ATR and H2AX Cooperate in Maintaining Genome Stability under Replication Stress

Chromosomal abnormalities are frequently caused by problems encountered during DNA replication. Although the ATR-Chk1 pathway has previously been implicated in preventing the collapse of stalled replication forks into double-strand breaks (DSB), the importance of the response to fork collapse in ATR-deficient cells has not been well characterized. Herein, we demonstrate that, upon stalled replication, ATR deficiency leads to the phosphorylation of H2AX by ATM and DNA-PKcs and to the focal accumulation of Rad51, a marker of homologous recombination and fork restart. Because H2AX has been shown to play a facilitative role in homologous recombination, we hypothesized that H2AX participates in Rad51-mediated suppression of DSBs generated in the absence of ATR. Consistent with this model, increased Rad51 focal accumulation in ATR-deficient cells is largely dependent on H2AX, and dual deficiencies in ATR and H2AX lead to synergistic increases in chromatid breaks and translocations. Importantly, the ATM and DNA-PK phosphorylation site on H2AX (Ser¹³⁹) is required for genome stabilization in the absence of ATR; therefore, phosphorylation of H2AX by ATM and DNA-PKcs plays a pivotal role in suppressing DSBs during DNA synthesis in instances of ATR pathway failure. These results imply that ATR-dependent fork stabilization and H2AX/ATM/DNA-PKcs-dependent restart pathways cooperatively suppress double-strand breaks as a layered response network when replication stalls.Genome maintenance prevents mutations that lead to cancer and age-related diseases. A major challenge in preserving genome integrity occurs in the simple act of DNA replication, in which failures at numerous levels can occur. Besides the mis-incorporation of nucleotides, it is during this phase of the cell cycle that the relatively stable double-stranded nature of DNA is temporarily suspended at the replication fork, a structure that is susceptible to collapse into DSBs.² Replication fork stability is maintained by a variety of mechanisms, including activation of the ATR-dependent checkpoint pathway.The ATR pathway is activated upon the generation and recognition of extended stretches of single-stranded DNA at stalled replication forks (1-4). Genome maintenance functions for ATR and orthologs in yeast were first indicated by increased chromatid breaks in ATR^-/- cultured cells (5) and by the “cut” phenotype observed in Mec1 (Saccharomyces cerevisiae) and Rad3 (Schizosaccharomyces pombe) mutants (6-9). Importantly, subsequent studies in S. cerevisiae demonstrated that mutation of Mec1 or the downstream checkpoint kinase Rad53 led to increased chromosome breaks at regions of the genome that are inherently difficult to replicate (10), and a decreased ability to reinitiate replication fork progression following DNA damage or deoxyribonucleotide depletion (11-14).In vertebrates, similar replication fork stabilizing functions have been demonstrated for ATR and the downstream protein kinase Chk1 (15-20). Several possible mechanisms have been put forward to explain how ATR-Chk1 and orthologous pathways in yeast maintain replication fork stability, including maintenance of replicative polymerases (α, δ, and ε) at forks (17, 21), regulation of branch migrating helicases, such as Blm (22-25), and regulation of homologous recombination, either positively or negatively (26-29).Consistent with the role of the ATR-dependent checkpoint in replication fork stability, common fragile sites, located in late-replicating regions of the genome, are significantly more unstable (5-10-fold) in the absence of ATR or Chk1 (19, 20). Because these sites are favored regions of instability in oncogene-transformed cells and preneoplastic lesions (30, 31), it is possible that the increased tumor incidence observed in ATR haploinsufficient mice (5, 32) may be related to subtle increases in genomic instability. Together, these studies indicate that maintenance of replication fork stability may contribute to tumor suppression.It is important to note that prevention of fork collapse represents an early response to problems occurring during DNA replication. In the event of fork collapse into DSBs, homologous recombination (HR) has also been demonstrated to play a key role in genome stability during S phase by catalyzing recombination between sister chromatids as a means to re-establish replication forks (33). Importantly, a facilitator of homologous recombination, H2AX, has been shown to be phosphorylated under conditions that cause replication fork collapse (18, 34).Phosphorylation of H2AX occurs predominantly upon DSB formation (34-38) and has been reported to require ATM, DNA-PKcs, or ATR, depending on the context (37-42). Although H2AX is not essential for HR, studies have demonstrated that H2AX mutation leads to deficiencies in HR (43, 44), and suppresses events associated with homologous recombination, such as the focal accumulation of Rad51, BRCA1, BRCA2, ubiquitinated-FANCD2, and Ubc13-mediated chromatin ubiquitination (43, 45-51). Therefore, through its contribution to HR, it is possible that H2AX plays an important role in replication fork stability as part of a salvage pathway to reinitiate replication following collapse.If ATR prevents the collapse of stalled replication forks into DSBs, and H2AX facilitates HR-mediated restart, the combined deficiency in ATR and H2AX would be expected to dramatically enhance the accumulation of DSBs upon replication fork stalling. Herein, we utilize both partial and complete elimination of ATR and H2AX to demonstrate that these genes work cooperatively in non-redundant pathways to suppress DSBs during S phase. As discussed, these studies imply that the various components of replication fork protection and regeneration cooperate to maintain replication fork stability. Given the large number of genes involved in each of these processes, it is possible that combined deficiencies in these pathways may be relatively frequent in humans and may synergistically influence the onset of age-related diseases and cancer.     

Cell Cycle-Dependent Role of MRN at Dysfunctional Telomeres: ATM Signaling-Dependent Induction of Nonhomologous End Joining (NHEJ) in G1 and Resection-Mediated Inhibition of NHEJ in G2

