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).
Mutations in the nbs gene cause telomeric fusion, chromosome breakage, and apoptosis: Most individuals homozygous or hemizygous for the nbs1 mutation die at late larval or pupal stages. In a few cases, mutants were observed dying close to the time of eclosion. These pharate adults consistently showed small and rough eyes. This trait is usually associated with high levels of chromosome instability and cell death and was previously observed in rad50 mutants (Ciapponi, 2004). Consistent with these results, it was also observed that the imaginal discs of nbs1 mutants were often small and misshapen and displayed apoptotic cells very frequently when stained by acridine orange (Ciapponi, 2006).
To directly assess the role of the nbs gene in the maintenance of chromosome stability, DAPI-stained preparations of colchicine-treated larval brains were examined from nbs1 mutants. This analysis revealed that homozygous nbs1 brains display high frequencies of both telomeric associations (TAs) and chromosome breaks. TAs were classified as double telomeric associations (DTAs) when they joined a pair of sister telomeres with another pair, and as single telomeric associations (STAs) when they conjoined a single telomere with either its sister telomere or another nonsister telomere. Since DTAs and STAs are thought to be generated during G1 and S-G2, respectively, each type of TA was considered as a single fusion event. Telomeric associations observed in nbs1 mutants involved all chromosome ends, as observed in rad50, mre11, UbcD1, Su(var)205, cav, and woc mutants. Surprisingly, the frequency of TAs observed in nbs1 mutant brains is approximately one-half of that found in either rad50Δ5.1 or mre11DC null mutants. This is not a consequence of a residual wild-type function of the nbs1 mutant allele; nbs1/nbs1 homozygotes and nbs1/Df(3L)AC1 hemizygotes display comparable frequencies of TAs, suggesting that the nbs1 mutation is functionally null (Ciapponi, 2006).
Although nbs1 mutants have fewer TAs than rad50 and mre11 mutants, the three types of mutants display similar frequencies of chromosome breaks. These breaks involved either one or both sister chromatids and were characterized by the simultaneous presence of both the acentric and the centric fragment. Thus, they were genuine chromatid and isochromatid breaks and not the consequence of a severed anaphase bridge generated by a TA. To substantiate the finding that mutations in the nbs gene affect mitotic chromosome integrity, the sensitivity of nbs1 mutants to the induction of chromosome breakage by X rays was determined. Since the Drosophila ATM kinase encoded by the tefu gene is thought to be in the same telomere protection pathway with the Mre11/Rad50 complex (Bi, 2004), the radiosensitivity of tefuatm6 mutants was examined; previous studies have shown that tefuatm6 is a functionally null allele. This analysis showed that nbs1 mutants irradiated with 1 Gy of X rays exhibit ~10-fold more chromosome breaks than the Oregon-R control irradiated with the same dose. Similar results were previously obtained by irradiating mre11 and rad50 mutants. However, the X-ray sensitivity of tefuatm6 mutants was substantially lower; they displayed only 4-fold more chromosome breaks than the Oregon-R controls (Ciapponi, 2006).
Nbs is required for HP1/HOAP localization at telomeres: To define the role of the nbs gene in telomere protection, it was asked whether nbs is required for proper localization of HP1 and HOAP at chromosome ends. HP1 and HOAP form a complex that protects Drosophila telomeres from fusion events (reviewed by Cenci, 2005). Because HP1 cannot be readily detected at mitotic chromosome ends, HOAP localization was examined in both mitotic and polytene chromosomes and HP1 localization was examined only in polytene chromosomes. The analysis of mitotic chromosomes showed that mutations in the nbs gene affect HOAP localization at telomeres. In nbs1 mutants, 45% of the telomeres not involved in fusions displayed a clear HOAP signal, whereas in the Oregon-R control the frequency of labeled telomeres was 89%. Consistent with these results, only 25% of the polytene chromosome telomeres from nbs1 mutants showed low but detectable HOAP and HP1 accumulations, while the remaining 75% of mutant telomeres did not exhibit any HP1/HOAP signal. In contrast, 96% of the Oregon-R polytene chromosome ends displayed strong HOAP/HP1 signals. These results differ from those obtained with rad50 and mre11 mutants. In mre11 and rad50 mutants, the frequencies of mitotic telomeres with detectable HOAP signals were 5.5 and 18.2%, respectively, and the polytene chromosome telomeres were always devoid of HP1/HOAP signals (Ciapponi, 2004). Thus, mutations in the nbs gene have milder effects on HP1/HOAP accumulation at telomeres than mutations in either mre11 or rad50, consistent with the relatively low frequency of TAs observed in nbs mutants (Ciapponi, 2006).
