To compare the in vivo functions of dATM and mei-41, their developmental expression patterns were examined. The dATM gene is expressed at relatively low levels throughout development and at much higher levels in adult females, when compared to males. Similarly, mei-41 is expressed at high levels in adult females and ovaries and is required for meiosis, suggesting a potential role for both genes in the female germ line (Song, 2004).
Telomeres are the stable ends of linear chromosomes in eukaryotes. These complex protein-nucleic acid structures are essential to maintain genomic stability and the integrity of linear chromosomes. A new mutation (tef for telomere fusion) was identified in Drosophila that causes a high frequency of end-to-end fusions of chromosomes during mitosis and meiosis. Linear chromosomal ends appear to be essential for fusions to take place. These fusions do not resolve, leading to cycles of chromosomal breakage and rejoining and severe genome rearrangements. The gene is essential for normal cell proliferation and mutant tissue shows significant apoptosis. This analysis suggests that the function encoded by the mutant gene is required to protect the linear ends of chromosomes (Queiroz-Machado, 2001).
To determine the function of atm, a genetic screen was carried out that isolated eight recessive lethal alleles. Seven of these alleles (atm1-7) have a nonconditional, pupal, lethal phenotype and one, atm8, is a temperature-sensitive hypomorphic allele. Hemizygous atm1-7 mutants die before emerging as adults, with variable morphological defects affecting the antennae, eyes, bristles, wings, and legs. The completely restrictive temperature for atm8 lethality is approximately 25°C. When atm8 mutants are raised at a semirestrictive temperature (24°C± 1°C), many can survive to adulthood with a similar range of morphological defects. The morphological defects, as well as atm mutant lethality, are rescued by introduction of a transgenic construct, P{atm+}, confirming that these phenotypes are specifically due to loss of atm function (Silva, 2004).
To determine when dATM activity is required for normal development, advantage was taken of the temperature-sensitive allele atm8 in a reciprocal temperature-shift experiment that established the temperature-sensitive period (TSP) for selected atm8 mutant phenotypes. The TSP for adult viability falls between the early third-larval instar and early pupal stage, the TSP for eye development is during the third-larval instar, and the TSP for wing and thoracic bristle development is during the pupal stage. These TSPs correspond to critical developmental stages of eye, wing, and bristle development. Requirements for dATM activity appear particularly stringent in developing neural tissues, because the structures most obviously affected in the atm mutants (eye, wing margin, thoracic bristles) are all involved in sensory perception (Silva, 2004).
Because movement disorders (ataxia) are a prominent symptom in patients with A-T, whether atm8 mutants exhibit signs of locomotor defects was examined. Climbing ability was examined in atm8 mutant and control flies for this purpose. Replicate tests were conducted for each fly, and the averages were used for deriving a ratio (mutant/control) that provided a measure of how well atm8 mutants climb, relative to matched controls. The average climbing ability of atm8 mutant males was markedly lower than normal (ratio of mutants/controls = 0.11). Interestingly, the atm8 mutant females performed even worse in this assay (mutants/controls = 0.01). The relative differences in climbing ability between mutants and controls did not change over a period of 4 weeks when the flies were maintained at the restrictive temperature of 29°C. Thus, the locomotor defects in atm mutants appear to be strictly developmental (Silva, 2004).
Because the temperature-sensitive period for normal eye development in atm8 mutants is during the third-larval instar, eye-antennal discs were examined at this stage of development. A structure called the morphogenetic furrow (MF) sweeps across the eye disc during the late-third instar, marking a transition between proliferating, undifferentiated cells (anterior to the furrow) and nonproliferating cells that are differentiating into distinct neuronal cell types, posterior to the furrow. The atm8 mutant-eye antennal discs are generally smaller than comparably aged controls. Conspicuously, atm8 mutant eye discs had large numbers of cells undergoing inappropriate apoptosis, marked by antibody staining for activated caspase-3. There were striking regional differences in the distribution of apoptotic cells in the mutant eye discs; these differences were also seen in discs stained with acridine orange staining as another marker of apoptosis. These apoptotic cells were primarily restricted to the anterior, proliferating region of the disc. There were relatively few apoptotic cells in the posterior region of the mutant discs, containing differentiating neuronal cells. Early neuronal patterning was markedly disrupted in mutant discs, presumably because so many cells were eliminated by earlier apoptosis that normal precluster formation was precluded (Silva, 2004).
The adult wing is also affected by loss of atm function, so atm8 mutant third-larval instar wing discs were examined for developmental defects. At this stage, cells in the wing disc are proliferating asynchronously except for a stripe of nondividing, differentiating cells along the presumptive wing margin of the disc. These cells express Cut, a Notch-induced transcription factor required for proper differentiation of the wing margin. A partial to complete loss of Cut expression was observed along the presumptive wing margin of atm8 mutant discs, a patterning defect that anticipates the wing margin defects seen in atm mutants later in development. Intriguingly, Cut expression was not obviously affected in the myoblast cells adjoining the presumptive dorsal thorax region of the atm mutant wing discs. The Cut expression defect in the developing wing margin could be due to inappropriate cell death in the proliferating imaginal cells, because spontaneous apoptosis also occurs throughout the atm8 mutant wing discs at this developmental stage. The spontaneous apoptosis and patterning defects in atm eye and wing imaginal discs suggest that cells are experiencing DNA damage, which is an established trigger for p53-dependent apoptosis in Drosophila (Silva, 2004).
Oogenesis and early embryogenesis are regulated by maternally acting genes in Drosophila. To determine if dATM activity is required during early stages of development, eggs derived from atm6 mutant-germline mosaic females, generated using the hs-FLP/FRT ovoD system, were examined. atm6 was molecularly characterized as a functional null allele. Maternal mutant atm6 eggs commonly have dorsal appendage defects, resembling a phenotype of homologous DSB repair-defective female-sterile mutants. The eggshells of atm6 mutant eggs also appear to be thinner than normal, suggesting that follicle cell development is compromised. Developing atm6 mutant embryos show dramatic mitotic defects during the rapid syncytial divisions of early embryogenesis. These mitotic defects include frequent spindle fusions and chromosomal bridges that could also be observed much earlier in development before the nuclei reach the surface of the embryo. Another frequently observed defect involved nuclei detaching from centrosomes and falling from the cortex (Silva, 2004).
The centrosome and nuclear fallout defect in atm mutants resembles a local DNA damage-induced response called 'centrosome inactivation', indicating that loss of dATM is associated with spontaneous DNA damage. Chk2 is an established transducer of ATM signaling in mammalian cells and Loki, the Drosophila Chk2 homolog, has been identified as a positive regulator of centrosome inactivation in Drosophila. Because centrosome inactivation occurs spontaneously in atm mutants, this result suggests that either a dATM-independent mechanism for activating Chk2 exists or a Chk2-independent mechanism for promoting centrosome inactivation was deployed (Silva, 2004).
In mammalian cells, the ATM and ATR signaling pathways negatively regulate Cdk1 in response to ionizing radiation, blocking mitosis while DNA is being repaired. Mei-41, the Drosophila ATR ortholog, is required for this response to DNA damage. To examine if dATM might also be required for this response, mitotic cells were labeled in atm mutant and control wing discs, with or without exposure to 40 Gy of γ irradiation. Mitotic cells were essentially absent 1 hr after ionizing radiation in both atm mutant and control discs, implying that dATM is dispensable for this premitotic-checkpoint response to acute DNA damage. dATM operates in a premitotic checkpoint, which is activated earlier in response to ionizing radiation than the measurements carried out in this study but the checkpoint response is fully engaged by 1 hr after irradiation (Silva, 2004 and references therein).
