048
  62. 62. Lavin MF. Ataxia-telangiectasia: from a rare disorder to a paradigm for cell signalling and cancer.Nat Rev Mol Cell Biol2008Oct;91075969
  63. 63. MatsuokaS.BABallifSmogorzewska. A.Mc DonaldE. R.3rd HurovK. E.LuoJ.etal. A. T. M.substrateA. T. R.analysisreveals.extensiveprotein.networksresponsive.toD. N. A.damageScience2007May 25;316582811606
  64. 64. ZouL.ElledgeS. J.SensingD. N. A.damagethrough. A. T. R. I. P.recognitionof. R. P.A-ssD. N. A.complexesScience2003Jun 6;300562515428
  65. 65. CallenE.JankovicM.WongN.ZhaS.ChenH. al.Essential role for DNA-PKcs in DNA double-strand break repair and apoptosis in ATM-deficient lymphocytes. Mol Cell2009May 15;34328597
  66. 66. SavitskyK.Bar-ShiraA.GiladS.RotmanG.ZivY.VanagaiteL.etal. A.singleataxia.telangiectasiagene.witha.productsimilar.toP. I.kinaseScience1995Jun 23;2685218174953
  67. 67. BoderE.Ataxia-telangiectasiaan.overviewKroc.Found Ser198519163
  68. 68. ShilohY.The ATM-mediated DNA-damage response: taking shape. Trends Biochem Sci2006Jul;31740210
  69. 69. UzielT.LerenthalY.MoyalL.AndegekoY.MittelmanL.ShilohY.Requirement of the MRN complex for ATM activation by DNA damage. EMBO J2003Oct 15;2220561221
  70. 70. Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation.Nature2003Jan 30;4216922499506
  71. 71. DanielJ. A.PellegriniM.LeeJ. H.PaullT. T.FeigenbaumL.NussenzweigA.Multiple autophosphorylation sites are dispensable for murine ATM activation in vivo. J Cell Biol2008Dec 1;183577783
  72. 72. al.Autophosphorylation at serine 1987 is dispensable for murine Atm activation in vivo. Nature2006Sep 14;44371082225
  73. 73. DifilippantonioS.CelesteA.Fernandez-CapetilloO.ChenH. T.ReinaSan.MartinB.Van al.Role of Nbs1 in the activation of the Atm kinase revealed in humanized mouse models. Nat Cell Biol2005Jul;7767585
  74. 74. SunY.XuY.RoyK.BDPriceD. N. A.damage-inducedacetylation.oflysine.30ofA. T. M.activatesA. T. M.kinaseactivity.Mol Cell Biol2007Dec;272485029
  75. 75. Lavin MF. ATM and the Mre11 complex combine to recognize and signal DNA double-strand breaks.Oncogene2007Dec 10;2656774958
  76. 76. al.Positional cloning of the gene for Nijmegen breakage syndrome. Nat Genet1998Jun;19217981
  77. 77. StewartG. S.MaserR. S.StankovicT.BressanD. A.KaplanM. I.JaspersN. al.TheD. N. A.double-strandbreak.repairgene. h. M. R. E.ismutated.inindividuals.withan.ataxia-telangiectasia-likedisorder.Cell1999Dec 10;99657787
  78. 78. BarlowC.Ribaut-BarassinC.ZwingmanT. A.PopeA. J.BrownK. D.OwensJ. W.etal. A. T. M.isa.cytoplasmicprotein.inmouse.brainrequired.toprevent.lysosomalaccumulation.Proc Natl Acad Sci U S A2000Jan 18;9728716
  79. 79. ShechterD.CostanzoV.GautierJ.Regulationof. D. N. A.replicationby. A. T. R.signalingin.responseto. D. N. A.intermediatesD. N.DNA replication by ATR: signaling in response to DNA intermediates. DNA Repair (Amst)2004Aug-Sep;3(8-9):901-8.