Nbs is required for Rad50 localization in interphase nuclei: Previous work has shown that in Drosophila mre11 mutants the rad50 gene is normally transcribed but the Rad50 protein is not detectable by Western blotting, suggesting that Rad50 is unstable in the absence of its binding partner Mre11 (Ciapponi, 2004). These results are consistent with studies in human cells showing that in MRE11 mutant cells there is no detectable expression of RAD50 and the expression of NBS1 is reduced (Stewart, 1999; Uziel, 2003; Stracker, 2004). However, mammalian NBS1 is not essential for the stability of the MRE11 and RAD50 proteins but is required to transport (or retain) these proteins in the nucleus (Carney, 1998; Maser, 2001; Difilippantonio, 2005). It was thus asked whether Drosophila Nbs is required for the stability and nuclear localization of its binding partner Rad50. Because Drosophila ATM is also involved in telomere protection, it was also asked whether this kinase is required for normal behavior of Rad50. Mutations in the nbs1 and tefuatm6 genes do not affect the stability of the Rad50 protein. However, examination of nbs1 mutant brains immunostained for Rad50 revealed that the Nbs protein is required for nuclear localization of Rad50. Whereas in wild-type brains Rad50 is concentrated in the nucleus, in nbs1 mutant brains Rad50 accumulates is the cytoplasm. Nonetheless, in 42% of the prometaphase/metaphase figures from nbs1 mutant brains, Rad50 is associated with the chromosomes; in wild-type brains, 63% of the mitotic figures are immunostained by the anti-Rad50 antibody. These results suggest that, in late G2, part of the cytoplasmic pool of Rad50 can enter the nucleus in the absence of Nbs. In tefuatm6 mutants, Rad50 is regularly enriched at both the interphase nuclei and the chromosomes; 60% of prometaphase/metaphase figures from tefuatm6 brains are immunostained by the anti-Rad50 antibody (Ciapponi, 2006).
Functional relationships among the nbs, rad50, tefu, and mei-41 genes: The partial exclusion of Rad50 protein from the nucleus of nbs brain cells raises the possibility that the telomere fusion phenotype observed in nbs mutants is simply the consequence of a reduced intranuclear concentration of Rad50. However, it is also possible that Nbs plays additional and more direct roles in Drosophila telomere protection. To obtain insight into the telomere-related functions of the Nbs protein, the interaction between the nbs1 mutation and mutations in other genes known to be involved in telomere protection were analyzed. Previous studies have shown that the frequencies of TAs observed in rad50 mre11 and mre11 tefu double mutants are approximately the same as those found in flies homozygous for the single mutations (Bi, 2004; Ciapponi, 2004). These results indicate that mre11, rad50, and atm are involved in a single pathway of Drosophila telomere protection. In contrast, epistasis analysis with mei-41 (that encodes an ATR homolog) showed that both mei-41 mre11 and mei-41 tefu double mutants exhibit frequencies of TAs significantly higher than those observed in strains bearing the single mutations (Bi, 2005). Similar results were obtained when the epistasis analysis was performed with mus-304, the Drosophila gene that encodes the ATR-interacting protein ATRIP (Brodsky, 2000); mus-304 mre11 and mus-304 tefu double mutants showed more TAs than the single mutants (Bi, 2005). These findings indicate that the Drosophila telomere protection pathway identified by the Rad50/Mre11 complex and the ATM kinase is different from the pathway specified by the ATR/ATRIP complex. Moreover, since mei-41 and mus-304 mutant flies do not exhibit telomere fusions, it has been suggested (Bi, 2005) that the ATR-controlled pathway is redundant (Ciapponi, 2006).
To analyze the epistasis relationships of the nbs1 mutation, rad50Δ5.1 tefuatm6, mei-4129D nbs1, rad50Δ5.1 nbs1, and nbs1 tefuatm6 double mutants were constructed and their phenotypes were compared with those of the corresponding single mutants; the mei-4129D mutant allele is functionally null, like the rad50Δ5.1, nbs1, and tefuatm6 mutations. The results of this analysis showed that the rad50Δ5.1 tefuatm6 double mutant has a frequency of TAs comparable to those observed in the single mutants, consistent with the finding that mre11 and tefu function in the same pathway of telomere protection (Bi, 2005). In contrast, the mei-4129D nbs1 double mutant displayed a higher frequency of TAs than either single mutant, in agreement with previous findings of Bi (2005). However, it was also found that the levels of TAs in nbs1 tefuatm6 and rad50Δ5.1 nbs1 double mutants are significantly higher than those observed in the corresponding single mutants. These results indicate that nbs, rad50, and tefu do not function in a single pathway for the protection of Drosophila telomeres (Ciapponi, 2006).