Increased numbers of apoptotic cells were observed in both controls and atm mutant wing discs, after larvae were exposed to ionizing radiation. Because the apoptosis response to ionizing radiation in Drosophila is thought to depend on p53, this result implies that p53-dependent apoptosis is functional in atm mutants. Indeed, expression of a dominant-negative p53 transgene suppresses spontaneous apoptosis in atm mutant wing discs, supporting the conclusion that the p53-dependent apoptosis response is intact (Silva, 2004).
atm mutants are extremely sensitive to ionizing radiation. Radiation sensitivity was assayed by comparing the adult viability of atm8 mutants, raised at either semirestrictive (24°C) or permissive (22°C) temperatures, after third-instar larvae were exposed to different doses of γ radiation. After taking into account the loss of adult viability caused by raising the mutants at 24°C, the data show that atm mutant viability is drastically compromised by exposure to ionizing radiation, even at the lowest dose of 1 Gy. Thus, it is concluded that dATM is required for cells to survive both spontaneous and induced DNA damage (Silva, 2004).
ATM has been implicated in two processes that are fundamental to chromosome integrity: repair of DNA double-strand breaks (DSBs) and telomere maintenance. To examine chromosome stability, larval neuroblasts from atm mutant and control larvae were examined; an astonishingly high rate of spontaneous chromosomal aberrations was observed in the mutants. The most frequent aberrations were telomere fusions, affecting >50% of metaphase spreads. Both single- and double-telomere associations (TA) were observed, including sister and nonsister fusions of homologous chromosomes, as well as fusions between nonhomologous chromosomes. Acentric chromosome fragments were also observed on rare occasions, and chromosomal transpositions involving the loss of whole chromosome arms. Dicentric chromosomes resulting from telomere fusions are inherently unstable, because the kinetochores can be captured by microtubules from opposite spindle poles, causing chromosome bridges that eventually rupture, triggering apoptosis. Consistent with this expectation, frequent chromosomal bridges were observed in atm mutant anaphase spreads. Collectively, these data implicate dATM in normal telomere maintenance and chromosome stability (Silva, 2004).
These results indicate that the primary function of dATM is in chromosome structural maintenance, without which proliferating cells are vulnerable to apoptosis. dATM is also implicated in an early premitotic checkpoint response to ionizing radiation, suggesting that ATM and ATR carry out temporally distinct cell cycle checkpoint functions as they do in mammals. Also in mammals, the DNA repair functions of ATM overlap with those of a functionally redundant, ATM-related kinase called DNA-PK. DNA-PK is not conserved in Drosophila, perhaps explaining why ATM is essential for morphological development in Drosophila but not in mammals (Silva, 2004).
Inappropriate apoptosis occurs predominantly in proliferating atm mutant cells, suggesting that dATM is required for repairing spontaneous DSBs arising during DNA replication. The extraordinary frequency of spontaneous telomere fusions in atm mutant neuroblasts also suggests a critical role for dATM in telomere maintenance in cycling cells. Drosophila telomeres are unusual because they are maintained by retrotransposition rather than by telomerase activity, as they are in other systems. Given this difference, it is very intriguing that ATM has now been linked to telomere maintenance in eukaryotic organisms as distantly related as yeast, Drosophila, and humans. The molecular mechanisms of ATM-dependent telomere maintenance have not yet been established in any organism, however, studies in yeast suggest that ATM/TEL1 activity prevents spontaneous chromosomal rearrangements involving telomeres during the repair of DSBs. The conserved MRN protein complex (comprised of Mre11, Rad50, and Nbs1) is required for all known ATM-dependent functions including telomere maintenance. Mutations affecting Drosophila mre11 and rad50 also result in pupal lethality and telomere fusions, consistent with these genes being involved in common DNA repair and chromosome structural maintenance functions. Recent genetic screens have identified a number of Drosophila genes of unknown function that are required for preventing spontaneous telomere fusions, many of which are probably also involved in ATM-dependent telomere maintenance functions (Silva, 2004 and references therein).
Drosophila atm mutants recapitulate major symptoms of ataxia telangiectasia including locomotor defects, sensitivity to ionizing radiation, and chromosome instability. Curiously, loss of ATM seems to affect developing neuronal tissues more than non-neuronal tissues, in both flies and humans. This correlation may reflect a relationship between telomere dysfunction and cell proliferation. Alternatively, specific developmental events may render certain cells more sensitive to loss of ATM function. Chromatin remodelling has been shown to activate ATM in cultured mammalian cells, making it tempting to speculate that neuron-specific chromatin remodelling processes might normally elicit ATM activity. Drosophila promises to be an excellent model for investigating the basic mechanisms of chromosome structural maintenance involving ATM, allowing these possibilities to be studied in a meaningful developmental context (Silva, 2004 and references therein).
The conserved ATM checkpoint kinase and the Mre11 DNA repair complex play essential and overlapping roles in maintaining genomic integrity. Genetic and cytological studies were conducted on Drosophila atm and mre11 knockout mutants. A telomere defect was discovered that was more severe than in any of the non-Drosophila systems studied. In mutant mitotic cells, an average of 30% of the chromosome ends engaged in telomere fusions. These fusions led to the formation and sometimes breakage of dicentric chromosomes, thus starting a devastating breakage-fusion-bridge cycle. Some of the fusions depended on DNA ligase IV, which suggests that they occurred by a nonhomologous end-joining (NHEJ) mechanism. Epistasis analyses results suggest that ATM and Mre11 might also act in the same telomere maintenance pathway in metazoans. Since Drosophila telomeres are not added by a telomerase, these findings support an additional role for both ATM and Mre11 in telomere maintenance that is independent of telomerase regulation (Bi, 2004).
The ATM checkpoint kinase and the Mre11 DNA repair protein function to maintain telomere integrity in yeast and mammals cells (reviewed in Ferreira, 2004). Whether or not they have a similar role in Drosophila, an organism that lacks a canonical telomerase, was investigated. Both atm and mre11 were knocked out by targeted mutagenesis. For atm, two different alleles were recovered by ends-in gene targeting, referred to here as atmstrong (atmstg) and atmweak (atmwk). For mre11, a complete knockout was achieved by deleting the entire Mre11 coding region by using ends-out gene targeting. Both atm and mre11 mutant animals were late pupal lethal. Proliferating tissues in the mutants experienced an excess amount of cell death. Both atm alleles failed to complement a chromosomal deficiency (Df(3R)hsc70-4Δ356) that deleted part of atm and the adjacent hsc70-4 gene. In addition, animals were generated that were homozygous for the deficiency and a wild-type hsc70-4+ transgene, thus rescuing the Hsc70-4 function. These animals were pupal lethal and displayed the same defects as atm knockout homozygotes. The pupal lethality of the mre11 deletion mutant was fully rescued by a wild-type mre11+ transgene (Bi, 2004).
To pinpoint the underlying genetic causes for the massive cell death, DAPI-stained mitotic chromosomes of neuroblasts were examined from the brains of third instar larvae. The most prominent cytological defect that was observed was chromosome end-to-end association (telomere attachment [TA]). A TA can involve any of the four chromosome pairs. TA was categorize into three different classes: single TA, double TA, and others. A single TA most likely occurred during S/G2 after telomeric DNA replication had completed, whereas a double TA could be derived from a single TA in G1 by replication. Besides widespread end-to-end attachments, severe genome instability was also observed in the forms of chromosome breakage, chromosome rearrangements, and gross aneuploidy. Chromosome breakage is grouped into two types: chromatid breaks and chromosome breaks. For example, a chromatid break was observed that involved only one of the two sisters. As a second example, an observed chromosome break involved both sister chromatids broken at an identical region and was likely the result of the replication of a single G1 break; such a G1 break could occur de novo. It could also occur as a result of a broken dicentric chromosome during the previous cell division. Interestingly, not only could the natural chromosome ends associate with each other in the mutants, they could also do so with broken ends, thus creating some of the chromosome rearrangements, which include nonreciprocal translocations, and terminal deficiencies. In addition, polyploid nuclei were observed undergoing chromosome condensation (Bi, 2004).
