Hus1-like: Biological Overview | References
Gene name - Hus1-like
Cytological map position - 82D6-82D6
Function - signaling
Symbol - Hus1-like
FlyBase ID: FBgn0026417
Genetic map position - 3R: 634,084..637,783 [-]
Classification - Hus1-like protein
Cellular location - nuclear
|Recent literature||Shaposhnikov, M., Proshkina, E., Shilova, L., Zhavoronkov, A. and Moskalev, A. (2015). Lifespan and stress resistance in Drosophila with overexpressed DNA repair genes. Sci Rep 5: 15299. PubMed ID: 26477511
DNA repair declines with age and correlates with longevity in many animal species. This study investigated the effects of GAL4-induced overexpression of genes implicated in DNA repair on lifespan and resistance to stress factors in Drosophila melanogaster. Stress factors included hyperthermia, oxidative stress, and starvation. Overexpression was either constitutive or conditional and either ubiquitous or tissue-specific (nervous system). Overexpressed genes included those involved in recognition of DNA damage (homologs of HUS1, CHK2), nucleotide and base excision repair (homologs of XPF, XPC and AP-endonuclease-1), and repair of double-stranded DNA breaks (homologs of BRCA2, XRCC3, KU80 and WRNexo). The overexpression of different DNA repair genes led to both positive and negative effects on lifespan and stress resistance. Effects were dependent on GAL4 driver, stage of induction, sex, and role of the gene in the DNA repair process. While the constitutive/neuron-specific and conditional/ubiquitous overexpression of DNA repair genes negatively impacted lifespan and stress resistance, the constitutive/ubiquitous and conditional/neuron-specific overexpression of Hus1, mnk, mei-9, mus210, and WRNexo had beneficial effects. This study demonstrates for the first time the effects of overexpression of these DNA repair genes on both lifespan and stress resistance in D. melanogaster.
The checkpoint proteins Rad9, Rad1 and Hus1 form a clamp-like complex which plays a central role in the DNA-damage-induced checkpoint response. This study addresses the function of the 9-1-1 complex in Drosophila. This study analyzed the meiotic and somatic requirements of hus1. For that purpose, a null allele of hus1 was created by imprecise excision of a P element found 2 kb from the 3' of the hus1 gene. It was found that hus1 mutant flies are viable, but the females are sterile. hus1 mutant flies are sensitive to hydroxyurea and methyl methanesulfonate but not to X-rays, suggesting that hus1 is required for the activation of an S-phase checkpoint. It was also found that hus1 is not required for the G2-M checkpoint and for post-irradiation induction of apoptosis. Subsequently the role of hus1 in activation of the meiotic checkpoint was studied and it was found that the hus1 mutation suppresses the dorsal-ventral pattering defects caused by mutants in DNA repair enzymes. Interestingly, it was found that the hus1 mutant exhibits similar oocyte nuclear defects as those produced by mutations in DNA repair enzymes. These results demonstrate that hus1 is essential for the activation of the meiotic checkpoint and that hus1 is also required for the organization of the oocyte DNA, a function that might be independent of the meiotic checkpoint (Abdu, 2007).
In many cell types specific checkpoint mechanisms exist that monitor the integrity of the chromosomes. These checkpoints coordinate cell cycle progression with DNA repair to ensure the distribution of accurate copies of the genome to daughter cells. If left unrepaired, chromosomal lesions can lead to genomic instability, a major contributing factor in the development of cancer and other genetic diseases. The DNA damage checkpoint response system involves a signal transduction pathway consisting of sensors, transducers and effectors. Damaged DNA is initially sensed by a complex consisting of Hus1, Rad1 and Rad9 and the associated protein Rad17. Computer modeling suggests that Rad9, Hus1 and Rad1 (also called the 9-1-1 complex) form a doughnut-like heteromeric proliferating cell nuclear antigen (PCNA) complex that can be loaded directly onto damaged DNA (Rauen, 2000; Venclovas, 2000; Bermudez, 2003). The signal transducers comprise four sets of conserved protein families. One family is composed of ATM and ATM-Rad3-related (ATR) proteins. Downstream of these proteins are two sets of checkpoint kinases, the Chk1 and the Chk2 kinases and their homologues. The fourth conserved family is that of the BRCT-repeat-containing proteins. Finally, a diverse range of effector proteins execute the function of the DNA damage response, which can lead to cell cycle arrest, apoptosis or activation of the DNA repair machinery (Abdu, 2007).