Cytological analyses showed that the rad50Δ5.1 and nbs1 single mutants and the nbs1 rad50Δ5.1 double mutant all exhibit comparable frequencies of chromosome breaks. Thus, although Nbs and Mre11/Rad50 make distinct contributions to telomere protection, the three components of the MRN complex appear to act in a single pathway to prevent spontaneous chromosome breakage. However, nbs1 tefuatm6, rad50Δ5.1 tefuatm6, mei-4129D nbs1, and mei-4129D rad50Δ5.1 double mutants displayed frequencies of chromosome breaks significantly higher than those observed in the corresponding single mutants. These results indicate that the MRN complex and the kinases ATM and ATR function in different pathways to prevent chromosome breakage (Ciapponi, 2006).
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).
The ends of eukaryotic chromosomes can be protected from end-to-end fusion by two distinct mechanisms. In most organisms, sequence-specific DNA binding proteins recognize telomere-specific sequences and protect telomeres from the activity of DNA repair systems. However, genetic studies in Drosophila have demonstrated that telomeres can also be protected from end-to-end fusion by an epigenetic mechanism. The telomeric DNA of Drosophila chromosomes is composed of retrotransposons and repetitive telomere-associated sequences. Terminal deletion chromosomes that completely lack these sequences can be recovered and propagated. The telomeres of these chromosomes are protected from fusion and do not induce DNA damage responses such as cell cycle arrest or apoptosis. These observations demonstrate that a sequence-independent mechanism can protect Drosophila chromosomes from telomere fusion and suggest that a similar mechanism contributes to protection of normal telomeres. The sequence-independent inheritance of telomere protection is conceptually similar to the epigenetic regulation of centromere function in which the function of a chromosomal domain is usually associated with a specific set of sequences, but can be stably transferred to alternative sequences. Thus, Drosophila telomere protection can be grouped with centromere function and gene expression as processes that can be regulated by an epigenetic mechanism (Oikemus, 2006 and references therein).
Two chromatin-associated proteins, HP1 and HOAP, are required for telomere protection and localize to the telomeres of both normal and terminally deleted chromosomes. The role of HP1 in the epigenetic inheritance of chromatin modifications during cell division suggests that a similar activity may contribute to telomere protection. Inheritance of chromatin modifications is often initiated or stabilized by specific chromosome features, such as binding sites for sequence-specific DNA binding proteins or repeat sequences at centromeres. The stable inheritance of terminally deleted chromosomes over many generations indicates that a feature of telomeres other than telomere-specific sequences can recruit or maintain HP1 and HOAP at telomeres (Oikemus, 2006 and references therein).
One signature of telomeres that might contribute to HP1 and HOAP recruitment is the chromosome end itself. Studies in yeast and mammalian cells have demonstrated that telomere protection requires proteins that act at broken chromosome ends during the cellular response to DNA damage; these include the ATM and ATR protein kinases and the Mre11/Rad50/NBS1 (MRN) DNA repair complex. Analysis of cells lacking telomerase and ATM suggests that ATM plays a particularly critical role in cells with short telomeres. Such cells may be least able to utilize sequence-specific mechanisms for telomere protection. In both budding and fission yeast, the combined loss of the ATM and ATR pathways results in severe telomere protection defects. In mammalian cell culture, acute inhibition of the MRN complex or of the ATM and ATR kinases also induces telomere fusions. Drosophila homologs of most DNA damage response genes have been described. The Drosophila telomere fusion (tefu) gene is required to prevent fusions in proliferating cells and is encoded by the Drosophila homolog of ATM. Mutations in the Drosophila DNA damage response genes tefu, mre11, and rad50 lead to partial loss of telomere protection and reduced recruitment of HP1 and HOAP to telomeres. Thus, a DNA damage response pathway contributes to the protection of Drosophila telomeres; however, HP1 and HOAP can also mediate some degree of telomere protection in the absence of this pathway (Oikemus, 2006 and references therein).
