Sensing of DNA Damage
Xiaohong H. Yang, Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, Massachusetts
Lee Zou, Massachusetts General Hospital Cancer Center and Department of Pathology, Harvard Medical School, Boston, Massachusetts
Maintenance of genomic stability is essential for the survival of cells, organisms, and species. Genomic stability relies on the complete and accurate transmission of genetic materials from mother cells to daughter cells and from one generation to the next. This task is daunting, however, because the genome is constantly challenged by numerous intrinsic and extrinsic stresses that damage deoxyribonucleic acid (DNA). If left untreated, such damage can destabilize the genome by introducing gene mutations, duplications, and chromosomal rearrangements such as deletions and inversions, which fortunately does not normally happen as cells evolve a complex damage sensing and signaling mechanism named the DNA damage and replication checkpoint. Checkpoint activation effectively pauses the progression of the cell cycle, allowing more time for the removal of DNA lesion. Moreover, activated checkpoint also regulates and coordinates a number of cellular processes including DNA repair, DNA replication, and chromatin remodeling to alleviate the stress on the genome. At the core of the checkpoint-signaling network, a family of phosphoinositide-3-kinase (PI-3K)-like kinases and their regulatory partners serve as both DNA damage sensors and initiators of checkpoint signaling. In this article, we will discuss in detail how DNA damage is sensed and processed by the DNA damage and replication checkpoint. We will also discuss how signals generated at sites of DNA damage are propagated and relayed through the checkpoint pathways to influence other cellular events.
The Biology of Checkpoint
Deoxyribonucleic acid (DNA) damage poses a constant and serious threat to the stability and integrity of the genome. DNA damage can develop from exposure to external sources such as chemical carcinogens, ultraviolet (UV) light, or X ray. It can also be caused by sources from within: metabolic byproducts, free radicals, and interference with duplication and segregation of genomic DNA. To maintain genomic stability in the face of DNA damage, an intricate and elaborate signaling pathway called the DNA damage and replication checkpoint is evolved. Remarkably, essential components of checkpoint are largely conserved throughout evolution, underlying the importance of this pathway and making the study of checkpoint simultaneously in different model systems rational. In eukaryotes ranging from human to yeast, two signaling pathways, which are initiated respectively by the ATM (ataxia telangiectasia mutated) kinase and the ATR (ATM- and Rad3-related) kinase, are the major guardians of genome stability (1). Although ATM primarily responds to double-stranded DNA breaks (DSBs), ATR regulates the response to a wide spectrum of DNA damage, especially those interfering with DNA replication (2). Through phosphorylation of signal transducer and effector proteins, ATM and ATR relay the DNA damage signals to downstream cellular processes. The effectors of ATM and ATR include proteins involved in cell-cycle transitions, DNA replication, DNA repair, chromatin remodeling, telomere maintenance, transcription control, and apoptosis. Collectively, the phosphorylation of these effectors enhances the ability of cells to repair and to overcome the encountered DNA damage. When the extent of damage reaches an intolerable level, activated checkpoint can also lead to programmed cell death.
What will happen if cells do not have a functional checkpoint? The ATR checkpoint is essential for the survival of cells and the embryonic development in mammals (3, 4), which is likely a result of the important function of ATR in coping with the intrinsic stresses during DNA replication. On the other hand, the ATM checkpoint is not essential, but its defects result in phenotypes associated with genomic instability, such as high cancer risk, premature aging, and neuron degeneration (2). Mutations in several other genes involved in the ATM or ATR checkpoint response, such as p53, Brca1, and Nbs1, have also been found to associate with cancer. Thus, it is clear that the ATM and the ATR checkpoint are crucial for survival at both cell and organism levels.
The Chemistry of DNA Damage Sensing
In eukaryotic cells, a prototypical checkpoint cascade consists of DNA damage sensors, signal initiators, signal transducers, and effectors. Similar to many other signal transduction cascades, the signaling of checkpoint pathways is primarily achieved by protein phosphorylation. ATM and ATR are believed to be the signal-initiating kinases in these pathways. Albeit related in sequence, ATM and ATR differ in their DNA damage specificity. ATM responds primarily to DSBs, a rare but acute threat to genomic stability. ATR, on the other hand, is more pleiotrophic and indispensable for checkpoint responses to damage caused by disturbance during normal DNA replication. With the help of distinct sets of DNA damage sensors that recognize specific DNA damage-induced structures (see below), ATM and ATR are localized to sites of DNA damage in cells and phosphorylate their substrates at these sites.
