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).

The MRN complex plays critical roles in the response to DNA damage and telomere maintenance in both yeast and mammalian systems (D'Adda Di Fagagna, 2004; Stracker, 2004; Zhang, 2005; Lee, 2006). Hypomorphic mutations in the Nbs and Mre11 genes lead to the Nijmegen breakage syndrome (NBS) and to ataxia telangiectasia-like disorder (ATLD), respectively. NBS and ATLD share common features, including chromosome instability, radiation hypersensitivity, immunological disorders, and cancer predisposition. However, while ATLD is characterized by cerebellar degeneration resulting in ataxia, NBS is characterized by microcephaly and growth retardation (Digweed, 2004; Stracker, 2004). These clinical differences are likely to reflect functional differences between the Nbs and Mre11 components of the human MRN complex (Ciapponi, 2006).

The components of the MRN complex have multiple and complex interactions with the two conserved protein kinases ATM (Tel1 in Saccharomyces cerevisiae) and ATR (Mec1 in S. cerevisiae). For example, it has been shown that the mammalian MRN complex acts both upstream and downstream of ATM in the DNA damage response. The complex mediates both ATM activation and ATM kinase activity by facilitating its binding to substrates (Uziel, 2003; J.-H. Lee, 2004, 2005; Cerosaletti, 2006). The MRN complex also enhances several ATR-dependent phosphorylation events (Stiff, 2005; Zhong, 2005). Moreover, it has been shown that ATM and ATR can phosphorylate the same substrates, including the Nbs protein (reviewed by Shiloh, 2003). Finally, there is evidence that the components of the MRN complex can act independently in mediating ATM activation and phosphorylation events (Cerosaletti, 2004; Lee, 2004). Mutations in ATM and ATR result in the genetic disorders ataxia telangiectasia (AT) and Seckel syndrome, respectively. AT has stronger but similar clinical features to those of ATLD, while Seckel patients have features that overlap NBS, including pronounced microcephaly (Ciapponi, 2006 and references therein).

Studies in mammalian cells have shown that the ATM and ATR kinases and the MRN complex are required for both chromosome integrity and proper telomere function. However, although these proteins have been extensively studied at the biochemical level, their functional relationships in the maintenance of chromosome stability have not been determined. Progress in understanding such relationships has been hampered because null mutations in the genes encoding the components of the MRN complex lead to early lethality in vertebrates. In contrast, thanks to the maternal effect that characterizes Drosophila development, null mutations in the mre11, rad50, and nbs genes cause lethality at late larval stages, allowing cytological analysis of dividing neuroblasts in larval brains. Previous studies have shown that mutations in the Drosophila mre11, rad50, nbs, and tefu (ATM) genes cause both telomeric fusions and chromosome breakage and that tefu and mei-41 (ATR) control redundant pathways of telomere protection. This study explored the role of the Drosophila nbs gene in both telomere protection and the maintenance of chromosome integrity. The results indicate that the Nbs protein and the Mre11/Rad50 complex make distinct contributions to telomere protection but function in a single pathway to prevent chromosome breakage (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-41^29D tefu^atm6 double mutants and tefu^atm6 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 (Bi, 2005). 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 (Ritchie, 2000). 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 (Cerosaletti, 2004; Lee, 2004). Moreover, while NBS1 localization to the human telomeres is restricted to the S phase, the MRE11/RAD50 complex (Zhu, 2000) remains associated with telomeres throughout the cell cycle (Ciapponi, 2006).

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 (Stiff, 2005). 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 (Bi, 2005) (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 (Carney, 1998; Maser, 2001; Cerosaletti, 2004; Lee, 2004; Difilippantonio, 2005; Zhang, 2005). 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 (reviewed by Stracker, 2004). 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).

GENE STRUCTURE

cDNA clone length - 2557 bps

Exons - 4

Bases in 3' UTR - 73

PROTEIN STRUCTURE

Amino Acids - 827

Structural Domains

The Drosophila Nbs protein consists of 827 amino acids and displays 23% identity and 38% similarity with its human NBS1 counterpart but has little if any sequence similarity to its yeast counterpart Xrs2. Drosophila Nbs contains the three conserved domains present in most Nbs-like proteins: the fork head-associated (FHA) and the BRCA1 C terminus (BRCT) domains at its N terminus and an MRE11-binding domain at the C-terminal region (Iijima, 2004). Cytological analysis of brains from larvae homozygous for l(3)67BDp, a lethal mutation mapping close to the nbs gene, revealed the presence of telomeric fusions and chromosome breakage. Sequencing of the nbs gene in the l(3)67BDp mutant strain showed a 238-bp deletion in exon 3, resulting in a truncated Nbs protein of 518 amino acids. This nbs mutant allele is identical to that recently described by Bi (2005) and will be henceforth designated as nbs¹ (Ciapponi, 2006).

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 (Oikemus, 2006).

date revised: 30 December 2006

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