Characterized here are the role of Nbs and the ATM and ATR DNA damage response pathways in the epigenetic protection of Drosophila telomeres. In humans, mutations in Nbs1 or ATM result in similar inherited syndromes. In both mammals and yeast, Nbs1 forms a complex with Mre11 and Rad50 (the MRN complex) that acts in the ATM pathway in response to DNA damage and is required for DNA repair and telomere function. This study demonstrates that Drosophila nbs is required for atm- and atr-dependent DNA damage responses including DNA repair. Drosophila mei-41 (the ATR homolog) and mus304 (the ATRIP homolog) act in parallel to the atm pathway in telomere protection; cells lacking both pathways fail to recruit HOAP to the telomeres of mitotic chromosomes and exhibit a severe telomere fusion phenotype. The telomere fusion defect in nbs mutants suggests that it acts in both the tefu and mei-41-mus304 telomere protection pathways and in the chromosome joining step. Advantage was taken of the severe telomere fusion phenotype in cells lacking both pathways to test the role of DNA damage response pathways in the sequence-independent protection of Drosophila telomeres. Analysis of these cells reveals that loss of telomeric HOAP and telomere fusions are not due to loss of telomeric sequences. Furthermore, these DNA damage response pathways are also required to protect the telomeres of terminally deleted chromosomes, directly demonstrating that the DNA damage response pathways are required for epigenetic regulation of telomere protection (Oikemus, 2006).
Drosophila Nbs is required for normal development: To identify genes that cooperate with atm/tefu in telomere protection, mutations were characterized in other Drosophila DNA damage response genes including nbs. Drosophila nbs encodes a protein with N-terminal FHA and BRCT domains and a short region of similarity to the Mre11 interaction domain encoded by human Nbs1. To identify mutations in Drosophila nbs, a collection of lethal mutations in the genetic region containing nbs were screened for pupal lethality and excess apoptosis during wing development, phenotypes previously described for Drosophila tefu, mre11, and rad50. Two mutations with these phenotypes failed to complement each other and their lethality was rescued by a transgene containing the nbs genomic region. Sequencing of these mutations revealed that l(3)67BDp1 (nbs1) contains a 238-base pair (bp) deletion and 1bp insertion that disrupts the open reading frame while l(3)67BDr1 (nbs2 ) introduces a stop codon that truncates the reading frame at amino acid position 685. Both of these mutations are predicted to eliminate the ability of Nbs to interact with Mre11 (Oikemus, 2006).
Flies homozygous for the nbs1 mutation die as pharate adults with rough eyes and missing or abnormal bristles. In tefu, mre11, or rad50 mutant flies, this phenotype is accompanied by increased genomic instability and apoptosis. tefu, but not mei-41 or mus304, is also required for rapid induction (within 4 h) of additional apoptosis by X-irradiation. The developing wings of nbs mutant animals also exhibit high levels of spontaneous apoptosis compared to wild-type animals. X-irradiation of these discs does not induce the rapid, large increase in apoptosis observed in wild-type discs. These results suggest that nbs acts in the tefu DNA damage response pathway to regulate apoptosis. Consistent with this conclusion, nbs tefu double mutant animals also exhibit high levels of apoptosis and fail to induce further apoptosis following irradiation (Oikemus, 2006).
To determine whether the elevated spontaneous apoptosis in these discs requires p53 or mnk (the Drosophila Chk2 homolog), apoptosis was examined in nbs p53 and nbs mnk double mutant discs. The Drosophila p53 and mnk genes are required for induction of apoptosis by X-irradiation. The Drosophila p53 gene was shown to be required for some, but not all, of the apoptosis observed in tefu mutant discs. Apoptosis is substantially reduced in nbs p53 and nbs mnk double mutant discs compared to nbs single mutants. Although p53 has been implicated in a variety of stress response pathways, Chk2 homologs appear to specifically function in DNA damage responses. Thus, these results suggest that the absence of nbs leads to apoptosis via activation of a DNA damage response. This response may be directly activated by unprotected telomeres or by chromosome breaks formed following telomere fusions. The regulation of this response must, however, differ from the regulation of apoptosis 4 h following X-irradiation, which requires wild-type nbs and tefu function (Oikemus, 2006).