Activated ATM and ATR phosphorylate proteins of various functions, two of which are the Chk1 and Chk2 kinases. Chk1 and Chk2 are pivotal signal transducers in the checkpoint pathways (5, 6). Phosphorylation and activation of Chk1 and Chk2 not only allow the checkpoint signals to reach additional effectors, but also provide an additional layer of signal regulation. A group of signal transducers, including Brca1, TopBP1, Claspin, 53BP1, and Mdc1, mediate the signaling from ATR and ATM to Chk1 and Chk2 in cells (7-15). These proteins are often referred to as the mediators. Many of the mediators are substrates of ATM or ATR, and they contain phosphopeptide-binding domains such as the breast cancer C-terminal (BRCT) and the forkhead-associated (FHA) domains. These properties of the mediators enable them to interact with other proteins in a phosphorylation-dependent and damage-regulated manner, thereby allowing the checkpoint signals to be relayed to downstream effectors. As a result of checkpoint activation, the cell cycle can be temporarily halted at the G1/S or the G2/M transitions, or even within the S phase. Furthermore, activated ATM, ATR, and the CHK kinases also phosphorylate proteins involved in DNA repair, DNA replication, chromatin remodeling, and gene expression to facilitate the removal of DNA lesions and to alleviate the stress on the genome.
DNA Damage Sensing by the ATM Pathway
In response to ionizing radiation or certain types of DNA- damaging agents (e.g., cisplatin), the activity of ATM is rapidly stimulated by DSBs (16). In undamaged cells, ATM molecules exist as inactive oligomers. Upon DNA damage, autophosphorylation of ATM on a Serine residue (Ser1981) transforms the latent oligomers into active monomers (Fig. 1) (17), which appears to be an essential step for ATM-mediated checkpoint activation as mutation of this residue renders the kinase functionless. It should be noted that ATM autophosphorylation can be triggered by very low doses of ionizing radiation or disruption of chromatin structure in the absence of detectable DSBs (17). It was hypothesized that alterations of chromatin structure might partially activate ATM. However, the exact mechanism that leads to ATM autophosphorylation remains unclear.
Autophosphorylated ATM is capable of phosphorylating non-DNA-bound substrates like p53, but the phosphorylation of other substrates at sites of DNA damage requires actions of the Mre11-Rad50-Nbs1 (MRN) complex (Fig. 1). In cells lacking Mre11 or Nbsl, ATM does not associate with chromatin nor does it phosphorylate its substrates such as Brca1 and Smcl (18). The MRN complex can directly associate with double-stranded DNA ends in vitro (19, 20), and it possesses a 3' to 5' exonuclease activity (21). Mre11, the nuclease of the complex, stably associates with Rad50 to form a globular DNA-binding head with two flexible arms comprising the coiled coils of Rad50 (19, 20). The Mre11-Rad50 complex can bind to DNA ends and tether DNA fragments through the arms of Rad50 (20, 22). Nbsl interacts with both Mre11 and ATM, and its ATM-binding domain in the C terminus is critical for the recruitment of ATM to DNA damage (23). Interestingly, a recent study using purified proteins demonstrated that the MRN complex is able to stimulate the phosphorylation of substrates by ATM in the presence of double-stranded DNA ends (24). These findings suggest that the MRN complex, which not only recruits ATM to DSBs but also directly stimulates its kinase activity, is an important DNA damage sensor of the ATM pathway.
The recognition of double-stranded DNA ends is only the first step of the DNA damage sensing by ATM. As soon as ATM is activated at the DNA ends, it phosphorylates the histone H2AX in the adjacent nucleosomes (Fig. 1) (25). Phos- phorylated histone H2AX recruits the mediator Mdc1 through an interaction with its BRCT domain (12, 26, 27), allowing Mdc1 to recruit additional ATM molecules with its FHA domain (26). This Mdc1-mediated recruitment mechanism enables ATM to phosphorylate distant histone H2AX molecules farther away from the DNA breaks, and eventually generate a large chromatin region with phosphorylated histone H2AX. Phos- phorylated histone H2AX may then bring in additional ATM substrates involved in DNA repair, chromatin remodeling, and checkpoint signaling to facilitate the functions of these proteins at sites of DNA damage (26).
In addition to phosphorylated histone H2AX, methylated histones H3 and H4 are also implicated in checkpoint signaling (28-30). It was shown that the mediator 53BP1 recognizes methylated histone H3 through its tandem Tudor domains (28). Interestingly, the methylation of histone H3 is not induced by DNA damage, which led to the hypothesis that a change of chromatin structure, which exposes the preexisting methylated site on histone H3, is induced at sites of DNA damage.