  80. 80. Lopez-ContrerasA. J.Fernandez-CapetilloO.TheA. T. R.barrierto.replication-bornD. N. A.damageD. N.DNA damage. DNA Repair (Amst) Dec 10;912124955
  81. 81. BrownE. J.BaltimoreD. A. T. R.disruptionleads.tochromosomal.fragmentationearlyembryonic.lethalityGenes Dev2000Feb 15;144397402
  82. 82. O’DriscollM.Ruiz-PerezV. L.WoodsC. G.JeggoP. A.GoodshipJ. A. A.splicingmutation.affectingexpression.ofataxia-telangiectasia.Rad3-relatedprotein. . A. T. R.resultsin.Seckelsyndrome.Nat Genet2003Apr;334497501
  83. 83. CimprichK. A.CortezD. A. T. R.anessential.regulatorof.genomeintegrity.Nat Rev Mol Cell Biol2008Aug;9861627
  84. 84. FanningE.KlimovichV.NagerA. R. A.dynamicmodel.forreplication.proteinA. . R. P. A.functionin. D. N. A.processingpathways.Nucleic Acids Res20063415412637
  85. 85. CortezD.GuntukuS.QinJ.ElledgeS. J. A. T. R.partnersA. T. R. I. P.incheckpoint.signalingScience2001Nov 23;294554717136
  86. 86. YangX. H.ZouL.Recruitment-Aof. A. T. R.Rad1T. R. I. P.9-1complexesto. D. N. A.damageMethods Enzymol200640911831
  87. 87. LeeJ.KumagaiA.DunphyW. G.TheRad.HusRadcheckpoint.clampregulates.interactionof.TopB. P.withA. T. R.J, Kumagai A, Dunphy WG. The Rad9-Hus1-Rad1 checkpoint clamp regulates interaction of TopBP1 with ATR. J Biol Chem2007Sep 21;282382803644
  88. 88. KumagaiA.LeeJ.YooH. Y.DunphyW. G.TopB. P.activates-Athe. A. T. R.complexT. R. I. P.Cell2006Mar 10;124594355
  89. 89. ToledoL. I.MurgaM.Gutierrez-MartinezP.SoriaR.Fernandez-CapetilloO. A. T. R.signalingcan.drivecells.intosenescence.inthe.absenceof. D. N. A.breaksGenes Dev2008Feb 1;223297302
  90. 90. CelesteA.Fernandez-CapetilloO.MJKruhlakPilch. D. R.StaudtD. al.HistoneH. A. X.phosphorylationis.dispensablefor.theinitial.recognitionof. D. N. A.breaksNat Cell Biol2003Jul;576759
  91. 91. Fernandez-CapetilloO.ChenH. T.CelesteA.WardI.RomanienkoP. J.MoralesJ. C.etal. D. N. A.damage-inducedG.checkpointM.activationby.histoneH. A. X..B. P.Nat Cell Biol2002Dec;4129937
  92. 92. MSHuenGrant. R.MankeI.MinnK.YuX.YaffeM. B.etal. R. N. F.transducesthe. D. N.A-damagesignal.viahistone.ubiquitylationcheckpointprotein.assemblyCell2007Nov 30;131590114
  93. 93. KolasN. K.ChapmanJ. R.NakadaS.YlankoJ.ChahwanR.SweeneyF. al.Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science2007Dec 7;3185856163740
  94. 94. HuyenY.ZgheibO.DitullioR. A.Jr GorgoulisV. G.ZacharatosP.PettyT. al.Methylatedlysine. .ofhistone. H.targets.toB. P.double-strandD. N. A.breaksNature2004Nov 18;432701540611
  95. 95. BotuyanM. V.LeeJ.WardI. M.KimJ. E.ThompsonJ. al.Structural basis for the methylation state-specific recognition of histone H4-K20 by 53BP1 and Crb2 in DNA repair. Cell2006Dec 29;1277136173
  96. 96. CelesteA.PetersenS.RomanienkoP. J.Fernandez-CapetilloO.ChenH. T.SedelnikovaO. al.Genomic instability in mice lacking histone H2AX. Science2002May 3;29655699227
  97. 97. WardI. M.DifilippantonioS.MinnK.MDMuellerMolina. J. R.YuX.etal. .cooperatesB. P.withp.asfunctionsa.haploinsufficienttumor.suppressorin.miceMolina JR, Yu X, et al. 53BP1 cooperates with 53and functions as a haploinsufficient tumor suppressor in mice. Mol Cell Biol2005Nov;25(22):10079-86.