DNA damage checkpoint and repair defects in nbs mutant cells: To further compare the function of nbs with tefu, mei-41, and mus304, cell cycle arrest and double-strand DNA break repair were examined. Previous studies have demonstrated that mei-41 is required for G2 arrest at both high (4,000 rads) and low (500 rads) doses of ionizing radiation, whereas tefu is primarily required at low doses, but not high doses. Dose-response curves confirm that mei-41 mutant discs fail to arrest in response to a range of irradiation doses whereas tefu mutant discs have a partial arrest phenotype at low doses, but not at 4,000 rads. Similar to mei-41, nbs is required for cell cycle arrest at all doses tested. These results demonstrate that nbs plays a tefu-independent role in cell cycle arrest and suggests that it acts in the atr-atrip pathway to mediate G2 arrest. A cell cycle arrest defect at low, but not high X-ray doses has been reported for tefu and mre11 mutant cells; however, it was observed that loss of nbs results in an arrest defect at high doses whereas loss of mre11 results in a partial arrest at high doses. nbs mus304 and nbs tefu double mutants also exhibit a cell cycle checkpoint phenotype at high doses; however, the reduced number of mitotic cells and smaller discs indicates that mitosis has been severely disrupted in the double mutants, making direct comparisons to single mutants problematic. It is concluded that nbs, mei-41, and mus304 are all essential for cell cycle arrest at high doses of X-irradiation whereas tefu is not (Oikemus, 2006).
mei-41, mus304, rad50, and mre11 are all required for DNA double-strand break (dsb) repair in Drosophila. The effect of nbs and tefu mutations on dsb formation and repair was examined in metaphase chromosomes from larval neuroblasts. Dsbs can arise as an indirect result of telomere fusion followed by chromosome breakage during mitosis. Broken chromosomes generated by this mechanism will generally retain their centromere. To analyze breaks due to mechanisms other than telomere fusion, the number of acentric chromosome fragments was analyzed. In untreated cells, these fragments may reflect a role in preventing formation of breaks during DNA replication. Both nbs and tefu are required to prevent the spontaneous accumulation of dsbs during normal cell cycles. However, nbs, mre11, mei-41, and mus304 mutant cells all have a more severe phenotype than tefu. Analysis of double mutant cells suggests that nbs and tefu act in parallel to mus304 to prevent accumulation of dsbs. nbs mutant cells also exhibit defective repair of X-irradiation-induced chromosome breaks, consistent with the role of Nbs in the MRN DNA repair complex and with analysis of Drosophila Mre11 and Rad50. Less severe dsb repair defects are seen following X-irradiation of tefu, mei-41, or mus304 mutant cells. Following irradiation, nbs tefu or nbs mus304 double mutant cells do not exhibit a greater defect than nbs single mutants, suggesting that nbs acts in both the tefu and mus304 pathways to mediate repair of induced DNA breaks (Oikemus, 2006).
In summary, nbs acts in both the tefu and mei-41-mus304 DNA damage response pathways. Double mutant analysis indicates that Drosophila nbs acts in common genetic pathways with tefu and mus304 during DNA repair. In addition, nbs has DNA damage response phenotypes in common with both tefu (defective induction of apoptosis) and mei-41-mus304 (defective induction of cell cycle arrest at high doses of X-irradiation). Although Nbs1 homologs are best known for their roles in DNA repair and signaling in the ATM pathway, human Nbs1 is also required for signaling by the ATR pathway. Thus, nbs has a conserved role in ATM- and ATR-ATRIP-dependent DNA damage responses (Oikemus, 2006).
Two DNA damage response pathways contribute to telomere protection: Metaphase larval neuroblasts were also used to examine the roles of different DNA damage response genes in telomere protection. Previous studies have demonstrated that Drosophila tefu, mre11, and rad50 mutant cells have a partial defect in telomere protection. Consistent with these results, nbs mutant animals exhibit a high frequency of cells with one or more fusions. These fusions are observed during both metaphase and anaphase. nbs tefu double mutant cells exhibit similar fusion rates as tefu single mutants, indicating that these genes act in a common telomere protection pathway. These results are consistent with results in Drosophila and other organisms, indicating that ATM and components of the MRN complex act in a common telomere protection pathway. Downstream targets of ATM in the mammalian DNA damage response pathway include Nbs1 and the checkpoint kinases CHK1 and CHK2. The Drosophila homologs of these kinases are encoded by the grp and mnk genes and are required for DNA damage-induced apoptosis and cell cycle arrest. Both telomere protection and chromosome break repair are normal in grp mnk double mutant cells, indicating that other targets of Drosophila tefu and nbs are responsible for their telomere protection and DNA repair functions (Oikemus, 2006).