A number of checkpoint proteins that were initially characterized in budding and fission yeast, have counterparts in Drosophila, Caenorhabditis elegans and mammals, demonstrating the conservation of these surveillance mechanisms. Several checkpoint proteins have been characterized in Drosophila, mainly the ATM and/or ATR and the Chk1 and/or Chk2 transducer family of proteins. An ATR homolog in Drosophila is encoded by mei-41. mei-41 is essential for the DNA damage checkpoint in larval imaginal discs and neuroblasts and for the DNA replication checkpoint in the embryo. mei-41 also has an essential role during early nuclear divisions in embryos. In addition, mei-41 plays important roles during meiosis, where it has been proposed to monitor double-strand-break repair during meiotic crossing over, to regulate the progression of prophase I, and to enforce the metaphase I delay observed at the end of oogenesis. Drosophila ATM and ATR orthologs are required for different functions. In Drosophila, recognition of chromosome ends by ATM prevents telomere fusion and apoptosis, by recruiting chromatin-modifying complexes to telomeres. It has also been shown that dATM and mei-41 have temporally distinct roles in G2 arrest after irradiation (Abdu, 2007 and references therein).
A Chk1 homolog in Drosophila is encoded by grapes. Similarly to mei-41, grapes is required to delay the entry into mitosis in larval imaginal discs after irradiation and to delay the entry into mitosis after incomplete DNA replication in the embryo. The Drosophila Chk2 homolog [also designated loki (lok) or Dmnk] regulates multiple DNA repair and apoptotic pathways following DNA damage. It plays an important role in a mitotic checkpoint in syncytial embryos and is important in centrosome inactivation. Like Mei-41, DmChk2 also plays an important role in monitoring double-strand-break repair during meiotic crossing over. Although understanding of the role of DNA damage proteins is increasing, there is still a lack of information on the function of the Drosophila PCNA-like complex, 9-1-1 (Abdu, 2007).
This study analyzes the interaction between the Drosophila Rad9, Hus1 and Rad1 proteins using a yeast two-hybrid assay. Interaction was detected between Hus1 and Rad9 or Rad1, but not between Rad9 and Rad1. This analysis focuses on the meiotic and somatic requirement of Hus1. A null allele of hus1 was created by imprecise excision of a P element. Sensitivity was observed of hus1 mutants to hydroxyurea (HU) and to methyl methanesulfonate (MMS) but not to X-ray irradiation. This implies that hus1 is required for the DNA replication checkpoint. The ability of a mutation in hus1 to suppress the eggshell polarity defects detected in mutants affecting double strand DNA repair enzymes demonstrates that it is required for the activation of the meiotic checkpoint that leads to a strong reduction in the translation of gurken mRNA. The similarity of the defects in the organization of the DNA in the oocyte nucleus between hus1 mutants and mutations in DNA repair enzymes suggest that hus1 may act upstream of the DNA repair machinery (Abdu, 2007).
Hus1 interacted with Rad1 or Rad9, however no interaction between Rad1 and Rad9 was observed. The yeast two hybrid system may not be sensitive enough to pick up the interaction, since possibly the interaction between these two proteins is more transient than the interaction between Hus1 and the other proteins. Similar results were seen in C. elegans (Hofmann, 2002) where these proteins interact in vivo (Abdu, 2007).