It has become clear that the sensing of DNA damage by the ATM pathway is a multistep process involving several independent sensing mechanisms. The ATM pathway recognizes not only DNA breaks but also protein modifications at sites of DNA damage. These distinct sensing mechanisms ensure that the signaling of the ATM pathway is tightly regulated at multiple levels.
Figure 1. Sensing of DSBs by the ATM checkpoint. DSBs trigger the autophosphorylation of ATM and transform the inactive ATM oligomers to active ATM monomers. The MRN complex is important for the recruitment and the activation of ATM at DSBs. ATM phosphorylates the histone H2AX adjacent to the DSBs, which in turn recruits the mediator Mdc1 and brings in additional ATM molecules.
Recruitment of ATR to DNA Damage
Unlike ATM, which strongly prefers DSBs, ATR is a broad-spectrum signal initiator. Various types of replication interference, such as those induced by UV irradiation or ribonucleotide reductase inhibitor Hydroxyurea (HU), strongly elicit the ATR pathway. This versatility and the pivotal role of ATR in cell viability and genomic stability has prompted an intensive investigation into the mechanism(s) by which ATR senses different types of DNA damage and activates the checkpoint.
In human cells, ATR exists in a stable complex with another essential checkpoint protein called ATRIP (3). In budding yeast Saccaramycies serevisiae, ATR’s homolog Mec1 also functions in a complex with Ddc2, the budding yeast ATRIP homolog (31-33). It has been shown that Mec1 can be activated in the cdc13 temperature-sensitive mutant as well as by the HO endonuclease (34, 35). Cdc13 is a telomere-binding protein. When mutated, telomere is unprotected and stretches of single-stranded DNA (ssDNA) are exposed because of cleavage by exonucleases (36). In the other scenario, ssDNA is also generated at the exonuclease-processed DSBs induced by HO ((37)). Increasing amounts of ssDNA and ATR activation were also observed when yeast replication forks are blocked by HU and UV treatments (Fig. 2) (38, 39). All these findings suggested a link between ssDNA and the activation of the ATR pathway.
Figure 2. Sensing of replication interference by the ATR checkpoint. Long stretches of ssDNA are generated at replication forks when they encounter DNA damage interfering with DNA polymerases. RPA-coated ssDNA is recognized by the ATR-ATRIP complex. The Rad17 complex recognizes the junctions of double- and single-stranded DNA and recruits 9-1-1 complexes to the stressed forks.
Replication protein A (RPA), a protein complex with affinity to ssDNA, is a key player in many types of DNA metabolisms such as DNA replication and DNA repair (40). In G2 yeast cells, depletion of RPA abolished the localization of Ddc2 to HO-induced DSBs (41), suggesting that RPA is important for the recruitment of Mec1 to sites of DNA damage. The distinct functions of RPA in DNA replication and checkpoint activation were demonstrated in rfa-t11, an RPA mutant strain proficient in DNA replication yet partially defective in checkpoint activation (41). In the African frog Xenopus egg extract, another model system used in the study of DNA replication and checkpoint, ATR associates with chromatin in a replication-dependent manner (42). Depletion of RPA, however, ceased the association of ATR with chromatin (43). In human cells, RPA is required for the localization of ATR to DNA damage-induced nuclear foci and the efficient phosphorylation of Chk1 (41). Collectively, these findings suggest that ssDNA coated with RPA might be a common structure recognized by ATR and ATRIP.
In a series of in vitro biochemical experiments aimed at recapitulating the initial steps of DNA damage sensing in human cells, the roles of ssDNA and RPA were directly analyzed (41). In these experiments, single- or double-stranded DNA of various lengths was biotinylated and immobilized on streptavidin-tagged magnetic beads. The association of purified ATR and ATRIP with immobilized DNA was analyzed in the presence or absence of RPA. It was found that 1) purified ATRIP protein was efficiently recruited to ssDNA only in the presence of purified RPA; 2) RPA confers ATRIP higher affinity to ssDNA than dsDNA (double-stranded DNA); and 3) the ATR-ATRIP complex, but not ATR alone, binds to ssDNA efficiently in the presence of RPA. Together, these experiments demonstrated that ssDNA coated with RPA is a structure that efficiently recruits ATR-ATRIP (Fig. 2). As RPA-coated ssDNA is commonly generated during different types of DNA repair and when DNA replication was interrupted, it is highly possible that RPA-coated ssDNA is the key structure that enables ATR-ATRIP to respond to a broad spectrum of DNA damage (Fig. 2). These findings, however, do not rule out the possibility that ATR-ATRIP can localize to specific types of DNA damage through alternative protein-protein or protein-DNA interactions.