Here, we address the role of the MRN (Mre11/Rad50/Nbs1) complex in the response to telomeres rendered dysfunctional by deletion of the shelterin component TRF2. Using conditional NBS1/TRF2 double-knockout MEFs, we show that MRN is required for ATM signaling in response to telomere dysfunction. This establishes that MRN is the only sensor for the ATM kinase and suggests that TRF2 might block ATM signaling by interfering with MRN binding to the telomere terminus, possibly by sequestering the telomere end in the t-loop structure. We also examined the role of the MRN/ATM pathway in nonhomologous end joining (NHEJ) of damaged telomeres. NBS1 deficiency abrogated the telomere fusions that occur in G₁, consistent with the requirement for ATM and its target 53BP1 in this setting. Interestingly, NBS1 and ATM, but not H2AX, repressed NHEJ at dysfunctional telomeres in G₂, specifically at telomeres generated by leading-strand DNA synthesis. Leading-strand telomere ends were not prone to fuse in the absence of either TRF2 or MRN/ATM, indicating redundancy in their protection. We propose that MRN represses NHEJ by promoting the generation of a 3′ overhang after completion of leading-strand DNA synthesis. TRF2 may ensure overhang formation by recruiting MRN (and other nucleases) to newly generated telomere ends. The activation of the MRN/ATM pathway by the dysfunctional telomeres is proposed to induce resection that protects the leading-strand ends from NHEJ when TRF2 is absent. Thus, the role of MRN at dysfunctional telomeres is multifaceted, involving both repression of NHEJ in G₂ through end resection and induction of NHEJ in G₁ through ATM-dependent signaling.Mammalian telomeres solve the end protection problem through their association with shelterin. The shelterin factor TRF2 (telomere repeat-binding factor 2) protects chromosome ends from inappropriate DNA repair events that threaten the integrity of the genome (reviewed in reference 32). When TRF2 is removed by Cre-mediated deletion from conditional knockout mouse embryo fibroblasts (TRF2^F/− MEFs), telomeres activate the ATM kinase pathway and are processed by the canonical nonhomologous end-joining (NHEJ) pathway to generate chromosome end-to-end fusions (10, 11).The repair of telomeres in TRF2-deficient cells is readily monitored in metaphase spreads. Over the course of four or five cell divisions, the majority of chromosome ends become fused, resulting in metaphase spreads displaying the typical pattern of long trains of joined chromosomes (10). The reproducible pace and the efficiency of telomere NHEJ have allowed the study of factors involved in its execution and regulation. In addition to depending on the NHEJ factors Ku70 and DNA ligase IV (10, 11), telomere fusions are facilitated by the ATM kinase (26). This aspect of telomere NHEJ is mediated through the ATM kinase target 53BP1. 53BP1 accumulates at telomeres in TRF2-depleted cells and stimulates chromatin mobility, thereby promoting the juxtaposition of distantly positioned chromosome ends prior to their fusion (18). Telomere NHEJ is also accelerated by the ATM phosphorylation target MDC1, which is required for the prolonged association of 53BP1 at sites of DNA damage (19).Although loss of TRF2 leads to telomere deprotection at all stages of the cell cycle, NHEJ of uncapped telomeres takes place primarily before their replication in G₁ (25). Postreplicative (G₂) telomere fusions can occur at a low frequency upon TRF2 deletion, but only when cyclin-dependent kinase activity is inhibited with roscovitine (25). The target of Cdk1 in this setting is not known.Here, we dissect the role of the MRN (Mre11/Rad50/Nbs1) complex and H2AX at telomeres rendered dysfunctional through deletion of TRF2. The highly conserved MRN complex has been proposed to function as the double-stranded break (DSB) sensor in the ATM pathway (reviewed in references 34 and 35). In support of this model, Mre11 interacts directly with DNA ends via two carboxy-terminal DNA binding domains (13, 14); the recruitment of MRN to sites of damage is independent of ATM signaling, as it occurs in the presence of the phosphoinositide-3-kinase-related protein kinase inhibitor caffeine (29, 44); in vitro analysis has demonstrated that MRN is required for activation of ATM by linear DNAs (27); a mutant form of Rad50 (Rad50S) can induce ATM signaling in the absence of DNA damage (31); and phosphorylation of ATM targets in response to ionizing radiation is completely abrogated upon deletion of NBS1 from MEFs (17). These data and the striking similarities between syndromes caused by mutations in ATM, Nbs1, and Mre11 (ataxia telangiectasia, Nijmegen breakage syndrome, and ataxia telangiectasia-like disease, respectively) are consistent with a sensor function for MRN.MRN has also been implicated in several aspects of DNA repair. Potentially relevant to DNA repair events, Mre11 dimers can bridge and align the two DNA ends in vitro (49) and Rad50 may promote long-range tethering of sister chromatids (24, 50). In addition, a binding partner of the MRN complex, CtIP, has been implicated in end resection of DNA ends during homology-directed repair (39, 45). The role of MRN in NHEJ has been much less clear. MRX, the yeast orthologue of MRN, functions during NHEJ in Saccharomyces cerevisiae but not in Schizosaccharomyces pombe (28, 30). In mammalian cells, MRN is not recruited to I-SceI-induced DSBs in G₁, whereas Ku70 is, and MRN does not appear to be required for NHEJ-mediated repair of these DSBs (38, 54). On the other hand, MRN promotes class switch recombination (37) and has been implicated in accurate NHEJ repair during V(D)J recombination (22).The involvement of MRN in ATM signaling and DNA repair pathways has been intriguing from the perspective of telomere biology. While several of the attributes of MRN might be considered a threat to telomere integrity, MRN is known to associate with mammalian telomeres, most likely through an interaction with the TRF2 complex (48, 51, 57). MRN has been implicated in the generation of the telomeric overhang (12), the telomerase pathway (36, 52), the ALT pathway (55), and the protection of telomeres from stochastic deletion events (1). It has also been speculated that MRN may contribute to formation of the t-loop structure (16). t-loops, the lariats formed through the strand invasion of the telomere terminus into the duplex telomeric DNA (21), are thought to contribute to telomere protection by effectively shielding the chromosome end from DNA damage response factors that interact with DNA ends, including nucleases, and the Ku heterodimer (15).H2AX has been studied extensively in the context of chromosome-internal DSBs. When a DSB is formed, ATM acts near the lesion to phosphorylate a conserved carboxy-terminal serine of H2AX, a histone variant present throughout the genome (7). Phosphorylated H2AX (referred to as γ-H2AX) promotes the spreading of DNA damage factors over several megabases along the damaged chromatin and mediates the amplification of the DNA damage signal (43). The signal amplification is accomplished through a sequence of phospho-specific interactions among γ-H2AX, MDC1, NBS1, RNF8, and RNF168, which results in the additional binding of ATM and additional phosphorylation of H2AX in adjacent chromatin (reviewed in reference 33). The formation of these large domains of altered chromatin, referred to as irradiation-induced foci at DSBs and telomere dysfunction-induced foci (TIFs) at dysfunctional telomeres (44), promotes the binding of several factors implicated in DNA repair, including the BRCA1 A complex and 53BP1 (33).In agreement with a role for H2AX in DNA repair, H2AX-deficient cells exhibit elevated levels of irradiation-induced chromosome abnormalities (5, 9). In addition, H2AX-null B cells are prone to chromosome breaks and translocations in the immunoglobulin locus, indicative of impaired class switch recombination, a process that involves the repair of DSBs through the NHEJ pathway (9, 20). Since H2AX is dispensable for the activation of irradiation-induced checkpoints (8), these data argue that H2AX contributes directly to DNA repair. However, a different set of studies has concluded that H2AX is not required for NHEJ during V(D)J recombination (5, 9) but that it plays a role in homology-directed repair (53). In this study, we have further queried the contribution of H2AX to NHEJ in the context of dysfunctional telomeres.Our aim was to dissect the contribution of MRN and H2AX to DNA damage signaling and NHEJ-mediated repair in response to telomere dysfunction elicited by deletion of TRF2. Importantly, since ATM is the only kinase activated in this setting, deletion of TRF2 can illuminate the specific contribution of these factors in the absence of the confounding effects of ATR signaling (26). This approach revealed a dual role for MRN at telomeres, involving both its function as a sensor in the ATM pathway and its ability to protect telomeres from NHEJ under certain circumstances.     