Several studies have investigated the role of hus1 during development. In mouse, Hus1 is an essential gene, since its inactivation results in mid-gestational embryonic lethality due to widespread apoptosis. Also, loss of Hus1 leads to an accumulation of genome damage (Weiss, 2000). Both fission and budding yeast that lack hus1 fail to arrest the cell cycle after DNA damage or blockage of DNA synthesis (Enoch, 1992; Weinert, 1994; Kostrub, 1998). In C. elegans, although hus1 is not absolutely required for embryonic survival, a significant fraction of hus1 embryos die during embryogenesis, probably because of genomic instability. Also, hus1 mutants fail to induce apoptosis and proliferation arrest following DNA damage and show increased sensitivity to DNA damage-induced lethality (Hofmann, 2002). The Drosophila hus1 is not an essential gene, although similarly to in C. elegans, the female mutants are sterile; this is probably due to the defects in the organization of the DNA within the oocyte nucleus (Abdu, 2007).
In order to test for a requirement for Drosophila hus1 in response to genotoxic stress, the survival rates of flies were studied after exposure to HU, MMS and IR during larval development; hus1 mutant flies were sensitive only to HU and MMS. This result suggests that hus1 is required for the activation of an S-phase checkpoint. It is possible that this requirement is due to a role of hus1 in Chk1 (Grapes) activation after genotoxic stresses that affect S phase. In yeast and mice, hus1 has been shown to be required for Chk1 activation after replicative stress (Bao, 2004; Weiss, 2003) In Drosophila, mutations affecting grapes and mei-41 fail to show a decrease in BrdU-staining after irradiation, indicating a defect in an S-phase checkpoint, and it would, therefore, seem likely that Hus1 signals to activate Grapes (Chk1) through Mei-41 during S phase. An increase in aneuploid nuclei in hus1 mutants after MMS treatment is consistent with a requirement for hus1 in the response to DNA damage caused during S phase as it has been suggested in budding yeast that spontaneous chromosome loss is primarily suppressed by functional S-phase checkpoints and not by G2-M checkpoints. Since the hus1 mutant still exhibits cell cycle arrest after irradiation, hus1 does not seem to be required for the G2-M checkpoint that is dependent on Mei-41. Rather, the data suggest that hus1 is only required for certain DNA damage situations, and not for the same spectrum as Mei-41 (Abdu, 2007).
Activation of a meiotic checkpoint, also known as the pachytene checkpoint, in response to the persistence of unrepaired DSBs appears to be a conserved regulatory feature common to yeast, worms, flies and vertebrates. However, a requirement for the 9-1-1 complex in activation of the meiotic checkpoint has only been demonstrated in budding yeast. It was found that mutations in the yeast Hus1 homologue, Mec3, and the Rad1 homologue, Ddc-1, abolish the pachytene checkpoint in budding yeast (Hong, 2000). In Drosophila, mutations in the spindle class of double-strand break (DSB) DNA repair enzymes, such as spn-A (RAD51), spn-B (XRCC3), spn-C (HEL308), spn-D (Rad51C) and okra (Dmrad54), affect dorsal-ventral patterning in Drosophila oogenesis and cause defects in the appearance of the oocyte nucleus. Interestingly, the defects in dorsal-ventral patterning and in the oocyte nucleus are dependent upon activation of a meiotic checkpoint (Ghabrial, 1999; Abdu, 2002; Staeva-Vieira, 2003). hus1 mutants are able to suppress the dorsal-ventral defects but not the defects in the organization of the DNA within the oocyte nucleus. The suppression of the DV patterning defects of spn-B mutants demonstrates that during meiosis Hus1 is required for the meiotic checkpoint in response to persistent DSBs. This finding is interesting in light of the fact that hus1 mutants are not IR sensitive or defective in somatic checkpoints after irradiation. Either there is a fundamental difference between germline and somatic DSBs and DSB response machinery, or the non-DSB lesions created during irradiation that are not present during meiotic recombination serve as triggers for an alternative sensing mechanism that does not require hus1 and is therefore still able to activate a checkpoint mechanism. The inability of hus1 mutants to suppress the karyosome phenotype along with the hus1 mutant phenotype by itself, demonstrates that hus1 is required for the organization of the oocyte DNA, a function that might be independent of the meiotic checkpoint (Abdu, 2007).