Consistent with atmwk being a weaker allele, an average atmwk/wk cell had about one fewer TA than an atmstg/stg cell. In fact, over 20% of the atmwk/wk nuclei had no TA, compared to 6% for atmstg/stg. An average atmwk/wk cell also had lower frequencies for both chromosome breakage and aneuploidy than an atmstg/stg cell. Cells from atmstg/stg and mre11−/− had similar numbers of TAs on average, which was similar to the rate in atmstg/Df(3R)hsc70-4Δ356 cells. In contrast, mre11−/− cells possessed more breaks and were more likely to be aneuploid than atmstg/stg cells. This excess of genome instability in mre11−/− cells was likely due to additional repair defects since Mre11 participates in multiple processes in DNA recombination and repair. In order to establish epistasis for ATM and Mre11 in telomere maintenance, mre11− and atmstg double homozygotes were generated by crossing. At the chromosomal level, a double mutant nucleus had a number of TAs similar to atmstg/stg but slightly more than mre11−/−. This lack of an additive effect from the double mutations led to the conclusion that ATM and Mre11 were in the same pathway for telomere protection, similar to their epistasis relationship in yeast (Ritchie, 2000). At the organismal level, double mutant animals died as late third instar larvae, which was a few days earlier than either of the singles. This earlier death suggested nonoverlapping functions for either protein. Indeed, the double mutation had an additive effect on the frequency of breaks, suggesting that the two proteins might contribute in parallel to prevent chromosome breakage (Bi, 2004).
In certain yeast double mutants involving tel1 (yeast atm) or mre11, telomere fusions were preceded by extensive telomeric DNA loss (Chan, 2003; Mieczkowski, 2003). Attempts were made to determine whether that also applied to Drosophila mutants. In none of the mutant combinations was a reduction observed of the overall abundance of the terminal HeT-A elements, which are the most abundant retro-transposable elements that make up the normal ends of Drosophila chromosomes (reviewed in Pardue, 2003). However, since the short telomeres in tel1 or mre11 yeast mutants were not reached until after about 80 cell divisions (Ritchie, 2000), it is possible that only cells undergoing mitosis would experience loss of telomeric DNA as a result of the Drosophila atm mutations. To address this issue, in situ hybridization was carried out on mitotic chromosomes. Telomeric HeT-A signals were often observed at the point where two chromosomes fused. This led to the conclusion that at least some of the fusions occurred without complete loss of telomeric DNA. Since fusions were also observed without HeT-A signals, it could not be ruled out that some of the fusions were preceded by DNA loss (Bi, 2004).
Telomere fusions can lead to the formation of dicentric bridges during anaphase of mitosis. For both mutants, bridges were observed in chromosome squashes of larval neuroblasts, but at a low frequency, possibly due to hypotonic treatment of the cell prior to squashing. To gain a more comprehensive understanding of the consequences that follow mitotic bridge formation, chromatin was stained with an antibody against a phosphorylated form of histone H3 specific to mitosis. This enabled the observation to be made that mitotic bridges were frequently observed in cells from all the proliferating tissues studied; this suggested that the telomere protection function of ATM and Mre11 was required for all proliferating tissues. On average, about 20% of the mitotic nuclei from the brain and imaginal discs displayed abnormal chromosome configurations. A few interesting points were noted: (1) multiple bridges were commonly observed, consistent with the mutant nucleus having multiple TAs; (2) bridges could persist without breakage, at least until telophase (they might be eventually severed by cytokinesis); (3) acentric fragments were observed left at the metaphase plate, most likely leading to aneuploidy; (4) broken bridges were frequently observed. Bridges were most often broken around the midpoint. However, asymmetrically broken bridges were also present, which would lead to a nonreciprocal translocation. Some bridges showed multiple constrictions indicative of chromatin breaks that might not involve DNA. This provides physical evidence supporting the idea that breakage of mitotic bridges is a feasible mechanism for the generation of internal breaks and supports the hypothesis that most of the gross chromosome rearrangements seen in certain yeast repair mutants arise as a result of a breakage-fusion-bridge cycle (Bi, 2004).
In both yeast and mammalian cells, covalent telomere fusions in certain mutant backgrounds often occur by NHEJ that require the specialized DNA ligase IV (reviewed in Ferreira, 2004). To test the requirement for ligase IV, mutations were generated in the Drosophila ligase IV (lig4) gene by mobilizing a nearby P element (EP0385); a mutation was recovered that deleted the first 147 codons (Bi, 2004).
At the chromosomal level, a lig4− atmstg nucleus had slightly fewer TA on average than an atmstg single mutant. This lack of a larger effect was verified with another lig4 deletion mutation. For the atmwk mutation, loss of Lig4 function significantly reduced the overall TA rate. Interestingly, different classes of TA responded differently to the mutation. The rate for double TAs was most sensitive, dropping from 0.98 to 0.46. This suggested that Lig4 was more important for attachments that occurred in G1 in which NHEJ activity predominates for both yeast, mammals, and perhaps, Drosophila. In contrast, the rate for intersister TAs was not at all affected. Since sisters are identical in sequence, an intersister TA could occur by a homologous recombination-based mechanism, which does not require Lig4. It is concluded that at least some of the TAs in atm mutants involved covalent joining of telomeric DNA. This was also supported by the behaviors of mitotic bridges described earlier. To explain the allele-specific response to the lig4 mutation, it is first suggested that NHEJ is not entirely dependent on Lig4 in Drosophila. It is then imagined that in an atmstg/stg cell, the half-life in which a telomere could participate in fusions is significantly longer due to the complete loss of ATM. A fusion prevented by the lig4 mutation would be carried out by other mechanisms, which would result in a somewhat constant overall TA frequency. Based on this model, it is predicted that some of the NHEJ-typed fusions in lig4−/− atmstg/stg cells would be channeled into fusions of other types, perhaps one that was based on sequence homology. Consistent with this, an increase was observed of over 20% for the rate of intersister TAs accompanied by a reduction of the rate for double TAs (Bi, 2004).
The association of human Mre11 with TRF2, a telomeric repeat binding protein, suggests that Mre11 may be a structural component of a normal telomere. However, Mre11 was not localized to telomeres in a mixed population of wild-type yeast cells. Therefore, it remains possible that both ATM and Mre11 regulate the telomere protection activity of other proteins. In an effort to identify such targets, focus was placed on the Drosophila HP1/ORC-associated protein (HOAP). HOAP is the only Drosophila protein that has been shown to exclusively localize to the ends of mitotic chromosomes (Cenci, 2003). A mutation in HOAP caused telomere attachments. In addition, the small HOAP protein (337 aa) contains six S/TQ sites, which are the preferred phosphorylation sites in known targets of ATM (reviewed in Abraham, 2001). It is attractive to postulate that ATM exerts its telomere protection function by regulating HOAP localization to the telomeres and that Mre11 may do so by regulating the kinase activity of ATM (Bi, 2004).
To test this model, immunolocalization was performed of HOAP in mitotic cells from the larval brains. In either atmstg or atmwk mutant cells, HOAP was localized to the end normally. This normal localization was also observed in atmstg/Df(3R)hsc70-4Δ356 cells. In addition, HOAP could be detected on 15% of the telomeres engaged in attachments. This is identical to the results from HOAP localization studies in fly mutants lacking HP1. Therefore, HOAP localization to mitotic telomeres did not depend on ATM. However, this localization was affected by the mre11 mutation. The overall chromosomal staining of HOAP was reduced in the mre11 mutant background, with only 40% of the nuclei displaying a HOAP signal on more than one chromosomes. In these nuclei, HOAP was often missing from majority of the chromosome ends. HOAP could still be localized to telomeres engaged in TAs. Another study also discovered telomere fusion and reduced HOAP localization in another mre11 mutation (Ciapponi, 2004). HOAP localization was also studied in the mre11 atmstg double mutant and an extent of HOAP localization similar to mre11 alone was observed. These results, particularly the ability to detect HOAP at some of the fusion junctions in both mutants, led to the conclusion that failure to recruit HOAP to the telomeres could not have been the cause for fusion. However, it cannot be ruled out that HOAP's telomere-protecting function, but not its telomere localization, still depends on ATM (Bi, 2004).