Requirement for the Phospho-H2AX Binding Module of Crb2 in Double-Strand Break Targeting and Checkpoint Activation

Activation of DNA damage checkpoints requires the rapid accumulation of numerous factors to sites of genomic lesions, and deciphering the mechanisms of this targeting is central to our understanding of DNA damage response. Histone modification has recently emerged as a critical element for the correct localization of damage response proteins, and one key player in this context is the fission yeast checkpoint mediator Crb2. Accumulation of Crb2 at ionizing irradiation-induced double-strand breaks (DSBs) requires two distinct histone marks, dimethylated H4 lysine 20 (H4K20me2) and phosphorylated H2AX (pH2AX). A tandem tudor motif in Crb2 directly binds H4K20me2, and this interaction is required for DSB targeting and checkpoint activation. Similarly, pH2AX is required for Crb2 localization to DSBs and checkpoint control. Crb2 can directly bind pH2AX through a pair of C-terminal BRCT repeats, but the functional significance of this binding has been unclear. Here we demonstrate that loss of its pH2AX-binding activity severely impairs the ability of Crb2 to accumulate at ionizing irradiation-induced DSBs, compromises checkpoint signaling, and disrupts checkpoint-mediated cell cycle arrest. These impairments are similar to that reported for abolition of pH2AX or mutation of the H4K20me2-binding tudor motif of Crb2. Intriguingly, a combined ablation of its two histone modification binding modules yields a strikingly additive reduction in Crb2 activity. These observations argue that binding of the Crb2 BRCT repeats to pH2AX is critical for checkpoint activity and provide new insight into the mechanisms of chromatin-mediated genome stability.DNA damage response is an essential cellular guard that protects the genetic material from a constant barrage of genotoxic agents. To ensure their survival after genomic insult, cells orchestrate a signaling cascade that leads to checkpoint-mediated cell cycle arrest and the repair of damaged DNA (16, 35). A failure in this process can have catastrophic cellular consequences leading to the development of numerous disorders such as cancer (18, 30, 32). Because of its intimate connection with human health, deciphering the molecular mechanisms of DNA damage response is of high interest (16, 20).Recently, histone posttranslational modification has emerged as one element that is critical for ensuring a faithful response to genomic challenge (7, 31). An octamer of the four core histones, H3, H4, H2A, and H2B, forms the core protein component of chromatin, and cells possess a considerable number of enzymes that target histones for posttranslation modification (21). These marks can impinge upon many aspects of DNA biology by acting to directly alter chromatin structure or by serving as a binding scaffold for the recruitment of regulatory factors (24).In the context of DNA damage response, one factor that is intimately linked with histone modification is the fission yeast DNA damage checkpoint protein Crb2. After genomic insult, DNA damage checkpoints function to halt cell cycle progression, ensuring sufficient time for lesion repair (16, 35). In the fission yeast Schizosaccharomyces pombe, regulating the transition from G₂ to mitosis (G₂/M) represents the major DNA damage checkpoint and Crb2 is essential for this activity (4, 34). Crb2 is a member of a family of checkpoint regulators that have been termed mediators because they are thought to transmit the checkpoint signal from damage-sensing ATM/ATR-related kinases to effector kinases, such as Chk1, that trigger cell cycle arrest (11, 25). Crb2 is closely related to budding yeast Rad9 and mammalian p53 binding protein 53BP1, which all share two distinct domains, a tandem tudor motif and a pair of C-terminal BRCT repeats (Fig. ​(Fig.1A)1A) (11, 25). Besides 53BP1, Crb2 also shares some functional similarities with other mammalian BRCT-containing checkpoint regulators, such as MDC1 and BRCA1 (11, 25). In response to ionizing irradiation (IR), the rapid accumulation of Crb2 and other checkpoint proteins can be readily visualized as nuclear foci that mark sites of double-strand breaks (DSBs) (9, 25). Understanding the mechanisms that govern this targeting has been an area of intense interest, and for Crb2 this accumulation requires two distinct histone marks: dimethylation of histone H4 lysine 20 (H4K20me2) and phosphorylated H2AX (pH2AX) (27, 36).Open in a separate windowFIG. 1.Crb2 pH2AX-binding mutations. (A) Top, schematic representation of Crb2 (not drawn to scale) with relevant mutations indicated. Bottom, protein sequence alignment of a portion of the BRCT phospho-binding motifs from Schizosaccharomyces pombe (sp) Crb2, human (h) 53BP1, human MDC1, and Saccharomyces cerevisiae (sc) Rad9. Identical residues are shaded black; similar residues are shaded gray. *, Crb2 phospho-binding residues. (B) The Crb2 BRCT domains specifically interact with pH2AX. Peptide pulldowns were performed as described in the text with C-terminal fission yeast H2A.1 peptides either unmodified or phosphorylated at Ser129 (see − or + pH2AX) and increasing amounts of the indicated recombinant Crb2 BRCT domain fragments (∼0.1 and 0.3 μM). After binding and washing, SDS-PAGE and Coomassie staining were used to visualize peptide-bound protein. A fraction of the total protein used for binding was also visualized (Input).Mono-, di-, and trimethyl H4K20 are conserved chromatin marks that are readily detectable in fission yeast and mammalian cells (29, 36). In fission yeast, the Kmt5 methylase catalyzes all three H4K20 methyl modifications and its inactivation, or mutation of its H4K20 substrate, severely diminishes Crb2 accumulation at DSBs and compromises checkpoint activity (10, 36). Note that as outlined by the unified nomenclature for the naming of histone lysine methyltransferases (2), the fission yeast H4K20 methylase previously known as Set9 (36) is now termed Kmt5. The requirement for H4K20 methylation is mediated by the tandem tudor domains of Crb2 that preferentially bind H4 tail peptides dimethylated at lysine 20 (3, 14). Tudor motif mutations impair Crb2 DSB targeting and genome integrity in a manner analogous to loss of Kmt5 activity, and dimethylation of H4K20, but not trimethylation, is required for Crb2 activity (10, 14, 42). The tudor domain of 53BP1 can also directly bind H4K20me2, and this recognition event is required for its accumulation at IR-induced DSBs (3, 23, 45).After DNA damage, serine 139 phosphorylation in the mammalian H2A variant H2AX, or a homologous site in canonical yeast H2A, specifically marks sites of genomic lesions (7, 12). The fission yeast genome encodes two H2A proteins, H2A.1 and H2A.2, which differ slightly in their primary amino acid sequence. Phosphorylation of S129 in H2A.1 and S128 in H2A.2 is collectively referred to as phosphorylated H2AX (pH2AX). The ATM/ATR family of PI3-like kinases that includes the fission yeast Rad3 and Tel1 enzymes catalyzes pH2AX (37). H2AX phosphorylation has a critical role in controlling both DNA repair and checkpoint activation in a variety of organisms from yeast to humans (7, 12). Central to its function is the ability of the pH2AX mark to coordinate the recruitment of a number of proteins to genomic lesions, and several factors can directly bind the modification (40). Serine-to-alanine substitutions at the H2AX phosphorylation site in fission yeast H2A (h2ax⁻) severely reduce Crb2 accumulation at IR-induced DSBs and compromise the ability of cells to maintain checkpoint cell cycle arrest in a manner very similar to loss of H4K20 methylation (10, 27).The mechanism underlying the control of Crb2 DSB targeting and checkpoint activation by pH2AX is not understood. Because BRCT domains are known phospho-binding motifs (13), the initial demonstration that pH2AX is required for Crb2 function suggested that direct binding to the modification by Crb2 is critical for checkpoint activity (27). Supporting this idea, it has been demonstrated that the Crb2 BRCT repeats directly and specifically bind pH2AX peptides (22). Structural and biochemical studies have also identified a conserved pH2AX-binding motif in the BRCT repeats of Crb2, budding yeast Rad9, and human MDC1 and 53BP1 (Fig. ​(Fig.1A)1A) (15, 22, 39). As would be expected, mutation of Crb2''s critical phospho-binding motif impairs cell survival after DNA damage (22). Unexpectedly though, loss of its pH2AX-binding activity did not significantly affect the ability of Crb2 to localize to IR-induced DSBs (22). Rather, mutation of the Crb2 pH2AX-binding motif altered the kinetics of Rad22 accumulation at DSBs and triggered a prolonged checkpoint arrest after IR exposure (22). From these observations it was suggested that binding of the Crb2 BRCT repeats to pH2AX is critical for aspects of DNA repair but is not central to Crb2 targeting and checkpoint activity (22).The apparent dispensability of its pH2AX-binding motif in controlling Crb2 localization to IR-induced DSBs (22) was a surprising observation because of the established requirement for the pH2AX modification (10, 27). The extended checkpoint delay seen in Crb2 pH2AX-binding mutants (22) was also unexpected because h2ax⁻ cells cannot maintain checkpoint-mediated cell cycle arrest (10, 27). The prolonged checkpoint arrest was also surprising because a defect in IR-induced Chk1 phosphorylation was observed in the same Crb2 pH2AX-binding mutants (22). For these reasons we sought to reevaluate the requirement for the pH2AX-binding module of Crb2 in controlling DNA damage checkpoint activity. We demonstrate that the critical phospho-coordinating residue of Crb2 is required for binding to pH2AX peptides, Crb2 accumulation at IR-induced DSBs, cell survival after DNA damage, and maintenance of checkpoint-mediated cell cycle arrest. The observed impairments are similar to that reported for abolishment of pH2AX or mutation of the H4K20me2 binding tudor motif of Crb2. Strikingly, a combined ablation of the two modification binding modules of Crb2 produces an additive impairment in checkpoint dysfunction and genome integrity. These results argue that recognition of pH2AX by its BRCT repeats is critical for Crb2 accumulation at genomic lesions and its subsequent checkpoint activity. These observations also corroborate the independent findings of Sofueva et al. (38), who have observed a similar requirement for Crb2 binding to pH2AX in controlling DSB targeting and checkpoint activity.     