In this study it has been shown that Drosophila Hus1 is required for both the meiotic and somatic DNA damage responses as well as demonstrating a novel role of Hus1 in the organization of the oocyte nuclear DNA. Whereas some of the functions of Hus1, such as binding to 9-1-1 complex members and an essential role in surviving genotoxic stress during S phase, appear to be conserved across the species studied so far, some Hus1 functions seem to be less conserved. In contrast to the findings in yeast, worms and mouse, fly Hus1 is not required for survival after irradiation. Finally, the karyosome defect of hus1 mutants demonstrates a role for Drosophila Hus1 in organizing the chromosomal DNA of the meiotic nucleus (Abdu, 2007).
DNA replication stress triggers the activation of Checkpoint Kinase 1 (Chk1) in a pathway that requires the independent chromatin loading of the ATRIP-ATR (ATR-interacting protein/ATM [ataxia-telangiectasia mutated]-Rad3-related kinase) complex and the Rad9-Hus1-Rad1 (9-1-1) clamp. Rad9s role in Chk1 activation is to bind TopBP1, which stimulates ATR-mediated Chk1 phosphorylation via TopBP1s activation domain (AD), a domain that binds and activates ATR. Notably, fusion of the AD to proliferating cell nuclear antigen (PCNA) or histone H2B bypasses the requirement for the 9-1-1 clamp, indicating that the 9-1-1 clamps primary role in activating Chk1 is to localize the AD to a stalled replication fork (Delacroix, 2007).
Genotoxic damage activates conserved checkpoint signaling pathways that maintain genomic stability by regulating cell cycle progression, triggering apoptosis, and influencing DNA repair. One pathway that is potently activated by replication stress leads to activation of Checkpoint Kinase 1 (Chk1), which promotes cell survival by blocking the firing of replication origins, preventing entry into mitosis, stabilizing stalled replication forks, and facilitating DNA repair. This pathway is initiated when the replicative DNA polymerases stall and large tracts of single-stranded DNA are created by the uncoupling of the replicative helicase from the advancing replication fork. The single-stranded DNA is then coated by replication protein A (RPA), which signals the independent recruitment of two checkpoint complexes: the ataxia-telangiectasia mutated (ATM)-Rad3-related kinase-ATR-interacting protein (ATR-ATRIP) complex and the Rad9-Hus1-Rad1 (9-1-1) complex. The ATRIP-ATR complex is bound to DNA by a direct interaction between ATRIP and RPA. In contrast, loading of the 9-1-1 complex requires several steps. First, DNA polymerase is recruited, which in turn recruits the clamp loader, Rad17-replication factor C (RFC) (You, 2002; Lee, 2003; Byun, 2005). Second, the Rad17-RFC then loads the proliferating cell nuclear antigen (PCNA)-like 9-1-1 complex onto chromatin in a reaction that is analogous to the loading of PCNA onto sites of DNA replication (Bermudez, 2003; Ellison, 2003). Although the binding of the ATRIP-ATR complex and the loading of the 9-1-1 complex occur independently of one another, both events are essential for optimal ATR-mediated Chk1 phosphorylation and activation (Melo, 2002; Delacroix, 2007 and references therein).
Despite the tremendous progress that has been made in deciphering the biochemical functions of the 9-1-1 complex and the in-depth understanding of the signals that lead to the loading of the 9-1-1 clamp, it has remained unclear how the chromatin-bound 9-1-1 complex initiates and propagates the Chk1-activating signal. Several studies have demonstrated that Rad9 orthologs in Schizosaccharomyces pombe, Saccharomyces cerevisiae, and humans interact with their respective TopBP1 orthologs (Cut4, Dpb11, and TopBP1). However, the significance of the Rad9-TopBP1 interaction in 9-1-1 function has not been explored. This study shows that the role of the 9-1-1 clamp is to recruit TopBP1, which then triggers ATR-mediated Chk1 phosphorylation. Thus, TopBP1 is a molecular bridge that links the independently recruited 9-1-1 and ATRIP-ATR complexes, leading to checkpoint activation (Delacroix, 2007).