Compared with mutations in the genes that encode the telomere protection proteins HOAP and HP1, mutations in tefu, nbs, mre11, and rad50 exhibit a significantly lower frequency of telomere fusions, indicating that there may be a tefu-nbs-independent pathway for telomere protection. In mammals, the ATR checkpoint kinase is recruited to sites of DNA damage by ATRIP and acts in parallel to the ATM kinase in the DNA damage response. In budding and fission yeast, disruption of both atm/atr homologs results in loss of telomere protection. Mutations in mei-41 or mus304 do not result in telomere protection defects. However, nbs mus304, tefu mus304, and tefu mei-41 double mutant animals all show higher rates of telomere fusion than the corresponding single mutants indicating that the mei-41-mus304 pathway acts in parallel to a tefu-nbs pathway to mediate telomere protection. The higher fusion frequency in tefu mei-41 double mutants compared with tefu mus304 double mutants may indicate that there is a small amount of mei-41 activity in the absence of mus304 (Oikemus, 2006).
DNA damage response genes regulate telomeric HOAP: The formation of telomere fusions requires two steps: (1) the failure of telomeric protein complexes, such as HP1-HOAP, to prevent telomeric DNA ends from being recognized as damage-induced ends and (2) the subsequent ligation of unprotected telomeres by DNA repair systems. To probe the role of DNA damage response pathways in the first step, HOAP localization was examined in individual mitotic cells (neuroblasts). Previously, it was shown that levels of the telomere protection proteins HP1 and HOAP are reduced at the telomeres of polytene chromosomes from tefu salivary gland cells, but that telomeric HOAP is not strongly reduced in mitotic chromosomes from neuroblasts; these results suggest that in the absence of tefu, neuroblasts utilize an alternative mechanism for HOAP localization. In contrast, both salivary glands and neuroblasts required mre11 and rad50 for normal HOAP localization (Oikemus, 2006).
The frequency of neuroblast telomeres with HOAP staining and the intensity of staining at those telomeres were examined in wild-type and mutant cells. Measurements of fluorescence intensity can be used to demonstrate that HOAP levels at individual telomeres are reproducibly increased or decreased in different genotypes. (However, it is noted that there may not be a linear relationship between the percent change of fluorescence observed and the percent change of telomeric HOAP protein levels.) Most wild-type, tefu, or mus304 mutant metaphase cells are HOAP positive; between 77% and 94% of these cells had HOAP signals at chromosome ends. Among the HOAP positive cells, between 66% and 72% of telomeres stained for HOAP. The fluorescence intensity of HOAP staining was similar at the telomeres of each of the major chromosome arms in both wild-type and mus304 mutant cells. However, the average intensity of HOAP staining was elevated in tefu mutant cells, indicating that although HOAP is still recruited to telomeres, the mechanism regulating HOAP levels at telomeres may be perturbed. A more severe effect on HOAP localization was observed in nbs mutant metaphases, with only 44% of metaphases displaying HOAP signals and only 30% of the telomeres in those cells staining for HOAP. This phenotype is similar to that reported for mre11 and rad50 mutant neuroblasts. At the few HOAP-positive telomeres that are present in nbs mutant cells, HOAP fluorescence staining intensity was elevated compared to wild type, similar to the HOAP staining at tefu mutant telomeres. Together with the genetic data indicating that tefu and nbs act in a common telomere protection pathway, these results suggest that an alternative pathway can maintain HOAP levels at telomeres, but that this pathway is much less efficient in nbs mutant cells (Oikemus, 2006).
Quantification of HOAP telomeric signals: Since mus304 nbs, mus304 tefu, and mei-41 tefu double mutant cells have more severe telomere fusion phenotypes than nbs or tefu single mutants, the mei-41-mus304 pathway is a clear candidate to recruit HOAP to telomeres in the absence of tefu or nbs. mus304 single mutant animals do not exhibit a defect in either the frequency of HOAP-positive telomeres or the intensity of HOAP staining at those telomeres. In contrast, no telomeric HOAP staining was detected in mus304 tefu or mus304 nbs double mutant cells. Thus, the mei-41-mus304 pathway partially compensates for the absence of tefu, limiting the severity of the tefu telomere fusion phenotype. Cells lacking both pathways exhibit loss of telomeric HOAP and a severe telomere fusion phenotype. In a report published while this work was in preparation, Bi (2005) also found that disruption of the Drosophila atm and atr pathways results in a high frequency of telomere fusions and loss of telomeric HOAP (Oikemus, 2006).
These results support the model that the Drosophila tefu and mei-41-mus304 DNA damage response pathways mediate telomere protection by recruiting or maintaining HOAP at telomeres. The more severe HOAP localization phenotype of nbs mutant cells compared with tefu mutant cells indicates that nbs has a tefu-independent role in telomere protection. As described above, the common DNA repair and damage response phenotypes of nbs with mei-41 and mus304 indicate that nbs also acts in the mei-41-mus304 DNA damage response pathway. Thus, one explanation for the lower frequency of HOAP positive telomeres in nbs compared to tefu mutant cells is that nbs mutations both disrupt the tefu telomere protection function and partially disable a compensatory telomere protection pathway mediated by mei-41-mus304 (Oikemus, 2006).