Silencing NFBD1/MDC1 enhances the radiosensitivity of human nasopharyngeal cancer CNE1 cells and results in tumor growth inhibition

NFBD1 functions in cell cycle checkpoint activation and DNA repair following ionizing radiation (IR). In this study, we defined the NFBD1 as a tractable molecular target to radiosensitize nasopharyngeal carcinoma (NPC) cells. Silencing NFBD1 using lentivirus-mediated shRNA-sensitized NPC cells to radiation in a dose-dependent manner, increasing apoptotic cell death, decreasing clonogenic survival and delaying DNA damage repair. Furthermore, downregulation of NFBD1 inhibited the amplification of the IR-induced DNA damage signal, and failed to accumulate and retain DNA damage-response proteins at the DNA damage sites, which leaded to defective checkpoint activation following DNA damage. We also implicated the involvement of NFBD1 in IR-induced Rad51 and DNA-dependent protein kinase catalytic subunit foci formation. Xenografts models in nude mice showed that silencing NFBD1 significantly enhanced the antitumor activity of IR, leading to tumor growth inhibition of the combination therapy. Our studies suggested that a combination of gene therapy and radiation therapy may be an effective strategy for human NPC treatment.Nasopharyngeal carcinoma (NPC) is a non-lymphomatous, squamous cell carcinoma that occurs in the epithelial lining of the nasopharynx, which is a prevalent tumor in people of southern Chinese ancestry in southern China and Southeast Asia, and the incidence is still increasing.¹ Although radiotherapy is routinely used to treat patients with NPC, local recurrences and distant metastasis often occur in 30–40% of NPC patients at advanced staged.² Thus, new therapeutic strategies are required to improve the poor prognosis of NPC.Among the various types of DNA damage, DNA double-strand breaks (DSBs) are the most serious and require elaborated networks of proteins to signal and repair the damage.³ It has recently been shown that the histone H2A variant H2AX specifically controls the recruitment of DNA repair proteins to the sites of DNA damage.⁴ H2AX is phosphorylated extensively on a conserved serine residue at its carboxyl terminus in chromatin regions bearing DSBs, which is mediated by members of the phos-phoinositide-3-kinase-related protein kinase (PIKK) family.^{5, 6} Of these PIKKs, ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylate H2AX in response to DSBs in a partially redundant manner.^{7, 8} NFBD1 (Nuclear Factor with BRCT Domain Protein 1), also known as MDC1 (mediator of DNA damage checkpoint protein 1), is a recently identified nuclear protein that regulates many aspects of the DNA damage-response pathway, such as intra-S phase checkpoint, G2/M checkpoint, spindle assembly checkpoint and foci formation of NBS/MRE/Rad50 (MRN complex), 53BP1 and BRCA1.^{9, 10, 11, 12, 13} Human NFBD1 comprises 2089 amino acid residues and has a predicted molecular weight of ∼220 kDa. Motifs found in the protein include an FHA (Forkhead Associated) domain, two BRCT (BRCA1 carboxy terminal) domains and around 20 in terminal repeats of ∼41 amino acid residues each.¹⁴ Following DNA damage, NFBD1 serves as a bridging molecule and directly interacts with ATM and phospho-H2AX (γ-H2AX) through its FHA and BRCT domains, respectively, which leads to the expansion of γ-H2AX region surrounding DNA strand breaks and provides docking sites for many DNA damage and repair proteins including the MRN complex, 53BP1, BRCA1, RNF8, RNF4 and so on, ensuring genomics stability.^{11, 15, 16, 17, 18} In mammalian cells, DSBs are mainly repaired by two mechanisms, homologous recombination (HR) or non-homologous end-joining (NHEJ).^{19, 20, 21} For NHEJ repair, it is estimated that following exposure to ionizing radiation (IR), 80–90% of the DSBs in G1 are rejoined with fast kinetics in a manner dependent upon the NHEJ core components, Ku, DNA-PKcs, XRCC4 and DNA ligase IV. In contrast, HR predominates in late S- and G2-phase cells, when the sister chromatid is available to act as the template, representing those normally repaired with slow kinetics, require Rad51, Rad52, Rad54, XRCC2, XRCC3, the Rad51 paralogs and the breast cancer susceptibility genes BRCA1 and BRCA2.^{22, 23, 24, 25, 26}Since NFBD1 contains protein–protein interaction domains, and participate in the DNA damage-response (DDR) pathway. However, the mechanism by which NFBD1 regulates so many aspects of the DNA damage-response pathway in NPC cells is not fully understood. In addition, the physiological function of NFBD1 in NPC cells has been not investigated. With these goals in mind, we generated NFBD1-knockdown NPC cells and studied the physiological function of NFBD1 in DDR.     