The checkpoint proteins, Rad9, Rad1, and Hus1 (9-1-1), form a complex which plays a central role in the DNA damage-induced checkpoint response. Drosophila hus1 has been shown to be essential for activation of the meiotic checkpoint elicited in double-strand DNA break (DSB) repair enzyme mutants. The hus1 mutant exhibits similar oocyte nuclear defects as those produced by mutations in these repair enzymes, suggesting that hus1 plays a role independent of its meiotic checkpoint activity. This study further analyzed the function of hus1 during meiosis. The synaptonemal complex (SC) was found to disassemble abnormally in hus1 mutants. Oocyte nuclear and SC defects of hus1 mutants can be suppressed by blocking the formation of DSBs, implying that the hus1 oocyte nuclear defects depend upon DSBs. Interestingly, eliminating checkpoint activity through mutations in DmChk2 but not mei-41 suppress the oocyte nucleus and SC defects of hus1, suggesting that these processes are dependent upon DmChk2 checkpoint activity. Moreover, in hus1, DSBs that form during meiosis are not processed efficiently, and this defect is not suppressed by a mutation in DmChk2. A genetic interaction was found between hus1 and the Drosophila brca2 homologue, which was shown to participate in DNA repair during meiosis. Together, these results imply that hus1 is required for repair of DSBs during meiotic recombination (Peretz, 2009)
When the integrity of the genetic material is compromised, the cell activates checkpoints that inhibit cell cycle progression, allowing for repair of the damaged DNA or, if unsuccessful, lead to cell death. The DNA damage checkpoint response involves a signal transduction pathway consisting of sensors, transducers and effectors. Hus1, Rad1 and Rad9 and the associated protein, Rad17 are thought to act as a sensor complex. The signal is transduced by ATM and ATM-Rad3-related (ATR) proteins along with Chk1 and Chk2 kinases. A wide range of effector proteins influence cellular fate following the DNA damage, among these are cell cycle arrest, apoptosis or activation of the DNA repair machinery. Various checkpoints exist, with each addressing a different type of DNA damage through the use of a specific set of signal transduction proteins (Peretz, 2009)
A meiotic recombination checkpoint, also known as the 'pachytene checkpoint' has been characterized in yeast. Meiotic recombination initiates with the generation of DNA double-strand breaks (DSBs) by the Spo11 endonuclease. These breaks are repaired via homologous strand exchange with sequences on a non-sister chromatid. A set of proteins monitors recombination and activates a checkpoint during late prophase I (pachytene) if the recombination repair process has not been completed. This checkpoint prevents segregation of homologous chromosomes until recombination is complete and ensures proper distribution of the genetic material to the gametes (Peretz, 2009)
A meiotic checkpoint similar to that described in yeast also exists in Drosophila. Several of the spindle class genes were previously found to encode proteins with homology to known DNA repair enzymes. Specifically, spindle-A (spn-A) encodes a Rad51-like protein, spindle-B (spn-B) encodes a XRCC3-like protein, spindle-C (spn-C) encodes a HEL308-like protein, spindle-D (spn-D) encodes a Rad51C-like protein and okra encodes a Rad54-like protein. These genes were shown to be required for the repair of recombination-induced DSBs during Drosophila oogenesis. Moreover, mutations in these genes lead to activation of a meiotic checkpoint, leading to the appearance of several defects during oogenesis. The most obvious phenotypes manifested are the dorsal-ventral (D-V) patterning defects of the egg, arising due to improper localization and translation of gurken mRNA. In addition, the hollow sphere of highly packed chromatin (also called the karyosome) that is characteristic of the wild-type oocyte nucleus is often fragmented or thread-like in appearance in the DNA repair enzyme mutants. These defects can be suppressed by blocking the formation of DSBs during meiosis through mutations in the spo11 homologue, mei-W68, or by eliminating the checkpoint through a mutations in mei-41 or DmChk2, the Drosophila homologues for ATR and Chk2, respectively (Peretz, 2009)