  98. 98. YooH. Y.KumagaiA.ShevchenkoA.DunphyW. G.Ataxia-telangiectasia mutated (ATM)-dependent activation of ATR occurs through phosphorylation of TopBP1 by ATM. J Biol Chem2007Jun 15;28224175016
  99. 99. KumagaiA.DunphyW. G.Claspina.novelprotein.requiredfor.theactivation.ofChk.duringa. D. N. A.replicationcheckpoint.responsein.Xenopusegg.extractsMol Cell2000Oct;6483949
  100. 100. GuardavaccaroD.PaganoM.Stabilizers and destabilizers controlling cell cycle oscillators. Mol Cell2006Apr 7;22114
  101. 101. Abraham RT.Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev2001Sep 1;1517217796
  102. 102. BartekJ.LukasJ.ChkChkkinases.incheckpoint.controlcancerCancer Cell2003May;354219
  103. 103. Smits VA, Reaper PM, Jackson SP.Rapid PIKK-dependent release of Chk1 from chromatin promotes the DNA-damage checkpoint response. Curr Biol2006Jan 24;1621509
  104. 104. JinJ.ShiroganeT.XuL.NalepaG.QinJ.ElledgeS. J.etal. S. C.Fbeta-TR. C. P.linksChk.signalingto.degradationof.theCdc.proteinA.phosphataseGenes Dev2003Dec 15;1724306274
  105. 105. XiaoZ.ChenZ.GunasekeraA. H.SowinT. J.RosenbergS. al.Chkmediates. S.arrestsG.throughCdc.degradationA.inresponse.toD. N.A-damagingagents.J, Rosenberg SH, Fesik S, et al. Chk1 mediates S and G2 arrests through Cdc25A degradation in response to DNA-damaging agents. J Biol Chem2003Jun 13;278242176773
  106. 106. BoutrosR.DozierC.DucommunB.The when and wheres of CDC25 phosphatases. Curr Opin Cell Biol2006Apr;18218591
  107. 107. CECanmanLim.DSCimprichK. al.Activation of the ATM kinase by ionizing radiation and phosphorylation of 53Science1998Sep 11;281(5383):1677-9.
  108. 108. ShiehS. Y.AhnJ.TamaiK.TayaY.PrivesC.The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate 53at multiple DNA damage-inducible sites. Genes Dev2000Feb 1;14(3):289-300.
  109. 109. GengY.EatonE. N.PiconM.RobertsJ. M.ASLundbergGifford. al.Regulation of cyclin E transcription by E2Fs and retinoblastoma protein. Oncogene1996Mar 21;126117380
  110. 110. FalckJ.MailandN.SyljuasenR. G.BartekJ.LukasJ.The-ChkA. T. M.CdccheckpointA.pathwayguards.againstradioresistant. D. N. A.synthesisNature2001Apr 12;41068308427
  111. 111. MailandN.FalckJ.LukasC.SyljuasenR. al.Rapid destruction of human Cdc25A in response to DNA damage. Science2000May 26;288547014259
  112. 112. BartekJ.LukasJ.Pathwaysgoverning. G.transitionS.theirresponse.toD. N. A.damageF. E. B.FEBS Lett2001Feb 16;490311722
  113. 113. Arias EE, Walter JC. Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells.Genes Dev2007Mar 1;215497518
  114. 114. Taylor WR, Stark GR.Regulation of the G2/M transition by 53Oncogene2001Apr 5;20(15):1803-15.