XLS (c9orf142) is a new component of mammalian DNA double-stranded break repair

Repair of double-stranded DNA breaks (DSBs) in mammalian cells primarily occurs by the non-homologous end-joining (NHEJ) pathway, which requires seven core proteins (Ku70/Ku86, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis, XRCC4-like factor (XLF), XRCC4 and DNA ligase IV). Here we show using combined affinity purification and mass spectrometry that DNA-PKcs co-purifies with all known core NHEJ factors. Furthermore, we have identified a novel evolutionary conserved protein associated with DNA-PKcs—c9orf142. Computer-based modelling of c9orf142 predicted a structure very similar to XRCC4, hence we have named c9orf142—XLS (XRCC4-like small protein). Depletion of c9orf142/XLS in cells impaired DSB repair consistent with a defect in NHEJ. Furthermore, c9orf142/XLS interacted with other core NHEJ factors. These results demonstrate the existence of a new component of the NHEJ DNA repair pathway in mammalian cells.Double-stranded DNA breaks (DSBs) are among the most cytotoxic DNA lesions for mammalian cells.¹ Effective repair of DSBs is essential for cellular survival and for suppression of potential deleterious chromosomal rearrangements.² Two main DNA repair pathways eliminate DSBs—homologous recombination (HR) or non-homologous end joining (NHEJ). HR utilises an undamaged copy of the chromosome as a template to direct repair, thus this restricts HR to the S and G2/M phases of the cell cycle, when such an extra chromosome copy is available.³ NHEJ performs the bulk of DSB repair in mammalian cells and in particular in during the G1 phase of the cell cycle, where the cells are completely dependent on NHEJ. NHEJ can be further subdivided into so-called classical NHEJ (c-NHEJ) and alternative NHEJ (alt-NHEJ).⁴ These DNA repair pathways utilise distinct protein components and also show different efficiencies of end ligation. In general, c-NHEJ is much more effective in end ligation than alt-NHEJ and can ligate most unrelated DNA ends directly or with minimal processing. In contrast alt-NHEJ requires short microhomologies between the DNA ends for ligation.⁵ C-NHEJ requires the following seven core proteins: Ku70/Ku86 dimers, DNA-PKcs (DNA-dependent protein kinase catalytic subunit), Artemis nuclease, XRCC4-like factor (XLF) and the XRCC4/ligase IV complex.^{6, 7} The DSB repair during c-NHEJ is initiated by the Ku dimer that senses the presence of free double-stranded DNA ends in cells and rapidly binds such ends with high affinity. DNA-bound Ku then recruits DNA-PKcs (DNA-PKcs/Ku70/Ku86 complex is termed DNA-PK holoenzyme), which has a protein kinase activity and is required for activation of the nuclease Artemis.⁸ Artemis, in turn, is responsible for DNA end processing in order to achieve DNA end structures suitable for ligation. The final step of c-NHEJ is the ligation of processed DNA ends by XRCC4/ligase IV complex. This final step is stimulated by XLF protein that interacts with XRCC4 forming long filamentous structures at DSBs to facilitate DNA end joining.^{9, 10} XRCC4 and XLF factors are distinct among NHEJ factors in that they share similar tertiary structure but show low primary sequence conservation.¹¹ Since the identification of XLF in 2006, no new core factors have been discovered.^{11, 12} Importantly, c-NHEJ is essential for proper development, as mutations in this pathway lead to immunodeficiency and defective neurogenesis in humans.⁷ It is therefore essential to fully decipher the identity of components for the c-NHEJ pathway and their regulation.In this study, proteomic analysis of DNA-PKcs-containing protein complexes identified an abundant previously uncharacterised protein c9orf142, which we have named c9orf142—XLS (XRCC4-like small protein). Structural modelling predicts XLS to be highly similar to XRCC4 and XLF, and depletion of XLS delays ionising radiation (IR)-induced DNA DSB repair. Moreover, XLS is associated with other core c-NHEJ factors. Our data strongly suggest that c9orf142/XLS represents a novel c-NHEJ component in mammalian cells.     

Hypoxia: A novel function for VIN3

VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.¹ Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).^2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,¹ and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).^4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),^4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.^4,7–10 In our recent paper¹¹ we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.Key words: arabidopsis, VIN3, FLC, hypoxia, vernalization, chromatin remodelling, survival     

Integrator3, a Partner of Single-stranded DNA-binding Protein 1, Participates in the DNA Damage Response

Single-stranded DNA-binding protein 1 (SSB1) plays an important role in the DNA damage response and maintenance of genomic stability. Here, by using protein affinity purification, we have identified Integrator3 (INT3) as a novel partner of SSB1. INT3 forms a complex with SSB1 by constitutively interacting with SSB1 regardless of DNA damage. However, following DNA damage, along with SSB1, INT3 relocates to the DNA damage sites and regulates the accumulation of TopBP1 and BRCA1 there. Moreover, INT3 controls DNA damage-induced Chk1 activation and G₂/M checkpoint activation. In addition, INT3 is involved in homologous recombination repair by regulating Rad51 foci formation following DNA damage. Taken together, these results demonstrate that INT3 plays a key role in the DNA damage response.The DNA damage response, including DNA damage checkpoint activation and DNA damage repair, ensures genomic stability under genotoxic stress. Among various types of DNA damage, DNA double-strand breaks (DSBs)³ are the most deleterious, easily causing chromosomal loss, fusion, and translocation. However, cells can sense and repair DNA DSBs by activating evolutionarily conserved pathways (1–3). Following DNA DSBs, ATM, ATR, and DNAPK, a family homologous to phosphoinositide 3-kinases (4, 5), are activated and phosphorylate histone H2AX at the DNA damage sites (6). Subsequently, phospho-H2AX (γH2AX) provides the platform for accumulation of a larger group of DNA damage response factors, such as MDC1, BRCA1, 53BP1, and TopBP1 (2, 7–9), at the DNA damage sites. Translocalization of these proteins to the DNA DSBs facilitates DNA damage checkpoint activation by activating downstream Chk1/Chk2 kinases, which arrest the cell cycle at G₁, S, or G₂ phase (10). In addition, it also enhances the efficiency of DNA damage repair by recruiting and stabilizing the DNA repair machinery at the DNA damage sites (11).Among these important mediators, single-stranded DNA (ssDNA)-binding proteins play important roles during the DNA damage response. For example, following DNA damage, the MRN complex recognizes DNA DSB ends and processes the blunt ends into ssDNA overhangs (12). The replication protein A (RPA) complex, a group of ssDNA-binding proteins, immediately coats these ssDNA overhangs and loads and activates the ATR·ATRIP complex at the DNA damage sites (13). Meanwhile, the RPA complex protects ssDNA from nucleolytic resection and facilitates Rad51 filament formation along ssDNA overhangs, which is a key step for homologous recombination repair (14). Moreover, RPA70 and RPA32 subunits in the complex could recruit several DNA damage response factors to the DNA damage sites that enhance the efficacy of DNA damage repair (15).Besides the RPA complex, several other ssDNA-binding proteins have been identified to participate in the DNA damage response recently. One of them is ssDNA-binding protein 1 (SSB1) (16). Human SSB1 is a 211-amino acid polypeptide with an N-terminal oligosaccharide/oligonucleotide-binding (OB) domain. It has been shown that SSB1 is phosphorylated by ATM and relocates to the DNA damage site following DNA DSBs. Loss of SSB1 impairs DNA damage-induced checkpoint activation and induces genomic instability. Like the RPA complex, SSB1 participates in homologous recombination by facilitating Rad51·ssDNA filament formation and stabilizing Rad51 at the DNA damage sites. Interestingly, SSB1 has a homolog SSB2 that contains an almost identical OB domain at the N terminus. However, the function of SSB2 in the DNA damage response is not clear yet.To examine the molecular mechanism and functional pathway of SSB1 and SSB2 in the DNA damage response, we have searched for functional partners of SSB1 and SSB2 by using protein affinity purification. We have found Integrator3 (INT3) to be a common partner of both SSB1 and SSB2. Like SSB1, following DNA damage, INT3 relocates to the DNA damage sites and regulates ATR activation. Moreover, INT3 not only participates in DNA damage checkpoint activation but also regulates homologous recombination repair. Taken together, we have found a novel mediator in the DNA damage response.     