hus1 mutant flies have been shown to be viable although the females are sterile. hus1 mutant flies are sensitive to hydroxyurea (HU) and to methyl methanesulfonate (MMS) but not to X-rays, suggesting that hus1 is required for the activation of an S phase checkpoint. Furthermore, hus1 is not required for the G2/M checkpoint or for post-irradiation induction of apoptosis. hus1 is able to suppress the D-V pattering defects caused by mutations in DNA repair enzymes. Interestingly, hus1 mutants are also characterized by a range of karyosome formation defects, much like mutants expressing defective DNA repair enzymes. These results suggested that during meiosis, hus1 is required for efficient activation of the meiotic checkpoint in response to persistent DSBs and is also essential for the organization of the oocyte DNA, a function that may be independent of the meiotic checkpoint (Peretz, 2009)
This study further analyzes the role of hus1 during meiosis; hus1 was found to be required for the efficient repair of DSBs during homologous recombination (HR) in meiosis. hus1 genetically interacts with brca2. It was also shown that non-repaired DSBs in the hus1 mutant lead to activation of a DmChk2 checkpoint. These results thus suggest that hus1 plays a role in the repair of meiotic DSBs (Peretz, 2009)
This study shows that the aberrant karyosome phenotype in the hus1 mutant is caused by defective homologous recombination (HR) repair. Histone γ-His2Av phosphorylation, a DSB marker, was dramatically increased in hus1 mutant flies and these persisted until later stages of oogenesis, as compared to wild-type flies. Additionally, blocking the formation of DSBs by using mei-W68 mutant flies suppressed the karyosome defect of hus1 mutant. The persistence of DSBs and karyosome defects in hus1 mutants resemble phenotypes found in flies with mutations in DNA repair enzymes of the spindle class genes. Taken together, these findings suggest that hus1 functions not only in activating the meiotic (pachytene) checkpoint but also in the repair of DSBs by HR during meiosis. Supportive of a role for hus1 in HR is the finding that reducing hus1expression in mouse cells by a siRNA approach decreases the efficiency of HR repair (Wang, 2006). Mammalian Rad9, a member of the Rad9-Hus1-Rad1 complex (9-1-1), interacts with Rad51, and inactivation of mammalian Rad9 results in decreased HR repair (Pandita, 2006). In yeast cells, it was shown that Rad17, the Rad9 homologue, and Rad24, the Rad17 homologue, are required for repair of DSBs during meiosis by facilitating proper assembly of the meiotic recombination complex containing Rad51, a protein which catalyzes DNA strand invasion. Therefore, it seems likely that the 9-1-1 complex as a whole could function during HR, this requires further examining (Peretz, 2009)
Interestingly, flies mutant for the recently identified Drosophila brca2 gene, are characterized by a highly penetrant karyosome defect, weakly ventralized eggs and persisting DBSs, implying a role for brca2 in homologous recombination repair. The abnormal D-V eggshell phenotype in mutants of DNA repair enzymes can be suppressed by mutations in brca2. This suggests that brca2 plays an additional role in transduction of the meiotic recombination checkpoint signal (Klovstad, 2008). It was reasoned that such a requirement for brca2 in activation of the checkpoint masks the strong eggshell ventralization phenotype normally characteristic of mutants of DNA repair enzymes (Klovstad, 2008). A similar rational could be applied to results with the hus1 mutant, where hus1 represents another protein with a dual function in both DNA repair and checkpoint activation during Drosophila meiosis. It is suggested that hus1 and brca2 thus represent a new class of proteins that serve a dual function in HR repair and in checkpoint activation during meiosis but whose mutant alleles do not show the full and/or strong repertoire of phenotypes of classic repair enzyme mutants (Klovstad, 2008). Interestingly, it was also reported that the Drosophila ATR homologue, mei-41, serves a dual function in DNA damage checkpoint and in facilitating the later stages of HR repair. mei-41 mutants also show a pattern of γ-His2Av staining in oocytes similar to that seen in DSB repair mutants, including delayed onset and persistence of foci into late pachytene (Joyce, 2009). The reduced or partial phenotypes displayed in mutants of these proteins (hus1, brca2 and mei-41), as compared to DNA repair enzyme mutants of the spindle class, may be indicative of a more regulatory natured role in repair, rather than a direct one. Thus, in DNA repair mutants a meiotic checkpoint is activated due to lack in repair of DSBs, while in mutants of regulatory genes (such as hus1, brca2 and mei-41) DSBs are also not repaired, however the checkpoint is not transduced properly leading to less pronounced phenotypes (Peretz, 2009)