The above results are consistent with the hypothesis that Mre11 might act at or around the telomere. It is imagined that the Mre11 nuclease processes the telomere into a special structure, which facilitates HOAP binding. Absent this structure, the efficiency of HOAP binding is reduced, and the fixative treatment in the immunostaining procedure might result in variable loss of telomere bound HOAP proteins. Based on the lack of effect of the atm mutations on HOAP localization, it is proposed that ATM is not an integral part of a telomere. Instead, it may exert its regulatory role as a kinase (Bi, 2004).
Not only are Drosophila telomeres not added by a telomerase, but it was also believed that the end of a Drosophila chromosome could consist of essentially any sequence. Therefore, Drosophila telomeres, unlike ones in yeast or mammals, lack the protection conferred by the canonical telomerase and various telomeric repeat binding proteins. This would render Drosophila telomeres more vulnerable to uncapping and make the telomere protection function of ATM and Mre11 more critical for cell survival (Bi, 2004).
The mechanisms by which ATM and Mre11 maintain telomere homeostasis are largely unclear. In the most advanced model from yeast, they recruit telomerase activity to the chromosome ends by altering the structure of the telomeric DNA (Chan, 2001; Tsukamoto, 2001). A more direct role of ATM was recently suggested that was based on a synergistic effect of an atm knockout with a telomerase knockout in causing telomere dysfunction in both yeast and mouse (Chan, 2003; Qi, 2003). This study provides evidence supporting such a hypothesis, and shows that Drosophila would be an excellent system for advancing such studies without the complication of telomerase (Bi, 2004).
Cells of metazoan organisms respond to DNA damage by arresting their cell cycle to repair DNA, or they undergo apoptosis. Two protein kinases, ataxia-telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR), are sensors for DNA damage. In humans, ATM is mutated in patients with ataxia-telangiectasia (A-T), resulting in hypersensitivity to ionizing radiation (IR) and increased cancer susceptibility. Cells from A-T patients exhibit chromosome aberrations and excessive spontaneous apoptosis. Drosophila has been used as a model system to study ATM function. Previous studies suggest that mei-41 corresponds to ATM in Drosophila; however, it appears that mei-41 is probably the ATR ortholog. Unlike mei-41 mutants, flies deficient for the true ATM ortholog die as pupae or eclose with eye and wing abnormalities. Developing larval discs exhibit substantially increased spontaneous chromosomal telomere fusions and p53-dependent apoptosis. These developmental phenotypes are unique to Drosophila ATM, and both Drosophila ATM and mei-41 have temporally distinct roles in G2 arrest after IR. Thus, ATM and ATR orthologs are required for different functions in Drosophila; the developmental defects resulting from absence of Drosophila ATM suggest an important role in mediating a protective checkpoint against DNA damage arising during normal cell proliferation and differentiation (Song, 2004).
Drosophila lines were generated with specific disruptions of the dATM gene. A previously generated P element insertion, EP(3)0859, located within the hsc70-4 (hsc4) gene, which is 546 bp upstream of dATM, disrupts hsc4 and exhibits homozygous lethality. In efforts made by others to generate a null allele of the hsc4 gene, this transposable element was mobilized, resulting in two different imprecise excision deletions of hsc4 including the dATM gene. These are referred to as Δ11 and Δ356. The endpoints of the deleted region were confirmed by PCR followed by sequence analysis. dATM was predicted to contain 24 exons: Δ11 flies lack the first 2 exons, whereas the first 8 exons are deleted in Δ356 flies. The flies were extensively out-crossed to wild-type flies to eliminate any additional mutations on the chromosome and then used for further analysis. The lethality resulting from the hsc4 mutation was rescued transgenetically with a 14 kb genomic fragment encompassing the complete hsc4 gene, thereby generating 'clean' loss-of-function alleles of dATM (Song, 2004).
The majority of flies with the genotype P[hsc4]; Δ11 and P[hsc4]; Δ356 exhibit pupal lethality. However, 12% (Δ11) and 15% (Δ356) of homozygous mutants eclosed and were viable, although they did not survive very long. Northern blot analysis of the 'escaper' females with the genotype P[hsc4]/Cyo; Δ11, P[hsc4]/Cyo; Δ356, and P[hsc4]/Cyo; EP(3)0859 revealed that the dATM transcript is substantially reduced in both Δ11 and Δ356 mutant flies, whereas it is present in EP(3)0859. Interestingly, Δ11/Δ356 transheterozygotes exhibit a higher percentage of viability, suggesting that there may be some complementation between the two alleles. Moreover, two additional dATM alleles that exhibit homozygous lethality were subsequently obtained from the Exelixis transposon disruption collection. These findings suggest that the Δ11 and Δ356 alleles are strong hypomorphic loss-of-function dATM alleles (Song, 2004).
dATM, mei-41, grp, and dChk2 are close orthologs of mammalian DNA damage checkpoint genes that are all highly expressed in Drosophila females relative to males. The encoded checkpoint protein kinases presumably respond to DNA double-strand breaks generated during meiotic recombination. Notably, meiotic recombination in Drosophila occurs only in females. Moreover, mammalian ATM and ATR localize to synapsed and unsynapsed regions of meiotic chromosomes, respectively, suggesting a role for these proteins during meiotic recombination. In Drosophila, most mei-41 alleles are recessive female sterile, and females homozygous for the more fertile hypomorphic mei-41 alleles exhibit reduced meiotic exchange. The Δ356 dATM mutant females do not lay eggs, whereas Δ11 females lay a very small number of eggs that do not hatch, indicating that dATM is required for female fertility. In mouse models, ATR is an essential gene, whereas ATM is dispensable for normal development and viability, although homozygous mutant mice exhibit immune defects, growth retardation, and sterility. In Drosophila, the mei-41(ATR) mutant was found to be maternal lethal and nullizygous dATM mutants were found to be lethal. Thus, unlike its mammalian counterpart, Drosophila ATM plays a critical developmental role (Song, 2004).
To determine the basis for these phenotypes, third-instar wing discs or eye discs were isolated and examined for morphology and for apoptosis by TUNEL staining. Although wing and eye disc morphology appears to be normal, dATM mutant discs are somewhat reduced in overall size and exhibit a substantial increase in TUNEL-positive cells with relatively few apoptotic cells detected within wild-type discs. To determine whether the excessive apoptosis is p53-dependent, a dominant-negative (dn) form of Dmp53 was introduced. The dn-Dmp53 transgene has been shown to block IR-induced apoptosis when expressed in the posterior part of the wing with engrailed-GAL4. Expression of dn-Dmp53 in dATM mutant wing discs results in a substantial decrease in apoptosis. Thus, loss of dATM function results in p53-mediated apoptosis during development. In a report by Bi (2004), spontaneous apoptosis in dATM mutant discs was found to occur even in a p53-zygotic mutant background, suggesting that the observed apoptosis is p53 independent. This apparent discrepancy in findings may reflect the presence of maternal Dmp53 product in larvae of the homozygous mutant background (Song, 2004).
In imaginal eye discs double-stained for TUNEL and the neuronal marker Elav, it was observed that the excessive spontaneous apoptosis seen in dATM mutants is largely restricted to the proliferating cells anterior to the morphogenetic furrow, and, in addition, there was evidence of reduced neuronal differentiation and abnormal patterning of differentiated cells. Thus, dATM appears to protect proliferating cells from apoptosis and consequently affects their capacity to differentiate (Song, 2004).