Roles of Replication Factor C (RFC)- and Proliferating Cell Nuclear Antigen (PCNA)-like Complexes in Damage Sensing
Although RPA-coated ssDNA is critical for the recruitment of ATR-ATRIP to sites of DNA damage, RPA-ssDNA alone is not sufficient for ATR-ATRIP to elicit a robust checkpoint response. The function of ATR-ATRIP requires additional regulatory proteins including the RFC-like Rad17 complex and the PCNA-like Rad9-Rad1-Hus1 (9-1-1) complex. During DNA replication, the RFC complex recognizes the primer-template junctions (the junctions of dsDNA and ssDNA) at replication forks and recruits ring-shaped PCNA complexes onto DNA in an ATP-dependent manner (44). Once loaded onto DNA, PCNA functions as a sliding clamp allowing DNA polymerases to stably associate with their template. Likewise, the PCNA-like 9-1-1 complex is recruited onto DNA by the RFC-like Rad17 complex (45). However, the recruitment of 9-1-1 occurs only after DNA damage, suggesting that the Rad17 complex can specifically recognize certain DNA structures induced by DNA damage.
Single-strand DNA generated at sites of DNA damage or stressed replication fork is always juxtaposed with junctions of ssDNA and dsDNA (Fig. 2). To investigate whether the Rad17 and 9-1-1 complexes recognize such DNA junctions, these complexes were expressed in engineered insect cells and purified using affinity chromatography. In several in vitro biochemical assays, the Rad17 complex recruited 9-1-1 complexes onto DNA structures with both ssDNA and dsDNA regions (46-49), and this recruitment was enhanced by RPA (48, 49). Interestingly, unlike RFC, which uses only the 3' double/single-strand DNA junctions to PCNA, the Rad17 complex can use the 5' double/single-strand DNA junctions to recruit 9-1-1 complexes (48, 49). This finding provides a possible explanation as to how Rad17 and 9-1-1 complexes are recruited to resected DSBs and telomeres that possess only 5' double/single-strand DNA junctions. As ssDNA gaps are observed at stressed replication forks, both 5' and 3' double/single-strand DNA junctions are induced by replication interference (Fig. 2). Together, these experiments suggest that the Rad17 and 9-1-1 complexes are sensors of the double/single-strand DNA junctions at sites of DNA damage and stressed DNA replication forks.
Similar to the DNA damage sensing by the ATM pathway, the sensing by the ATR pathway is also a multistep process. ATR-ATRIP directly recognizes RPA-coated ssDNA, whereas the Rad17 and 9-1-1 complexes recognize the junctions of ssDNA and dsDNA. Additional checkpoint regulators may also contribute to the sensing of different DNA structures at sites of DNA damage. The colocalization of ATR-ATRIP and its regulators on damaged DNA may enable the ATR to be activated by these regulators and to phosphorylate its substrates. Consistent with this idea, TopBP1, a protein that interacts with the 9-1-1 complex, stimulates the kinase activity of ATR-ATRIP in vitro (50). In summary, the ability to recognize certain shared DNA structures at sites of DNA damage is most likely the key for the versatility of the ATR pathway. Furthermore, the involvement of multiple sensors in this pathway may enable it to distinguish different types of DNA damage and generate a signal accordingly.
Processing of DNA Damage
Many types of DNA damage that efficiently elicit the ATR checkpoint interfere with DNA replication, suggesting that DNA replication forks may play a particularly important role in the processing of DNA damage to structures recognizable to the ATR checkpoint (e.g., ssDNA and junctions of ssDNA and dsDNA). Accumulating evidence has suggested that the uncoupling of DNA helicases and DNA polymerases at progressing replication forks may lead to increased amounts of ssDNA at the forks (51). The stalling of DNA polymerases can directly result from DNA lesions themselves or from those recognized or processed by specific repair proteins. For example, xeroderma pigmentosum group A (XPA), a protein involved in the recognition of UV-induced DNA damage, is required for the activation of ATR checkpoint by UV during S phase (52).
In addition to the DNA damage that interferes with replication, the ATR checkpoint is also elicited by DSBs. These DSBs, however, need to be first recognized and processed by specific factors. It was recently shown that the activation of ATR by ionizing radiation, but not that by replication stress, requires ATM and the MRN complex (53-55). In the absence of ATM or the MRN complex, RPA can no longer localize to DSBs, indicating that the formation of ssDNA at DSBs is compromised. The processing of DSBs also requires CDK activity and is restricted to the S and G2 phases of the cell cycle. Thus, even with distinct DNA structure specificities, the ATM and the ATR checkpoints may function together to mediate a coordinated response to DNA breaks in the replicating or replicated genome.