Chromosomal Translocations Caused by Either Pol32-Dependent or Pol32-Independent Triparental Break-Induced Replication

Double-strand breaks (DSBs) are harmful DNA lesions that can generate chromosomal rearrangements or chromosome losses if not properly repaired. Despite their association with a number of genetic diseases and cancer, the mechanisms by which DSBs cause rearrangements remain unknown. Using a newly developed experimental assay for the analysis of translocations occurring between two chromosomes in Saccharomyces cerevisiae, we found that a single DSB located on one chromosome uses a short homologous sequence found in a third chromosome as a bridge to complete DSB repair, leading to chromosomal translocations. Such translocations are dramatically reduced when the short homologous sequence on the third chromosome is deleted. Translocations rely on homologous recombination (HR) proteins, such as Rad51, Rad52, and Rad59, as well as on the break-induced replication-specific protein Pol32 and on Srs2, but not on Ku70. Our results indicate that a single chromosomal DSB efficiently searches for short homologous sequences throughout the genome for its repair, leading to triparental translocations between heterologous chromosomes. Given the abundance of repetitive DNA in eukaryotic genomes, the results of this study open the possibility that HR rather than nonhomologous end joining may be a major source of chromosomal translocations.Genomic instability can be a source of cell death and cancer (3, 34). It is usually manifested as mutations and chromosomal rearrangements, including translocations, deletions, inversions, and duplications, often leading to gene fusions that may play a key role in the initial steps of tumorigenesis and subsequent cancer development (41, 42, 44). It is widely assumed that all rearrangement events are initiated by DNA double-strand breaks (DSBs) (1, 27, 49). In somatic cells, DSBs generally occur during DNA replication or by the action of environmental agents, such as genotoxic chemicals or ionizing radiation. Nevertheless, the mechanisms responsible for chromosomal rearrangements are not completely understood.Eukaryotic cells have evolved two main pathways to repair DSBs: nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ involves processing of the two break ends so that either addition or deletion of nucleotides can occur prior to ligation and, consequently, mutations may be introduced. The involvement of NHEJ in the generation of chromosomal rearrangements has been extensively studied (9, 32, 62), and a number of NHEJ-mediated chromosomal rearrangements associated with cancer have been reported (4, 35). HR requires significant amounts of homology between the broken DNA ends and the intact DNA sequence to be used for repair. For this reason, HR is typically error free. However, HR can also be a source of chromosomal rearrangements when occurring between DNA repeats located in different chromosomes (33, 54).HR may occur via different mechanisms, depending on the nature of the DNA ends, the location of the homologous partners, and the length of homology (reviewed in reference 46). After a chromosome break, cells carry out a genome-wide search for homologous sequences that are used as template for the repair of the broken chromosome. When both ends of the DSB (two-ended DSB) are homologous to sequences present in an intact chromosome, HR may proceed by DSB repair (DSBR) or synthesis-dependent strand annealing (SDSA) mechanisms (reviewed in reference 21). When only one end of the DSB (one-ended DSB) is homologous to sequences elsewhere in the genome, break-induced replication (BIR) becomes the alternative HR repair mechanism, as shown for yeast (39). Whereas HR repair of two-ended DSBs is completed with the capture of the second end, repair of one-ended DSBs relies on DNA synthesis primed by the invading end all the way to the end of the chromosome or until it encounters a barrier (43).In yeast, both DSBR/SDSA and BIR require the DSB repair genes of the RAD52 epistasis group, including the RAD51 strand exchange factor and the RAD52 single-stranded DNA binding protein (reviewed in reference 30). However, RAD51 may be dispensable in BIR occurring between particular substrates, such as inverted repeats (5, 12, 58). BIR requires additional specific replication proteins, such as Pol32, one of the subunits of DNA polymerase δ that is dispensable for replication and DSB repair mechanisms other than BIR (36).To gain further insight into the mechanisms of chromosomal translocations, we devised an intron-based chromosomal translocation assay in Saccharomyces cerevisiae, in which a DSB is generated in a single chromosome by the HO endonuclease. We show that DSB-induced translocations occur via triparental recombination events. A short homologous sequence in a third chromosome serves as a bridge template for HR events occurring between two nonhomologous chromosomes. The triparental HR events that occur in our assays give rise mainly to reciprocal translocations that require Rad52, Rad51, and importantly, Pol32. Rad59, as well as the nonreplicative DNA helicase Srs2, are also required, although to a lesser extent, whereas Ku70 or Mus81 endonuclease play no role. We conclude that BIR-mediated triparental recombination could be a major mechanism for chromosomal translocations in eukaryotic cells.     

Exonuclease Function of Human Mre11 Promotes Deletional Nonhomologous End Joining