  115. 115. HayflickL.The Limited in Vitro Lifetime of Human Diploid Cell Strains. Exp Cell Res1965Mar;3761436
  116. 116. Harley CB, Futcher AB, Greider CW.Telomeres shorten during ageing of human fibroblasts. Nature1990May 31;345627445860
  117. 117. ParrinelloS.SamperE.KrtolicaA.GoldsteinJ.MelovS.CampisiJ.Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell Biol2003Aug;587417
  118. 118. SerranoM.LinA. W.MEMc CurrachBeach. D.LoweS. W.Oncogenic ras provokes premature cell senescence associated with accumulation of 53and p16INK4a. Cell1997Mar 7;88(5):593-602.
  119. 119. LinA. W.BarradasM.StoneJ. C.van AelstL.SerranoM.LoweS. W.Premature senescence involving 53and p16 is activated in response to constitutive MEK/MAPK mitogenic signaling. Genes Dev1998Oct 1;12(19):3008-19.
  120. 120. MichaloglouC.VredeveldL. C.MSSoengasDenoyelle. C.KuilmanT.van der HorstC. M.etal. B. R. A. F.BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature2005Aug 4;43670517204
  121. 121. ZhuJ.WoodsD.Mc MahonM.BishopJ. M.Senescence of human fibroblasts induced by oncogenic Raf. Genes Dev1998Oct 1;121929973007
  122. 122. d’Adda diFagagna. F.ReaperP. M.Clay-FarraceL.FieglerH.CarrP.VonZglinicki. T.etal. A. D. N. A.damagecheckpoint.responsein.telomere-initiatedsenescence.Nature2003Nov 13;42669631948
  123. 123. MartensU. M.ChavezE. A.PoonS. S.SchmoorC.LansdorpP. M.Accumulation of short telomeres in human fibroblasts prior to replicative senescence. Exp Cell Res2000Apr 10;25612919
  124. 124. al.Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature2006Nov 30;44471196337
  125. 125. Di al.Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. Nature2006Nov 30;444711963842
  126. 126. MalletteF. A.Gaumont-LeclercM. F.FerbeyreG.TheD. N. A.damagesignaling.pathwayis. a.criticalmediator.ofoncogene-induced.senescenceGenes Dev2007Jan 1;211438
  127. 127. HalazonetisT. D.GorgoulisV. G.BartekJ.Anoncogene-induced. D. N. A.damagemodel.forcancer.developmentScience2008Mar 7;319586813525
  128. 128. SerranoM.TheI. N.K4aA. R. F.locusin.murinetumorigenesis.Carcinogenesis2000May;2158659
  129. 129. PollackM.PhaneufS.DirksA.LeeuwenburghC.The role of apoptosis in the normal aging brain, skeletal muscle, and heart. Ann N Y Acad Sci2002Apr;95993107
  130. 130. MiyashitaT.ReedJ. C.Tumorsuppressor. p.isa.directtranscriptional.activatorof.thehuman.baxgene.Cell1995Jan 27;8022939
  131. 131. NakanoK.VousdenK. H. P. U. M. A. a.novelproapoptotic.geneis.inducedby.p5Mol Cell2001Mar;7368394
  132. 132. al.Noxaa. B.H3-onlymember.ofthe.Bclfamilycandidatemediator.ofp53-induced.apoptosisScience2000May 12;288546810538
  133. 133. Martinou JC, Green DR.Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol2001Jan;21637
  134. 134. FerriK. F.KroemerG.Organelle-specific initiation of cell death pathways. Nat Cell Biol2001Nov;3(11):E25563
  135. 135. CampisiJ.Aging and cancer cell biology, 2007. Aging Cell2007Jun;632613
  136. 136. InomataK.AotoT.BinhN. al.Genotoxic stress abrogates renewal of melanocyte stem cells by triggering their differentiation. Cell2009Jun 12;1376108899
  137. 137. TedeschiA.Di GiovanniS.The non-apoptotic role of 53in neuronal biology: enlightening the dark side of the moon. EMBO Rep2009Jun;10(6):576-83.