There is a good correlation between the levels of telomeric HOAP and the frequency of telomere fusion, except in nbs, mre11, and rad50 mutants. In yeast and mammalian cells, some DNA repair genes are required to both maintain telomere protection and to promote joining of unprotected telomeres. The observed telomere fusion frequency in nbs mutant cells may reflect the combined effects of decreased telomere protection and inefficient fusion of unprotected telomeres. Although the loss of nbs has a more severe effect than tefu on telomeric HOAP, nbs and tefu mutant cells have similar telomere fusion frequencies. nbs mutations have a more severe effect on repair of DNA breaks, suggesting that nbs mutant cells may also have reduced joining of unprotected telomeres. Consistent with a role for nbs in fusion of unprotected telomeres, nbs mus304 mutant cells have a lower telomere fusion frequency than tefu mus304 mutant cells, despite undetectable levels of telomeric HOAP in both genotypes. Similarly, nbs cav double mutant cells have a lower telomere fusion frequency than tefu cav double mutant cells (Oikemus, 2006).
In summary, DNA damage response genes are essential for the telomeric localization of the protection protein HOAP. Analysis of DNA repair, telomere fusions and HOAP localization suggests that the telomere fusion frequency reflects a combination of defective protection and reduced fusion of unprotected chromosomes. Although these results do not rule out the possibility that DNA damage response genes are also required for modification of HP1 and HOAP complexes at telomeres, they strongly suggest that recruitment or maintenance of these complexes to telomeres is critical for telomere protection (Oikemus, 2006).
DNA damage response pathways are required for epigenetic protection of telomeres: In many organisms, telomere-specific sequences are required to recruit proteins that prevent chromosome end fusion. Loss of these sequences in cells that do not express telomerase or that are mutant for DNA damage response genes can result in telomere fusions. However, in Drosophila, the stable protection of terminally deleted chromosomes from telomere fusion suggests that a sequence-independent mechanism acts to protect the telomeres of normal chromosomes. Given the requirement of the tefu and mei-41-mus304 DNA damage response pathways for telomere protection, it is proposef that recognition of chromosome ends contributes to this epigenetic phenomenon. One prediction of this model is that cells lacking these pathways will exhibit telomere fusion without loss of telomeric DNA sequences such as HeT-A. HeT-A sequences should not be lost simply as a secondary effect of unprotected telomeres since telomere fusions in cells lacking HP1 function still retain these sequences. A second prediction is that terminal deletion chromosomes lacking telomeric sequences will still fuse in the absence of the DNA damage response pathways. This observation would rule out the possibility that the epigenetic mechanism for protection of terminal deletions utilizes an alternative mechanism to recruit HP1 and HOAP that is independent of the DNA damage response pathways. These predictions can be evaluated in animals with the extreme telomere fusion phenotype associated with loss of both the tefu and mei-41-mus304 DNA damage response pathways (Oikemus, 2006).
To test the first prediction, the telomere-specific retrotransposon HeT-A was analyzed at individual telomeres of DNA damage response defective cells by fluorescence in situ hybridization. Measurements of fluorescence intensity can be used to demonstrate that HeT-A levels at individual telomeres are reproducibly increased or decreased in different genotypes. (However, it is noted that there may not be a linear relationship between the percent change of fluorescence observed and the percent change of telomeric HeT-A DNA.) Previously, telomere fusions in tefu mutant cells were shown to retain at least some HeT-A sequences. However, these studies only examined mutants with mild telomere fusion phenotypes and were less thorough than the analysis presented in this study. Because the number of HeT-A copies per telomere can vary between strains, particularly in strains with altered HP1 function, HeT-A signals at free chromosome ends in homozygous mutant animals were compared to chromosome fusion sites in the same cells and to free chromosome ends in an appropriate heterozygous parental strain. HeT-A is still present at free telomeres and at chromosome fusion sites in tefu, nbs, tefu mus304, and nbs mus304 homozygous mutant cells. For each genotype, both the frequency and intensity of HeT-A staining at chromosome fusions is equal to or greater than that observed at the free chromosome ends, indicating that loss of telomere-specific sequences does not correlate with telomere fusion in cells with defective DNA damage response pathways. Note that if a HeT-A-positive telomere fuses with another HeT-A-positive telomere, the intensity of staining will increase; if it fuses with a HeT-A-negative telomere or a chromosome break, the intensity should be the same. Different genotypes exhibit different relative intensities of HeT-A staining at chromosome fusions compared to free ends. These differences may reflect different frequencies of telomere-telomere fusions versus telomere-break fusions or differences in the precise mechanism of telomere fusion. Nonetheless, the observation that the staining intensity at fusions is equal to or greater than the intensity at free chromosome ends demonstrates that loss of these sequences is not required for fusion in any of these genotypes (Oikemus, 2006).