DNA double-stranded breaks (DSBs) are lethal if not repaired and are highly mutagenic if misrepaired. Nonhomologous end joining (NHEJ) is one of the major DSB repair pathways and can rejoin the DSB ends either precisely or with mistakes. Recent evidence suggests the existence of two NHEJ subpathways: conservative NHEJ (C-NHEJ), which does not require microhomology and can join ends precisely; and deletional NHEJ (D-NHEJ), which utilizes microhomology to join the ends with small deletions. Little is known about how these NHEJ subpathways are regulated. Mre11 has been implicated in DNA damage response, thus we investigated whether Mre11 function also extended to NHEJ. We utilized an intrachromosomal NHEJ substrate in which DSBs are generated by the I-SceI to address this question. The cohesive ends are fully complementary and were either repaired by C-NHEJ or D-NHEJ with similar efficiency. We found that disruption of Mre11 by RNA interference in human cells led to a 10-fold decrease in the frequency of D-NHEJ compared with cells with functional Mre11. Interestingly, C-NHEJ was not affected by Mre11 status. Expression of wild type but not exonuclease-defective Mre11 mutants was able to rescue D-NHEJ in Mre11-deficient cells. Further mutational analysis suggested that additional mechanisms associated with methylation of Mre11 at the C-terminal glycine–arginine-rich domain contributed to the promotion of D-NHEJ by Mre11. This study provides new insights into the mechanisms by which Mre11 affects the accuracy of DSB end joining specifically through control of the D-NHEJ subpathway, thus illustrating the complexity of the Mre11 role in maintaining genomic stability.DNA double-stranded breaks (DSBs)³ can be produced in physiological and genotoxic processes. Improper repair or failure to repair DSBs can lead to gene deletions, duplications, translocations, and missegregation of large chromosome fragments, which may result in gene dosage imbalance, cancer development, or cell death (1–3). Historically, two distinct pathways have been described which ensure that DSBs are repaired: nonhomologous end joining (NHEJ) and homologous recombination (HR). During HR, the damaged chromosome interacts via synapsis with an undamaged DNA molecule with which it shares extensive sequence homology, usually its sister chromatid (4, 5). HR is most active in the late S and G₂ phases of the cell cycle. In contrast, NHEJ is active throughout the cell cycle and requires little or no DNA homology during repair; thus, it is traditionally considered an error-prone repair pathway (6, 7). However, accumulating evidence from recent studies suggests that there exists an error-free NHEJ subpathway (8, 9).Two types of end-joining reactions can be defined operationally. The first one, which may be called conservative NHEJ (C-NHEJ), is characterized by the precise joining of short, overhanging, complementary ends. Proteins including Ku70/Ku80 and XRCC4 (10–12) are associated with this highly efficient pathway, whereby most ends are rejoined successfully without any alteration of the DNA sequence (8). The alternative pathway for NHEJ is the highly mutagenic and deletional NHEJ (D-NHEJ), which results in short deletions after use of imperfect microhomology of about 1–10 bp at the repair junctions. D-NHEJ activity has been demonstrated in the budding yeast Saccharomyces cerevisiae. In addition, D-NHEJ is independent of Rad52, Rad1, or Ku80 but depends on Mre11 in yeast (13, 14). However, the genetic determinants of this subpathway have not been well established in mammalian cells.Mre11 is the core subunit of the Mre11·Rad50·Nbs1 complex (called the MRN complex), which is conserved throughout all kingdoms of life. The MRN complex is a central player in most aspects of the cellular response to DSBs, including HR, NHEJ, telomere maintenance, and DNA damage checkpoints (15–17). Loss of Mre11 results in increased radiosensitivity and chromosomal instability (17). Patients with germ line mutations of Mre11 have clinical presentations similar to those of ataxia telangiectasia patients (ataxia telangiectasia-like disorder) (18).After DNA damage, the MRN complex is recruited to the sites of damage via zinc hooks at the ends of the long, flexible arms of Rad50 (19, 20). Mre11 contains both single-stranded DNA endonuclease and 3′-5′ exonuclease activities in vitro, but in vivo Mre11 is also implicated in 5′-3′ DSB resection. The MRN complex also interacts with BRCA1 and CtIP, which may be essential for DSB end resection to generate 3′ overhanging single-stranded DNA during initiation of HR (21, 22).Mre11 has an N-terminal nuclease domain, which contains five phosphoesterase motifs, and a C-terminal glycine–arginine-rich domain (GAR). Arthur et al. (23) showed that an H85L mutation completely abrogated exonuclease activity, whereas binding to Rad50 and Nbs1 was retained. Complementation of ataxia telangiectasia-like disorder cells with this mutant, called Mre11-3, restored the localization of the MRN complex to DSBs in IR-induced foci (23, 24). Methylation of the GAR region has also been shown to be important for the DNA binding and exonuclease activity of Mre11 in vitro (25, 26). Both the crystal structure of yeast Mre11 and data from conditional knock-out mice (Mre11^H129N/Δ) reveal that the nuclease activity of Mre11 is required for HR repair of DSBs (22, 27). However, the role of Mre11 in NHEJ is not well defined (27, 28). Most recently, Mre11 was reported to support NHEJ in mammalian cell (29–31). However, whether Mre11 regulates both NHEJ subpathways or only D-NHEJ is controversial, and the mechanisms by which Mre11 is involved in NHEJ remain to be established.To address these questions, we have established a system that can analyze the accuracy and efficiency of rejoining of two adjacent DSB ends at chromosomal level in human embryonic kidney 293 (HEK293) cells. We show here that Mre11 siRNA knockdown in these cells results in significant reduction of the overall NHEJ efficiency. Upon sequencing the repair junctions, we found that Mre11 siRNA knockdown suppressed D-NHEJ by ∼10-fold, reflected by a reduction of small deletions in the repair junction, but it had no effect on the efficiency of C-NHEJ. Mutation of Mre11 in either the phosphoesterase domain (Mre11-3) or the GAR region (Mre11-R/A) to produce abnormal exonuclease activity impaired the D-NHEJ pathway only. The D-NHEJ deficiency is significantly more severe in cells with Mre11-R/A than that in cells with Mre11-3. Therefore, our data suggest that Mre11 is required specifically for D-NHEJ repair of DNA DSBs and that its exonuclease activity is at least one of the important mechanisms for this DNA end joining subpathway.     

PTIP Regulates 53BP1 and SMC1 at the DNA Damage Sites

Although PTIP is implicated in the DNA damage response, through interactions with 53BP1, the function of PTIP in the DNA damage response remain elusive. Here, we show that RNF8 controls DNA damage-induced nuclear foci formation of PTIP, which in turn regulates 53BP1 localization to the DNA damage sites. In addition, SMC1, a substrate of ATM, could not be phosphorylated at the DNA damage sites in the absence of PTIP. The PTIP-dependent pathway is important for DNA double strand breaks repair and DNA damage-induced intra-S phase checkpoint activation. Taken together, these results suggest that the role of PTIP in the DNA damage response is downstream of RNF8 and upstream of 53BP1. Thus, PTIP regulates 53BP1-dependent signaling pathway following DNA damage.The DNA damage response pathways are signal transduction pathways with DNA damage sensors, mediators, and effectors, which are essential for maintaining genomic stability (1–3). Following DNA double strand breaks, histone H2AX at the DNA damage sites is rapidly phosphorylated by ATM/ATR/DNAPK (4–10), a family homologous to phosphoinositide 3-kinases (11, 12). Subsequently, phospho-H2AX (γH2AX) provides the platform for accumulation of a larger group of DNA damage response factors, such as MDC1, BRCA1, 53BP1, and the MRE11·RAD50·NBS1 complex (13, 14), at the DNA damage sites. Translocalization of these proteins to the DNA double strand breaks (DSBs)³ facilitates DNA damage checkpoint activation and enhances the efficiency of DNA damage repair (14, 15).Recently, PTIP (Pax2 transactivation domain-interacting protein, or Paxip) has been identified as a DNA damage response protein and is required for cell survival when exposed to ionizing radiation (IR) (1, 16–18). PTIP is a 1069-amino acid nuclear protein and has been originally identified in a yeast two-hybrid screening as a partner of Pax2 (19). Genetic deletion of the PTIP gene in mice leads to early embryonic lethality at embryonic day 8.5, suggesting that PTIP is essential for early embryonic development (20). Structurally, PTIP contains six tandem BRCT (BRCA1 carboxyl-terminal) domains (16–18, 21). The BRCT domain is a phospho-group binding domain that mediates protein-protein interactions (17, 22, 23). Interestingly, the BRCT domain has been found in a large number of proteins involved in the cellular response to DNA damages, such as BRCA1, MDC1, and 53BP1 (7, 24–29). Like other BRCT domain-containing proteins, upon exposure to IR, PTIP forms nuclear foci at the DSBs, which is dependent on its BRCT domains (16–18). By protein affinity purification, PTIP has been found in two large complexes. One includes the histone H3K4 methyltransferase ALR and its associated cofactors, the other contains DNA damage response proteins, including 53BP1 and SMC1 (30, 31). Further experiments have revealed that DNA damage enhances the interaction between PTIP and 53BP1 (18, 31).To elucidate the DNA damage response pathways, we have examined the upstream and downstream partners of PTIP. Here, we report that PTIP is downstream of RNF8 and upstream of 53BP1 in response to DNA damage. Moreover, PTIP and 53BP1 are required for the phospho-ATM association with the chromatin, which phosphorylates SMC1 at the DSBs. This PTIP-dependent pathway is involved in DSBs repair.     