  138. 138. CicaleseA.BonizziG.CEPasiFaretta. al.The tumor suppressor 53regulates polarity of self-renewing divisions in mammary stem cells. Cell2009Sep 18;138(6):1083-95.
  139. 139. BudanovA. V.KarinM. p.targetgenes.sestrinsestrinconnect.genotoxicstress.signalingm. T. O. R.Cell2008Aug 8;134345160
  140. 140. AlexanderA.CaiS. L.KimJ.NanezA.SahinM.MacLean. K. H.etal. A. T. M.signalsto. T. S. C.inthe.cytoplasmto.regulatem. T. O. R. C.inresponse.toR. O. S.Proc Natl Acad Sci U S A Mar 2;107941538
  141. 141. GorgoulisV. G.VassiliouL. al.Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature2005Apr 14;434703590713
  142. 142. BraigM.LeeS.LoddenkemperC.RudolphC.PetersA. al.Oncogene-induced senescence as an initial barrier in lymphoma development. Nature2005Aug 4;43670516605
  143. 143. ColladoM.GilJ.EfeyanA.GuerraC.SchuhmacherA. al.Tumourbiology.senescencein.premalignanttumours.Nature2005Aug 4;436(7051):642.
  144. 144. LordC. J.AshworthA.Targeted therapy for cancer using PARP inhibitors. Curr Opin Pharmacol2008Aug;843639
  145. 145. ChenZ.XiaoZ.GuW. Z.XueJ.BuiM. al.SelectiveChk.inhibitorsdifferentially.sensitizep53-deficient.cancercells.tocancer.therapeuticsInt J Cancer2006Dec 15;11912278494
  146. 146. MurgaM.BuntingS.MontanaM. F.SoriaR.MuleroF.CanameroM.etal. A.mousemodel.of-SeckelA. T. R.showsembryonic.replicativestress.acceleratedaging.Nat Genet2009Aug;4188918
  147. 147. ToledoL. I.MurgaM.ZurR.SoriaR.RodriguezA.MartinezS.etal. A.cell-basedscreen.identifiesA. T. R.inhibitorswith.syntheticlethal.propertiesfor.cancer-associatedmutations.
  148. 148. MurgaM.CampanerS.Lopez-ContrerasA. J.ToledoL. I.SoriaR.MontanaM. al.Exploiting oncogene-induced replicative stress for the selective killing of Myc-driven tumors. Nat Struct Mol Biol Dec;181213315
  149. 149. GarinisG. A.van der HorstG. T.VijgJ.HoeijmakersJ. H. D. N.DNA damage and ageing: new-age ideas for an age-old problem. Nat Cell Biol2008Nov;101112417
  150. 150. LombardD. B.ChuaK. F.MostoslavskyR.FrancoS.GostissaM.AltF. W. D. N. A.repairgenome.stabilityagingCell2005Feb 25;1204497512
  151. 151. RossiD. J.BryderD.SeitaJ.NussenzweigA.HoeijmakersJ.WeissmanI. L.Deficienciesin. D. N. A.damagerepair.limitthe.functionof.haematopoieticstem.cellswith.ageNature2007Jun 7;44771457259
  152. 152. SedelnikovaO. A.HorikawaI.ZimonjicD. B.PopescuN. C.BonnerW. M.BarrettJ. C.Senescing human cells and ageing mice accumulate DNA lesions with unrepairable double-strand breaks. Nat Cell Biol2004Feb;6216870
  153. 153. Rossi DJ, Jamieson CH, Weissman IL.Stems cells and the pathways to aging and cancer. Cell2008Feb 22;132468196
  154. 154. Fernandez-CapetilloO.Intrauterine programming of ageing. EMBO Rep Jan;111326