In addition to the high level of spontaneous apoptosis observed in dATM mutants, in neuroblasts from dissected brain imaginal discs of dATM mutants, a substantial fraction of mitotic cells exhibit evidence of chromosomal damage, particularly, telomere fusions. Among metaphase chromosomal spreads, both single and double telomere associations were observed, including fusions between sister chromatids as well as between homologous and nonhomologous chromosomes. Occasionally, several chromosomes were fused together to form a chromosome 'chain'. Thus, dATM appears to provide a protective function required for maintenance of telomeres and chromosome structure. Bi (2004) reports that some of the telomere fusions are dependent on ligase IV and are, therefore, likely to occur by a nonhomolgous end-joining mechanism. These observations are of particular interest, because telomere abnormalities have also been observed in mammalian A-T cells. Moreover, it is likely that the presence of dicentric chromosomes, which are unstable, leads to the observed excessive spontaneous apoptosis in dATM mutant tissues during development (Song, 2004).
Like mammalian cells, Drosophila cells respond to DNA damage by undergoing cell cycle arrest. In response to IR, cells of wild-type larval discs exhibit delayed entry into mitosis with increased apoptosis. In mammals, the IR-induced G2 arrest is temporally regulated by the different checkpoint kinases, with ATM playing an important role in the initiation of G2 arrest and ATR playing a more prominent role in maintaining the G2 arrest. Moreover, Chk1 and Chk2 have temporally distinct roles in initiating and maintaining G2 arrest, respectively (Song, 2004 and references therein).
To examine the temporal roles of ATM and mei-41 in Drosophila, mitosis (as an indicator of cell cycle arrest) was examined at various time points after irradiation. Wild-type wing discs had very few mitotic cells 25 min after irradiation, and no mitotic cells were detected at 1 hr postirradiation. The wing disc cells remained arrested and then reentered the cycle approximately 6 hr later. Interestingly, dATM mutant wing discs had significantly more mitotic cells 25 min postirradiation as compared to wild-type discs. At later time points, G2 arrest occurred normally in dATM mutant wing discs. In mei-41 mutant wing discs, cells continued to cycle throughout the time points tested. These results suggest that dATM is involved in the early phase of G2 arrest and mei-41 has a major role in late response. The observed temporal differences in the IR-mediated checkpoint are consistent with those found in mammals (Song, 2004).
Potential functional redundancy between dATM and mei-41 was examined by generating double mutants. Flies harboring mutations in both genes exhibit the same excessive level of spontaneous apoptosis seen in the dATM mutant larval tissues and no apparent increase in apoptosis following IR; however, most of the third-instar larvae exhibited black 'growths', characteristic of so-called melanotic tumors. The underlying basis for these growths is not known, but it suggests that there is some developmental context in which these two genes function redundantly. The mechanisms underlying the distinct and overlapping functions of dATM and mei-41/ATR are likely to involve the upstream activation signals and downstream effector substrates for these closely related kinases; as such, the Drosophila model provides a genetically tractable system that should prove useful in dissecting their function in an in vivo context (Song, 2004).
In conclusion, the function of Drosophila ATM was examined and its role was compared with that of mei-41/ATR during normal development and DNA damage checkpoint responses. Both dATM and mei-41 are highly expressed in female adults and are required for female fertility. Unlike mei-41, flies deficient for dATM exhibit a substantial increase in spontaneous p53-dependent apoptosis and telomere fusions in developing tissues, leading to lethality or to viable flies with malformed adult tissues. It is presumed that dATM deficiency leads to the accumulation of DNA damage during normal cellular replication and differentiation and that this culminates in p53 activation. Although some ATM functions in mammals are mediated by the Chk2 kinase, the essential developmental role of dATM is apparently not mediated by dChk2, which is a nonessential gene, indicating that other ATM substrates are required. In addition to their distinct developmental requirements, dATM and mei-41/ATR perform temporally distinct functions in the DNA damage response to ionizing radiation. Together with the characterization of ATM and ATR functions in mammalian systems, these studies of the Drosophila orthologs point to evolutionarily conserved pathways involving two closely related proteins that together regulate genomic integrity during normal development and in response to genotoxic stress (Song, 2004).
Terminal deletions of Drosophila chromosomes can be stably protected from end-to-end fusion despite the absence of all telomere-associated sequences. The sequence-independent protection of these telomeres suggests that recognition of chromosome ends might contribute to the epigenetic protection of telomeres. In mammals, Ataxia Telangiectasia Mutated (ATM) is activated by DNA damage and acts through an unknown, telomerase-independent mechanism to regulate telomere length and protection. The Drosophila homolog of ATM is encoded by the telomere fusion (tefu) gene. In the absence of ATM, telomere fusions occur even though telomere-specific Het-A sequences are still present. High levels of spontaneous apoptosis are observed in ATM-deficient tissues, indicating that telomere dysfunction induces apoptosis in Drosophila. Suppression of this apoptosis by p53 mutations suggests that loss of ATM activates apoptosis through a DNA damage-response mechanism. Loss of ATM reduces the levels of heterochromatin protein 1 (HP1) at telomeres and suppresses telomere position effect. It is proposed that recognition of chromosome ends by ATM prevents telomere fusion and apoptosis by recruiting chromatin-modifying complexes to telomeres (Oikemus, 2004).
Drosophila atm is required to protect telomeres from fusion. HP1 and HOAP localize to the telomeres of polytene chromosomes, as well as other sites, and are required for telomere protection in mitotic cells. Immunostaining was used to examine the distribution of HP1 and HOAP on wild-type and atm- polytene chromosomes. HP1 staining at the chromocenter, fourth chromosome, and several euchromatic sites is unaffected by loss of atm, whereas HP1 staining is reduced at most atm- telomeres. At the tip of chromosome 2R, similar levels of HP1 staining at an internal site (cytological position 60F) can be observed in wild-type and mutant chromosomes, whereas HP1 is specifically reduced at the telomere of the mutant chromosome. The normal levels of HP1 at sites other than the telomere indicate that the lack of telomere staining at atm- chromosomes is not due to differences in chromosome preparations or to global changes in chromatin structure in atm- cells. Rather, atm is specifically required to recruit or maintain HP1 to chromosome ends (Oikemus, 2004).
Immunostaining of the same chromosomes for HOAP reveal reduced staining at the telomeres of most atm- chromosomes compared with wild type. Similar decreases in HP1 and HOAP localization at telomeres are seen in atmDelta356/ Df(3R)PG4 and atmtefu/Df(3R)PG4 animals, indicating that this phenotype is not allele specific. Quantification of the fluorescence intensity associated with HOAP and HP1 staining further demonstrates that there is a reproducible reduction at atm- telomeres compared with wild type. In contrast, HP1 staining at an internal chromosomal site (60F) is not reduced (Oikemus, 2004).
HP1 promotes heterochromatin formation, in part, by recruiting histone-modifying enzymes. To probe whether atm mutations alter chromatin at the telomeres of mitotic cells, telomere position effect (TPE) at three telomeres was examined. When a white reporter gene is placed adjacent to telomere-associated sequences (TAS), gene expression is silenced. At each site tested, TPE is partially suppressed by mutations in atm. In transgenes inserted at nontelomeric genomic positions, placement of the TAS from the telomere of chromosome arm 2L next to the white reporter gene is sufficient to silence white expression. Unlike TAS in their normal location adjacent to telomeres, silencing by a nontelomeric TAS is not affected by atm mutations. These results indicate that the suppression of TPE is due to the specific action of atm on gene expression near telomeres (Oikemus, 2004).