Percent chromosomes with HeT-A staining: The frequency and intensity of HeT-A staining was also compared at the free chromosome ends of mutant cells and the corresponding parental strain. The frequency of HeT-A staining at chromosome ends in homozygous nbs, nbs mus304, and tefu mus304 mutant cells is lower than in cells from the corresponding heterozygous strains. Although this decrease could reflect removal of telomeric sequences in homozygous mutant animals, two other factors are likely to contribute. First, defective DNA repair generates chromosome ends without telomeric sequences. Several of these mutations result in high levels of spontaneous breaks. Second, progression of cells with telomere fusions through mitosis generates anaphase bridges and chromosome breaks via the fusion/bridge/break cycle. In one example, an internal site of HeT-A (the original fusion site) was adjacent to a chromosome end without HeT-A (the break site). Chromosome ends with adjacent internal HeT-A sites are found in all mutant cells with telomere fusions. The overall frequency of breaks resulting from fusion is underestimated by this analysis since some broken chromosomes will not include the original fusion site. Thus, chromosome breaks can account for the increased number of ends without HeT-A staining. However, at those chromosome ends that are HeT-A positive, the intensity of staining is equal to or greater than in the corresponding heterozygous cells. Combined with the analysis of HeT-A staining at fusion sites, these results indicate that the fusion phenotype of single or double mutants in the DNA damage response pathways is not due to loss of telomeric sequences (Oikemus, 2006).
A second prediction of the end-recognition model for Drosophila telomere protection is that both normal and terminally deleted chromosomes will exhibit similar frequencies of fusion in cells lacking the DNA damage response pathways. The stable protection of terminally deleted chromosomes in wild-type cells suggests that the telomeres of normal chromosomes are also protected by sequence-independent mechanism; however, it is also possible that terminally deleted chromosomes acquire an alternative mechanism for telomere protection and that the DNA damage response pathways must act in conjunction with a sequence-specific mechanism. To address this possibility, fusion rates of a normal and a terminally deleted X chromosome were examined in tefu mus304 double mutant cells. Previous experiments have demonstrated that the telomere protection gene UbcD1 is required to prevent fusion of terminally deleted chromosomes. In tefu mus304 double mutant cells, a normal and a terminally deleted X chromosome fused to the sister or to heterologous chromosomes at a high frequency. The fusion frequency is similar but lower than observed with a normal X chromosome; this difference may indicate that the terminally deleted chromosome is slightly less sensitive to the loss of DNA damage signaling pathways. Nonetheless, the frequent fusion of terminally deleted chromosomes in tefu mus304 double mutant cells directly demonstrates that the DNA damage response pathways act in an epigenetic mechanism for telomere protection (Oikemus, 2006).
It is concluded DNA damage response genes have evolutionarily conserved roles in telomere function. Unprotected telomeres are recognized by these pathways and elicit a variety of cellular responses including apoptosis and end-to-end fusion of chromosomes. However, these same pathways are also required to promote telomere protection. The Drosophila atm and atr-atrip DNA damage response pathways act in an epigenetic mechanism to mediate telomere protection. In cells lacking both pathways, the chromatin-associated protein HOAP is not recruited to telomeres and both normal and terminally deleted chromosomes undergo fusion at a high frequency. Furthermore, fusion of normal telomeres occurs without loss of telomere-specific sequences. Taken together, these results support an end-recognition model in which DNA damage response proteins recognize a DNA structure at the chromosome end and recruit or stabilize the telomere protection proteins HP1 and HOAP at telomeres; in turn, these proteins act to prevent the ligation of chromosome ends by DNA repair enzymes and the activation of p53-dependent apoptosis. In other organisms, a similar epigenetic mechanism may act in conjunction with sequence-specific protection mechanisms or may be utilized to promote protection of critically short telomeres, which are least able to utilize sequence-specific binding proteins (Oikemus, 2006).
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date revised: 30 December 2006
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