  155. 155. Pickart CM.Ubiquitin enters the new millennium. Mol Cell2001Sep;83499504
  156. 156. HaglundK.DikicI.Ubiquitylation and cell signaling. EMBO J2005Oct 5;241933539
  157. 157. SchwartzA. L.CiechanoverA.Targeting proteins for destruction by the ubiquitin system: implications for human pathobiology. Annu Rev Pharmacol Toxicol2009497396
  158. 158. Pickart CM, Cohen RE.Proteasomes and their kin: proteases in the machine age. Nat Rev Mol Cell Biol2004Mar;5317787
  159. 159. Messick TE, Greenberg RA.The ubiquitin landscape at DNA double-strand breaks. J Cell Biol2009Nov 2;187331926
  160. 160. GrabbeC.DikicI.Functional roles of ubiquitin-like domain (ULD) and ubiquitin-binding domain (UBD) containing proteins. Chem Rev2009Apr;1094148194
  161. 161. Bekker-JensenS.RendtlewDanielsen. J.FuggerK.GromovaI.NerstedtA.LukasC.etal. H. E. R. C.coordinatesubiquitin-dependent.assemblyof. D. N. A.repairfactors.ondamaged.chromosomesNat Cell Biol Jan;12(1):80-6; sup 112
  162. 162. SandersS. L.PortosoM.MataJ.BahlerJ.AllshireR. C.KouzaridesT.Methylation of histone H4 lysine 20 controls recruitment of Crb2 to sites of DNA damage. Cell2004Nov 24;119560314
  163. 163. ShaoG.LilliD. R.Patterson-FortinJ.ColemanK. A.MorrisseyD. E.GreenbergR. A.TheRap8.B. R. C. R. N. F.Ubc13-dependentubiquitination.eventsat. D. N. A.doublestrand.breaksProc Natl Acad Sci U S A2009Mar 3;1069316671
  164. 164. ShaoG.Patterson-FortinJ.MessickT. E.FengD.ShanbhagN.WangY.etal. M. E. R. I. T.controlsB. R. C. A.Rapcomplexintegrity.recruitmentto. D. N. A.double-strandbreaks.Genes Dev2009Mar 15;23674054
  165. 165. YuX.FuS.LaiM.BaerR.ChenJ. B. R. C. A.ubiquitinatesits.phosphorylation-dependentbinding.partnerCt. I. P.Genes Dev2006Jul 1;201317216
  166. 166. BrzovicP. S.LissounovA.ChristensenD. E.HoytD. W.KlevitR. E. A.UbcH5/ubiquitin.noncovalentcomplex.isrequired.forprocessive. B. R. C.A1-directedubiquitination.Mol Cell2006Mar 17;21687380
  167. 167. ReidL. J.ShakyaR.ModiA. P.LokshinM.ChengJ. T.JasinM.etal. E.ligaseactivity.ofB. R. C. A.isnot.essentialfor.mammaliancell.viabilityor.homology-directedrepair.ofdouble-strand. D. N. A.breaksProc Natl Acad Sci U S A2008Dec 30;105522087681
  168. 168. ShakyaR.ReidL. J.ReczekC. R.ColeF.EgliD.LinC. S.etal. B. R. C. A.tumorsuppression.dependson. B. R. C. T.phosphoproteinbinding.butnot.itsE.ligaseactivity.Science Oct 28;33460555258
  169. 169. DrostR.BouwmanP.RottenbergS.BoonU.SchutE.KlarenbeekS.etal. B. R. C. A. R. I. N. G.functionis.essentialfor.tumorsuppression.butdispensable.fortherapy.resistanceCancer Cell Dec 13;206797809
  170. 170. ZhuQ.PaoG. M.HuynhA. M.SuhH.TonnuN.NederlofP. M.etal. B. R. C. A.tumoursuppression.occursvia.heterochromatin-mediatedsilencing.Nature Sep 8;477736317984