In other organisms, loss of telomere protection can be due to the attrition or degradation of telomere repeat sequences. In Drosophila, it is possible to recover terminal deletions that remove all telomere-specific sequences. However, these observations do not rule out the possibility that telomere-specific sequences contribute to telomere protection or TPE at normal Drosophila telomeres. In fact, the number of telomere repeats has been shown to influence some forms of TPE (Mason, 2003). To test whether the telomere defects in atm- animals could be due to loss of telomere sequences, fluorescent in situ hybridization was performed using a probe to the Het-A retrotransposon, which is specific to telomere DNA. Hybridization was performed with wild-type and atm- diploid and polytene chromosomes. In mitotic chromosomes from diploid neuroblast cells, the levels of Het-A hybridization are variable, but not significantly different between wild-type and atm mutant cells. In polytene chromosomes, HeT-A sequences are strongly detected at two telomeres of both wild-type and atm- chromosomes. Previous analysis of HP1 mutants demonstrated that telomere-specific sequences were still present at chromosome fusion sites (Fanti, 1998). In atm mutant cells, Het-A hybridization is also detected at sites of chromosome fusion. These results indicate that the reduction of telomeric HP1-HOAP and the fusion of telomeres in atm- cells is not a direct or indirect result of telomere sequence loss (Oikemus, 2004).
Both wild-type and terminally deleted Drosophila chromosomes are protected from telomere fusion and are capped with the telomere-protection proteins HP1 and HOAP (Biessmann, 1988; Fanti, 1998; Cenci, 2003). These results indicate that sequence-independent mechanisms can recruit and maintain telomere protection complexes to chromosome ends. This study has demonstrated that Drosophila atm/tefu is required to prevent chromosome end fusions, to regulate levels of HP1 and HOAP at telomeres, and to promote telomere-position effect. It is also found that atm is required for induction of apoptosis by ionizing radiation. Given the conserved role of ATM family proteins in recognizing DNA breaks, it is suggested that Drosophila ATM protects telomeres by recognizing chromosome ends and recruiting chromatin-modifying proteins to those ends (Oikemus, 2004).
To date ATM protein has not been directly detected at Drosophila telomeres. However, on the basis of results in mammalian cells, it may be necessary to develop antibodies specific for activated forms of ATM to probe ATM activity at telomeres (Bakkenist, 2003). However, several observations presented here indicate that Drosophila ATM acts at telomeres to prevent chromosome fusions. (1) The chromosome rearrangements observed are consistent with a defect in telomere protection rather than translocations due to defective DNA repair or replication. Most chromosome fusions occur near the ends of chromosome arms, and this study demonstrates that the fused chromosomes still contain telomeric DNA sequences. (2) A high frequency of acentric chromosome fragments is not observed during metaphase. In animals mutant for other damage-signaling genes such as the Drosophila homologs of ATR and ATRIP, acentric chromosome fragments are often observed during metaphase, suggesting that these mutations cause a defect in DNA repair or replication that is not observed in atm- animals. (3) Circular chromosomes do not undergo rearrangements in atmtefu mutant animals (Queiroz-Machado, 2001), strongly indicating that chromosome fusions are due to fusion of existing chromosome ends rather than the creation of new chromosome breaks. (4) ATM is specifically required for localization of HP1 to telomeres but not centromeric or euchromatic sites. (5) Loss of atm suppresses silencing by telomere-associated sequences when they are adjacent to telomeres, but not when they are at euchromatic sites (Oikemus, 2004).
The telomere fusion defect seen in atm- animals is consistent with a partial defect in telomere protection. Whereas ~80% of atm- metaphases contain a chromosome fusion, >95% of metaphases from animals lacking HP1 or HOAP contain a fusion (Fanti, 1998; Cenci, 2003). Furthermore, in some cells lacking HP1 or HOAP, nearly all telomeres appear to be fused. This extreme phenotype has not been observed in atm mutant nuclei. Consistent with a partial defect in telomere protection, the levels of HP1 and HOAP at polytene telomeres are found to be reduced, but not eliminated, in atm- animals. In mitotic cells, formation of repressive chromatin is also partially disrupted. The interpretation of these results is that reduced and variable levels of HP1 at the telomeres of atm- animals are sufficient to protect some, but not all telomeres from fusion. The results also indicate that another pathway, possibly involving other DNA damage-response proteins, must contribute to HP1 and HOAP localization, TPE, and telomere protection (Oikemus, 2004).
The direct target of ATM at telomeres is unclear. The decrease in HP1 and HOAP levels at atm- telomeres is not due to a loss of telomere sequences; wild-type and atm- chromosomes exhibit similar levels of a telomere-specific retrotransposon sequence as assayed by FISH, and even sites of fusion retain this sequence. This result is consistent with previous demonstrations that the sequences at chromosome ends are not required for telomere protection or for telomeric localization of HP1 and HOAP. Instead, ATM is likely to affect the interaction of HP1 and HOAP with telomeres by regulating the formation of the HP1-HOAP complex or by modification of telomeric chromatin. Other proteins in the DNA damage-response pathway may act with ATM to maintain telomere protection. Although Chk1, Chk2, and p53 are targets of mammalian ATM during the DNA damage response (Shiloh, 2003), Drosophila homologs of these proteins do not appear to be required for telomere protection; animals lacking one or more of these genes do not exhibit the high levels of apoptosis associated with loss of ATM (Brodsky, 2004). Mutations in homologs of other ATM targets such as NBS1 or SMC1 have not been described in Drosophila (Oikemus, 2004).
Recruitment of HP1 and HOAP by ATM is likely to alter chromatin structure at telomeres. HP1 plays a conserved role in heterochromatin formation, histone modification, and gene silencing . In Drosophila, both HP1 and HOAP are required for gene silencing at pericentric heterochromatin. In addition, HP1 is required for gene silencing near fourth chromosome and terminally deleted telomeres, and for repression of P-element transposition by subtelomeric P-element insertions. HP1 homologs are also associated with telomere function in other eukaryotes. In mammals, all three HP1 homologs are found at telomeres, and loss of histone H3 methylases leads to reduced levels of HP1 homologs at telomeres as well as elongated telomeres (Garcia-Cao, 2004). In contrast, overexpression of mammalian HP1 homologs is associated with decreased telomere length (Sharma, 2003). The fission yeast homolog of HP1 is not required for telomere protection, but does regulate telomere length, telomere clustering, and telomeric gene silencing. Interestingly, as in Drosophila telomere protection, some aspects of telomere function in fission yeast are controlled by an epigenetic mechanism. Together, these observations indicate that a requirement for HP1 in telomere function and chromatin structure is conserved, but that its precise role at the telomere may differ among organisms (Oikemus, 2004 and references therein).
Regulation of telomere chromatin structure is also a conserved function of ATM-like kinases. Fission yeast Rad3 and budding yeast Mec1 are required for gene silencing at telomeres (Matsuura, 1999; Craven, 2000) and mutations in human ATM are associated with altered nucleosomal periodicity at telomeres (Smilenov, 1999). The conserved role of ATM-kinases in telomere protection and telomeric chromatin structure suggests that these functions might be linked. The finding that Drosophila ATM is required for TPE and HP1-HOAP localization to telomeres demonstrates one mechanism by which ATM can influence telomere chromatin. It is possible that in organisms that utilize sequence-specific binding proteins such as TRF2 to protect telomeres, regulation of telomeric heterochromatin by ATM and HP1 plays a minor role in protection of normal telomeres, but a more important role at short telomeres that cannot recruit sufficient levels of TRF2. Such a model might explain the synergistic telomere defects seen in cells lacking both telomerase and ATM (Ritchie, 1999; Tsukamoto, 2001; Chan, 2003; Wong, 2003). The lack of an obvious TRF2 homolog may explain why ATM and HP1 play such striking roles in the protection of Drosophila telomeres (Oikemus, 2004).
In addition to preventing chromosome end fusion by DNA repair enzymes, telomere protection is required to prevent activation of DNA damage responses, including the induction of p53-dependent apoptosis and senescence. This analysis of the cellular effects of ATM loss indicates that induction of p53-dependent apoptosis is a conserved consequence of unprotected telomeres in metazoans. Because these unprotected telomeres lead to anaphase bridges and chromosome breaks, p53 may be directly activated by unprotected telomeres or may be activated by subsequent chromosome breaks. Drosophila ATM is required for the induction of apoptosis following IR. Because the spontaneous apoptosis in atm- animals is, by definition, ATM independent, a different pathway must be able to activate Drosophila p53 in response to unprotected telomeres. Similarly, loss of mammalian ATM reduces, but does not eliminate p53-dependent apoptosis in response to unprotected telomeres (van Steensel, 1998; Takai, 2003; Wong, 2003). Other DNA damage-response pathways may activate Drosophila p53 in the absence of ATM (Oikemus, 2004).