Role of ATM and the Damage Response Mediator Proteins 53BP1 and MDC1 in the Maintenance of G2/M Checkpoint Arrest

ATM-dependent initiation of the radiation-induced G₂/M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G₂ phase are repaired by DNA nonhomologous end joining (NHEJ), while ∼15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G₂/M checkpoint is maintained in irradiated G₂ cells, in light of our current understanding of G₂ phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1^−/− and MDC1^−/− cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage.DNA double-strand breaks (DSBs) activate the DNA damage response (DDR), a coordinated process that functions to enhance survival and maintain genomic stability. The DDR includes pathways of DSB repair and a signal transduction response that activates apoptosis and cell cycle checkpoint arrest and influences DSB repair (15). DNA nonhomologous end joining (NHEJ) and homologous recombination (HR) represent the major DSB repair mechanisms, NHEJ being the major mechanism in G₀/G₁, while both processes function in G₂ (9, 32). Ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) are related phosphoinositol 3-kinase-like kinases (PIKKs) that regulate the DNA damage signaling response. ATM is activated by DSBs, while ATR is activated at single-strand (ss) regions of DNA via a process that involves ATRIP-replication protein A (RPA)-ssDNA association. Ionizing radiation (IR) induces DSBs, base damage, and ss nicks. Since neither base damage nor ss nicks activate ATR, IR-induced signaling in the G₁ and G₂ phases is predominantly ATM dependent (3, 29). In S phase, ATR can be activated by both endogenous and exogenously induced lesions following replication fork stalling/collapse (8).Recent work has shown that in G₂ phase, DSBs can undergo resection via an ATM-dependent process generating ssDNA regions that can activate ATR following RPA association (11). ATR activation at resected DSBs is coupled to loss of ATM activation (11). Although ATM and ATR share overlapping substrates, there is specificity in their signaling to the transducer kinases; ATM uniquely phosphorylates Chk2, while ATR phosphorylates Chk1. Phosphorylation of either Chk1 or Chk2 causes their activation. Critical targets of Chk1/Chk2 are the Cdc25 phosphatases, which regulate the cyclin-dependent kinases (Cdks), including Cdk1, the regulator of mitotic entry (18). Collectively, these studies suggest that two components of ATM-dependent signaling to the G₂/M checkpoint machinery can occur: ATM-Chk2 signaling at unresected DSBs and ATM-ATR-Chk1 signaling at resected DSBs.Although much is known about the mechanism leading to G₂/M checkpoint activation, few studies have addressed how arrest is maintained and how release coordinates with the status of DSB repair. We examine here the maintenance of checkpoint arrest during the immediate phase of DSB repair. We do not address the issue of checkpoint adaptation, a distinct phenomenon which occurs after prolonged checkpoint arrest (22). Further, we focus on the process maintaining arrest in irradiated G₂-phase cells and do not consider how arrest is maintained in irradiated S-phase cells that progress into G₂ phase. (Previous studies have shown that while G₂/M arrest is ATM dependent at early times post-IR, at later times it becomes ATR dependent as S-phase cells progress into G₂ phase [2, 33].) To focus on mechanisms maintaining ATM-dependent signaling in G₂-phase cells, we use aphidicolin (APH) to prevent S-phase cells from progressing into G₂ during analysis. We, thus, examine checkpoint maintenance in cells irradiated in G₂ phase and do not evaluate arrest regulated by ATR following replication fork stalling. The basis for our work stems from two recent advances. First, we evaluate the impact of ATM-mediated ATR activation in the light of recent findings that resection occurs in G₂ phase (11). Second, we consider the finding that NHEJ represents the major DSB repair mechanism in G₂ and that a 15 to 20% subset of DSBs, representing those that are rejoined with slow kinetics in an ATM-dependent manner, undergo resection and repair by HR (3, 25). Thus, contrary to the notion that HR represents the major DSB repair pathway in G₂ phase, it repairs only 15 to 20% of X- or gamma-ray-induced DSBs and represents the slow component of DSB repair in G₂ phase. Given these findings, several potential models for how checkpoint arrest is maintained in G₂ can be envisaged. A simple model is that the initial signal generated by IR is maintained for a defined time to allow for DSB repair. Such a model appears to explain the kinetics of checkpoint signaling in fission yeast after moderate IR (17). In mammalian cells, the duration of arrest depends on dose and DSB repair capacity (6). Thus, it is possible that the status of ongoing repair is communicated to the checkpoint machinery to coordinate timely release with the process of DSB repair. Here, we consider the impact of resection leading to ATM-ATR-Chk1 signaling versus ATM-Chk2 signaling from nonresected DSBs and how they interplay to maintain rather than initiate checkpoint arrest.Mediator proteins, including 53BP1 and MDC1, assemble at DSBs in an ATM-dependent manner, but their roles in the DDR are unclear. Cells lacking 53BP1 or MDC1 are proficient in checkpoint initiation after moderate IR doses, leading to the suggestion that these proteins are required for amplification of the ATM signal after exposure to low doses but are dispensable after high doses, when a robust signal is generated, even in their absence (7, 16, 28, 31). Despite their apparent subtle role in ATM signaling, cells lacking these mediator proteins display significant genomic instability (19). We thus also examine whether the mediator proteins contribute to the maintenance of checkpoint arrest.We identify two ATM-dependent processes that contribute to the maintenance of checkpoint arrest in G₂-phase cells: (i) ATR-Chk1 activation at resected DSBs and (ii) a process that involves sustained signaling from ATM to Chk2 at unrepaired DSBs. Further, we show that 53BP1 and MDC1 are required for maintaining checkpoint arrest, even following exposure to high radiation doses due to roles in ATR-Chk1 activation and sustained ATM-Chk2 signaling, and that this contributes to their elevated genomic instability.     

Cell Migration from Baby to Mother

Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood-brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood-brain barriers and the persistence and distribution of fetal cells in the mother.Key Words: fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migrationMicrochimerism is the presence of a small population of genetically distinct and separately derived cells within an individual. This commonly occurs following transfusion or transplantation.^1–3 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)^4–7 and from the mother to the fetus.^8–10 Similar exchange may also occur between monochorionic twins in utero.^11–13 There is increasing evidence that fetomaternal microchimerism persists lifelong in many child-bearing women.^7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy-acquired microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells.Trophoblasts were the first zygote-derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.¹⁵ Later, trophoblasts were also observed in the maternal circulation.^16–20 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.^21,22 These fetal cell types included lymphocytes,²³ erythroblasts or nucleated red blood cells,^24,25 haematopoietic progenitors^7,26,27 and putative mesenchymal progenitors.^14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,^29–31 trophoblast release does not appear to occur in all pregnancies.³² Likewise, in mice, fetal cells have also been reported in maternal blood.^33,34 In the mouse, fetomaternal transfer also appears to occur during all pregnancies.³⁵