  171. 171. Moldovan GL, D’Andrea AD.How the fanconi anemia pathway guards the genome. Annu Rev Genet20094322349
  172. 172. WangW.Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins. Nat Rev Genet2007Oct;81073548
  173. 173. MatsushitaN.KitaoH.IshiaiM.NagashimaN.HiranoS.OkawaK.etal. A.FancD2-monoubiquitin.fusionreveals.hiddenfunctions.ofFanconi.anemiacore.complexin. D. N. A.repairMol Cell2005Sep 16;1968417
  174. 174. Yeh ET. SUMOylation and De-SUMOylation: wrestling with life’s processes.J Biol Chem2009Mar 27;2841382237
  175. 175. GalantyY.BelotserkovskayaR.CoatesJ.PoloS.MillerK. M.JacksonS. P.MammalianS. U. M. O.E3-ligasesP. I. A. S.promoteP. I. A. S.responsesto. D. N. A.double-strandbreaks.Nature2009Dec 17;46272759359
  176. 176. MorrisJ. al.TheS. U. M. O.modificationpathway.isinvolved.inthe. B. R. C. A.responseto.genotoxicstress.Nature2009Dec 17;462727588690
  177. 177. Potts PR.The Yin and Yang of the MMS21-SMC5/6 SUMO ligase complex in homologous recombination. DNA Repair (Amst)2009Apr 5;84499506
  178. 178. al.Ubcmms21-mediatedsumoylation.counteractsrecombinogenic.eventsat.damagedreplication.forksCell2006Nov 3;127350922
  179. 179. BenkiraneM.SardetC.CouxO.Lessons from interconnected ubiquitylation and acetylation of 53think metastable networks. Biochem Soc Trans Feb;38(Pt 1):98-103.
  180. 180. MomandJ.ZambettiG. P.OlsonD. C.GeorgeD.LevineA. J.Themdm.oncogeneproduct.formsa.complexwith.thep.proteininhibitsp53-mediated.transactivationCell1992Jun 26;697123745
  181. 181. ItoA.LaiC. H.ZhaoX.SaitoS.HamiltonM. al.p30P-mediatedC. B.p.acetylationis.commonlyinduced.byp53-activating.agentsinhibitedby. M. D. M.E. M. B.EMBO J2001Mar 15;206133140
  182. 182. ShiX.KachirskaiaI.YamaguchiH.WestL. E.WenH.WangE. al.Modulationof. p.functionby. S. E.T8-mediatedmethylation.atlysine.38Mol Cell2007Aug 17;27463646
  183. 183. IvanovG. al.Methylation-acetylation interplay activates 53in response to DNA damage. Mol Cell Biol2007Oct;27(19):6756-69.
  184. 184. Tjeertes JV, Miller KM, Jackson SP.Screen for DNA-damage-responsive histone modifications identifies H3K9Ac and H3K56Ac in human cells. EMBO J2009Jul 8;2813187889
  185. 185. van AttikumH.GasserS. M.Crosstalk between histone modifications during the DNA damage response. Trends Cell Biol2009May;19520717
  186. 186. YuanJ.PuM.ZhangZ.LouZ.HistoneH.K.acetylationis.importantfor.genomicstability.inmammals.Cell Cycle2009Jun 1;811174753
  187. 187. Fernandez-CapetilloO.MurgaM.Why cells respond differently to DNA damage: a chromatin perspective. Cell Cycle2008Apr 15;789803

Written By

Andres Joaquin Lopez-Contreras and Oscar Fernandez-Capetillo

Submitted: April 17th, 2012 Published: September 6th, 2012