In yeast, insects, and mammals, ATM-kinases are required to activate cellular responses to DNA ends generated by exogenous DNA damage, but also to suppress activation of these pathways by telomeres. Specific recognition of telomere sequences by telomere repeat-binding proteins provides one means to distinguish telomeric DNA ends from damage-induced DNA breaks. However, this mechanism is not sufficient to explain the epigenetic regulation of telomere protection in Drosophila. The requirement of ATM to recruit HP1 and HOAP to Drosophila telomeres suggests that recognition of chromosome ends contributes to chromatin-mediated telomere protection. This model may help explain how terminally deleted chromosomes can be stably inherited without any telomere-specific sequences. Future studies should reveal which other damage response proteins help ATM protect telomeres, what their targets are at telomeres, and how these proteins distinguish between damage-induced DNA ends and the natural ends of chromosomes (Oikemus, 2004).
In higher eukaryotes, the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) checkpoint kinases play distinct, but partially overlapping, roles in DNA damage response. Yet their interrelated function has not been defined for telomere maintenance. The two proteins control partially redundant pathways for telomere protection in Drosophila: the loss of ATM (encoded by telomere fusion) leads to the fusion of some telomeres, whereas the loss of both ATM and ATR (encoded by mei-41) renders all telomeres susceptible to fusion. The ATM-controlled pathway includes the Mre11 and Nijmegen breakage syndrome complex but not the Chk2 kinase, whereas the ATR-regulated pathway includes its partner ATR-interacting protein but not the Chk1 kinase. This finding suggests that ATM and ATR regulate different molecular events at the telomeres compared with the sites of DNA damage. This compensatory relationship between ATM and ATR is remarkably similar to that observed in yeast despite the fact that the biochemistry of telomere elongation is completely different in the two model systems. Evidence is provided suggesting that both the loading of telomere capping proteins and normal telomeric silencing require ATM and ATR in Drosophila and it is proposed that ATM and ATR protect telomere integrity by safeguarding chromatin architecture that favors the loading of telomere-elongating, capping, and silencing proteins (Bi, 2005).
This study defines two partially redundant pathways, regulated by ATM and ATR, respectively, that ensure complete protection of Drosophila telomeres. It is further suggested that two other proteins, Meiotic recombination 11 (Mre11) and Nbs) belong to the same ATM-regulated pathway, whereas ATR-Interacting Protein (ATRIP or Mutagen-sensitive 304) participates in the ATR-controlled pathway. This conclusion would be consistent with the pathway components defined in yeasts. Based on the facts that Drosophila tefu mutants have widespread telomere fusions that eventually lead to lethality, whereas mei-41 mutants are viable with no apparent telomeric defects, it is proposed that ATM is more important in regulating telomere protection, with ATR playing a backup role. In mei-41 mutants, ATM may fully compensate ATR's absence on telomeres to ensure complete capping throughout the cell cycle. In contrast, ATR in tefu mutants may partially compensate for the loss of ATM because it is a less efficient regulator of capping as suggested earlier. This partial redundancy leads to the fusion of some telomeres (Bi, 2005).
ATM, ATR, and their cofactors are known to control multiple checkpoints in response to DNA damage and abnormal telomeres. Perhaps cells with uncapped telomeres are allowed to continue cycling because of defective checkpoints and that leads to the fusion of these telomeres. This possibility is considered unlikely based on several observations. (1) In cav1 mutant (see caravaggio), telomere fusion occurs at a high rate even in the presence of a full complement of checkpoint genes. (2) No exacerbated telomere dysfunction is found in either the cav1 tefu (atm) or the cav1 mei-41 (atr) double mutant. (3) Mutations in Drosophila chk1 or chk2 did not affect existing telomere defects in either tefu or mre11 mutant, suggesting that checkpoints jointly controlled by these effectors and the respective upstream kinases do not normally respond to dysfunctional telomeres. Therefore, the contribution to the telomere dysfunction from checkpoint defects is likely small in the cases studied (Bi, 2005).
Cytological analyses suggest that one of the functions of ATM and ATR at Drosophila telomeres is to facilitate the loading of telomere capping and silencing proteins, such as HOAP (Caravaggio) and HP1, and they do so in a partially redundant fashion. In the case of HOAP's binding to mitotic telomeres, either ATM or ATR is sufficient for its normal loading. When both kinases are absent, HOAP can no longer be detected at the telomeres. It was interesting that there were more telomere fusions in either tefu mei-41D9 or tefu mus304D1/D3 than in either mre11 or nbs1 cells, yet normal HOAP signals were detected on fusion-free telomeres for only the first two genotypes. It is known that the presence of HOAP at chromosome ends is not sufficient to prevent fusion, which suggests that the loss of other capping proteins could also lead to fusion. That may be the case in tefu mei-41D9 and tefu mus304D1/D3 cells. It is also known that the absence of HOAP at any particular telomere does not necessarily lead to fusion, suggesting other capping proteins can sometimes compensate for HOAP's function. That idea, in contrast, may apply to the situation in cells deficient for the Mre11 complex (Bi, 2005).
The mechanism by which ATM and ATR maintain telomere integrity remains largely unclear. Because the conserved kinase domain of both ATM and ATR is essential for normal telomere function in S. cerevisiae, ATM and ATR likely exert their function by protein phosphorylation. Neither Chk1 nor Chk2 was involved in telomere protection in Drosophila, a situation similar to S. pombe, which suggests that ATM and ATR modify a different set of proteins at telomeres. ATM and ATR may regulate HOAP's capping activity by directly phosphorylating HOAP. However, no evidence was recovered that HOAP is phosphorylated in WT cells. Therefore, ATM and ATR may indirectly regulate HOAP's ability to bind telomeres, perhaps by modulating telomeric structure. The results support the hypothesis that ATM and ATR have a conserved function at the telomeres that is independent of telomerase. Because Drosophila telomeres are not elongated by a telomerase, the fly may be an excellent system for studying the roles of ATM and ATR in telomere protection, uncoupled from their roles in telomere elongation (Bi, 2005).
Two protein kinases ATM and ATR as well as the Mre11/Rad50/Nbs (MRN) complex, which contains two highly conserved proteins Mre11 and Rad50 and a third less-conserved component, Nbs/Xrs2 (also known as nibrin), play critical roles in the response to DNA damage and telomere maintenance in mammalian systems. The primary function of the MRN complex is to sense DNA strand breaks and then to amplify the initial signal and convey it to downstream effectors, such as ATM, p53, Nbs1 (as a target of ATM), SMC1 and Brca1, that regulate cell cycle checkpoints and DNA repair. Mre11-Rad50 can bind DNA and that Mre11 possesses a nuclease activity that can process these ends. Nbs stimulates the DNA binding and nuclease activity by Mre11-Rad50. In vivo, Nbs is responsible for translocating the MRN complex to the nucleus and relocalizing the complex to the sites of DSBs following irradiation. The MRN complex is also required for activation of the S-phase checkpoint following DNA damage (Ciapponi, 2006).