Tools and Techniques
The sensing of DNA damage by the checkpoint has been characterized both in cells and in cell-free systems by using biochemical approaches. Several methods are commonly used to introduce different types of DNA damage in cells (56). For instance, to generate DSBs in cultured cells, cells can be treated with IR (ionizing radiation), laser beam, or therapeutic compounds such as cisplatin and bleomycin. To introduce replication stress to cells, cells are treated with UV or replication inhibitors such as HU and aphidicolin. It should be noted that the actual DNA damage induced by these methods are likely heterogeneous. For example, IR induces not only DSBs but also single-strand DNA breaks and other types of DNA lesion.
Many checkpoint-mediated protein phosphorylation events can be detected by Western blotting with phospho-specific antibodies or mobility shift on protein gels due to the modification. As a result, the phosphorylational status of these proteins has been exploited as markers for checkpoint signaling in cells. Moreover, cell-cycle stage-specific checkpoint responses can now be monitored by stage-specific assays such as the G1, intra-S, and G2/M checkpoint assays (57-59). Checkpoint kinase activities can be measured by using immunoprecipitation and in vitro kinase assays. Immnofluorescence, chromatin fractionation, and chromatin immunoprecipitation are commonly used methodologies to analyze the recruitment of checkpoint proteins to sites of DNA damage.
Several cell-free assay systems have been used to dissect the mechanisms of DNA damage sensing in vitro. The most extensively applied system is the one using Xenopus extracts. As DNA replication can occur efficiently in Xenopus extracts, this system has been used to analyze how replication interference is sensed by checkpoint sensors (60, 61) and how activated checkpoint regulates DNA replication. The checkpoint can also be elicited in a replication-independent manner by various synthetic DNA structures in Xenopus extracts (9, 62). The checkpoint response in Xenopus extracts is often monitored by the phosphorylation of various checkpoint proteins. The association of checkpoint proteins with damaged chromatin or DNA is used to monitor the recognition of DNA damage in Xenopus extracts.
In vitro biochemical assays with purified proteins are developed to demonstrate the direct recognition of damage-induced DNA structures by checkpoint sensors (41). Various checkpoint proteins have been successfully expressed in bacteria, yeast, insect, and mammalian cells and have been purified biochemically. The association of checkpoint proteins with various DNA structures can be captured and visualized by using biotinylated DNA or DNA labeled with radioactive isotopes. Finally, in vitro kinase assay is performed to measure the effects of DNA on the activity of checkpoint kinases and the phosphorylation of their substrates.
Since Hartwell and Weinert first observed that DNA damage led to cell-cycle arrest in budding yeast and proposed the concept of the DNA damage checkpoint (63), the checkpoint field has evolved quickly into a leading area of both basic biological research and cancer and disease-oriented research. DNA damage and replication checkpoint, as we now know, is an indispensable and evolutionarily conserved cellular process. In all species, checkpoint is an integral part of cell survival and organism development by maintaining genomic stability against continuous insults from both outside and inside. Instability of the genome in human cells is often both a prelude and a hallmark of cancer, a debilitating disease affecting millions and the leading cause of death in many countries including the United States. In fact, mutations (p53, Brcal, Chk2) or abnormality of the checkpoint pathway have been found repeatedly to associate with cancer or other cancer-prone genetic disorders. A thorough understanding of the chemical and biochemical steps that lead to checkpoint activation and its impact on other cellular processes will surely generate much insight into tumorigenesis at the molecular, cellular, and organ level. The study of the mechanism of action and specificity of the checkpoint kinases will also help us design safer and better drugs to fight cancer. Indeed, several inhibitors of the checkpoint kinases are already in various stages of preclinical and clinical studies. From a more fundamental standpoint, we still know very little about the actual events leading up to the sensing of DNA damage and the activation of ATM and ATR. The biochemical and functional roles of a number of checkpoint proteins remain virtually unknown. Furthermore, from examining the disease symptoms and phenotypes of knockout mice, it is obvious that defects of checkpoint intercept with development. Yet, how checkpoint influences development and the difference among specific organs and systems is not clear at all. In summary, the DNA damage and replication checkpoint is a fundamental and elaborate cellular process. It protects cells from being genetically compromised by DNA damage. Uncovering the hidden secrets of the checkpoint mechanism will lead us closer to cracking the codes of cancer and other genetic and developmental diseases.
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Chemistry of DNA Damage Repair
Cell Cycle Checkpoint
Signal Transduction by Posttranslational Modifications
Chemical Biology of Cancer