It has been shown that mutations in the Drosophila mre11 and rad50 genes cause both telomere fusion and chromosome breakage. This study analyzed the role of the Drosophila nbs gene in telomere protection and the maintenance of chromosome integrity. Larval brain cells of nbs mutants display telomeric associations (TAs) but the frequency of these TAs is lower than in either mre11 or rad50 mutants. Consistently, Rad50 accumulates in the nuclei of wild-type cells but not in those of nbs cells, indicating that Nbs mediates transport of the Mre11/Rad50 complex in the nucleus. Moreover, epistasis analysis revealed that rad50 nbs, tefu (ATM) nbs, and mei-41 (ATR) nbs double mutants have significantly higher frequencies of TAs than either of the corresponding single mutants. This suggests that Nbs and the Mre11/Rad50 complex play partially independent roles in telomere protection and that Nbs functions in both ATR- and ATM-controlled telomere protection pathways. In contrast, analysis of chromosome breakage indicated that the three components of the MRN complex function in a single pathway for the repair of the DNA damage leading to chromosome aberrations (Ciapponi, 2006).
This study shows that the wild-type function of the Drosophila nbs gene is required to maintain chromosome integrity and to prevent telomere fusion. The results indicate that the nbs, mre11, and rad50 genes function in single pathway for the repair of spontaneous DNA lesions leading to chromosome breakage. In addition, it was found that double mutants affecting a single component of the MRN complex and either the ATM or the ATR kinase exhibit more chromosome breaks than the corresponding single mutants. The simplest interpretation of these results is that the two kinases function in multiple pathways for the repair of the DNA damage leading to chromosome breakage and that some of these pathways do not include the MRN complex. The finding that mei-4129D tefuatm6 double mutants and tefuatm6 single mutants display similar frequencies of chromosome breaks indicates that the ATM and ATR play redundant roles in the protection from spontaneous chromosome breakage. However, tefu and mei-41 mutants are four- and eightfold more sensitive than wild type to the X-ray induction of chromosome breakage, respectively. Thus, ATR may play a principal role in the repair of the lesions leading to chromosome breaks, with ATM playing a backup role (Ciapponi, 2006).
Recent work has shown that ATM and ATR/ATRIP function in different but redundant pathways of Drosophila telomere protection, with ATM playing an essential role and ATR compensating for the loss of ATM activity. This study shows that the frequencies of TAs observed in nbs tefu and rad50 nbs double mutants are significantly higher than those observed in the corresponding single mutants. An interpretation of these findings is that the Nbs protein functions in a telomere protection pathway that is different from either the ATR/ATRIP or the ATM/Rad50/Mre11 pathway. Alternatively, Nbs could function in both the ATM- and ATR-controlled pathways. These results are at odds with those obtained in budding yeast, where Tel1 (the ATM homolog), Rad50, Mre11, and Xrs2 (the NBS homolog) function in a single pathway of telomere maintenance. However, they are consistent with several results obtained in human cells, showing that the NBS and the MRE11/RAD50 components of the MRN complex can function independently. For example, it has been shown that NBS1 and the MRE11/RAD50 complex have separate roles in both ATM activation and ATM-mediated phosphorylation events. Moreover, while NBS1 localization to the human telomeres is restricted to the S phase, the MRE11/RAD50 complex remains associated with telomeres throughout the cell cycle (Ciapponi, 2006 and references therein).
The results suggest a model for the role of Nbs in Drosophila telomere protection. This model is based on the assumption that Nbs can facilitate both ATR- and ATM-mediated phosphorylation events, as recently shown in mammalian systems. It is proposed that Nbs is involved in both the Rad50/Mre11/ATM and the ATR/ATRIP telomere protection pathways. Nbs would mediate the transport of the Rad50/Mre11 complex in the nucleus in the Rad50/Mre11/ATM pathway and facilitate certain ATR-mediated phosphorylation events in the ATR/ATRIP pathway. Taking into account that the ATR/ATRIP telomere protection pathway is redundant (Bi, 2005), the model can explain the results of the epistasis analysis. It is speculated that in nbs mutants both pathways are partially impaired, resulting in a relatively low frequency of TAs. In rad50 nbs and tefu nbs double mutants, the Rad50/Mre11/ATM pathway would be disrupted and the ATR/ATRIP pathway partially impaired, resulting in TA frequencies higher than those found in the single mutants. Finally, in mei-41 tefu, mus-304 tefu, mei-41 rad50, and mei-41 mre11 double mutants, both pathways would be disrupted, resulting in very high frequencies of TAs (Ciapponi, 2006).
An aspect of the phenotype that is difficult to explain is the pattern of HOAP localization in different mutants and double mutants. In the mre11 and rad50 mutants, most mitotic telomeres are devoid of the HOAP protein. In nbs mutants, the frequency of telomeres with detectable HOAP accumulations is lower than in wild type but higher than in either the mre11 or the rad50 mutant, consistent with a reduced intranuclear concentration of the Rad50/Mre11 complex. tefu (ATM) and mei-41 (ATR) single mutants have normal HOAP concentrations at mitotic telomeres (Bi, 2004) but in mei-41 tefu double mutants telomeres lack the HOAP protein (Bi, 2005). Normal HOAP accumulations at mitotic telomeres were also found in Su(var)205 (HP1) and woc mutants that display very high frequencies of TAs, indicating that the presence of HOAP at chromosome ends is not sufficient to ensure proper telomere protection. An interpretation of these results is rather difficult, mainly because the current knowledge of the Drosophila telomere components is largely incomplete. HOAP localization at telomeres may be mediated, not only by the Rad50/Mre11 complex, but also by a factor that needs to be phosphorylated by both ATM and ATR. When this factor is not phosphorylated at its ATM-dependent site(s), telomeres are deprotected even if they accumulate normal amounts of HOAP. However, when this factor is not phosphorylated in both its ATM- and ATR-dependent sites, telomeres lose their ability to recruit HOAP. This factor cannot be HOAP itself, as recent work (Bi, 2005) has shown that the HOAP protein is not phosphorylated in a wild-type background (Ciapponi, 2006).
This study has shown that the Drosophila Nbs protein is required for transport of Rad50 in the nucleus and for prevention of telomere fusion and chromosome breakage. In addition, the results indicate that Nbs can act independently of the Rad50/Mre11 complex. Remarkably, all these features of the Drosophila Nbs protein are shared by its human counterpart. The ATLD disorder caused by hipomorphic mutations in the MRE11 gene and NBS have many overlapping features but are clinically distinct. NBS patients are characterized by microcephaly and developmental delay, while ATLD patients exhibit a mild ataxia telangiectasia-like phenotype with no microcephaly and no developmental delay. Given the functional similarities within Drosophila and human NBS proteins, it is likely that further studies on the Drosophila MRN complex will help to elucidate the molecular basis of the clinical differences between ATLD and NBS (Ciapponi, 2006 and references therein).
Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. The roles were examined of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. It is proposed that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms (Oikemus, 2006).
Alternative pre-mRNA splicing is a major mechanism utilized by eukaryotic organisms to expand their protein-coding capacity. To examine the role of cell signaling in regulating alternative splicing, the splicing of the Drosophila TAF1 pre-mRNA was analyzed. TAF1 encodes a subunit of TFIID, which is broadly required for RNA polymerase II transcription. TAF1 alternative splicing generates four mRNAs, TAF1-1, TAF1-2, TAF1-3, and TAF1-4, of which TAF1-2 and TAF1-4 encode proteins that directly bind DNA through AT hooks. TAF1 alternative splicing was regulated in a tissue-specific manner and in response to DNA damage induced by ionizing radiation or camptothecin. Pharmacological inhibitors and RNA interference were used to demonstrate that ionizing-radiation-induced upregulation of TAF1-3 and TAF1-4 splicing in S2 cells is mediated by the ATM (ataxia-telangiectasia mutated) DNA damage response kinase and checkpoint kinase 2 (CHK2), a known ATM substrate. Similarly, camptothecin-induced upregulation of TAF1-3 and TAF1-4 splicing is mediated by ATR (ATM-RAD3 related) and CHK1. These findings suggest that inducible TAF1 alternative splicing is a mechanism to regulate transcription in response to developmental or DNA damage signals and provide the first evidence that the ATM/CHK2 and ATR/CHK1 signaling pathways control gene expression by regulating alternative splicing (Katzenberger, 2006; Full text of article).
Reference names in red indicate recommended papers.
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