It was also found that a mutation in the DmChk2 gene was able to suppress the karyosome and SC disassembly defects observed in hus1 mutant egg chambers, although DSBs persisted in these double mutants. This implies that in flies lacking hus1, DSBs are not repaired and this, in turn, leads to the activation of a DmChk2-dependent checkpoint. A mei-41 mutation was, however, unable to suppress these karyosome and SC defects. Similar results were reported for brca2 mutants, where the karyosome defects were attributed to activation of DmChk2 checkpoint but not of mei-41dependent one (Klovstad, 2008). Supporting evidence to the inability of mei-41 to suppress hus1 karyosome defects is the finding that mei-41 (Joyce, 2009) but not DmChk2 (this study) mutants show defects in processing DSBs during meiosis. The activation of a DmChk2-dependent checkpoint in hus1 mutants could be due to activation of DmChk2 by the other upstream checkpoint kinase, ATM. At this point, using the karyosome suppression assay, it was not possible to test whether ATM is the upstream activator, since atm mutants themselves present karyosome defects. It will be interesting to test whether atm, as hus1 and brca2, has a dual role in activation of the meiotic checkpoint and in HR repair (Peretz, 2009)
Since Brca2 co-immunoprecipitated Rad9, a member of the 9-1-1 complex, (Klovstad, 2008) and this study demonstrated a dual function for hus1 in meiosis, which was similar to that of brca2, it was decided to test whether hus1 and brca2 genetically interact. Defects in oocyte localization and determination, which were found to be characteristic of other DNA repair enzymes, were used as an indirect outcome of DSB repair in the oocyte. A mutation in brca2 was shown to strongly enhance the oocyte localization and determination defects found in the hus1 mutants. The finding that both hus1 and brca2 mutants show defects in DSB repair in the oocyte and that Brca2 physically interacts with Rad9, a part of the 9-1-1 complex (Klovstad, 2008), suggest that hus1 and brca2 may be a part of the same pathway of HR repair (Peretz, 2009)
However, the genetic interaction between hus1 and brca2 in HR repair in the germarium pro-nurse cells could be interpreted in a different manner. In wild-type, in region 2a of the germarium some of the pro-nurse cells contained DSBs as revealed by γ-His2Av staining. In later stages of meiosis, region 2b-3, the DSBs were restricted only to the oocyte, suggesting that there is a mechanism that ensures the restriction of DSBs to the oocyte and prevents these breaks in pro-nurse cells. It was found that in the double mutant flies for hus1 and brca2, but not in the single mutants, all of the germaria nurse cells showed γ-His2Av staining, suggesting that the nurse cells throughout the germarium have DSBs. These results point towards the role of hus1 and brca2 in DSB repair both in the pro-nurse cells and the oocyte. Such defects in DSB repair in the nurse cells and the oocyte could be the cause for the apoptosis of egg chambers in brca2;hus1 double mutant flies. The finding that the defects in DSB repair in the pro-nurse cells were detected only in the double brca2;hus1 mutants but not in the single ones, suggests that in this process hus1 and brca2 could act in parallel or in redundant pathways. Since it was shown that mei-41 mutants do cause a persistence of γ-His2Av foci in the oocyte but not in the nurse cells (as in the hus1 mutant), it will be interesting to study the complex interactions between mei-4, brca2 and hus1 in this process (Joyce, 2009). Altogether, these results lead to the identification of Hus1 as a protein with a dual role in activation of the meiotic checkpoint and in HR repair during meiosis (Peretz, 2009)
Search PubMed for articles about Drosophila hus1
Abdu, U., Brodsky, M. and Schüpbach, T. (2002). Activation of a meiotic checkpoint during Drosophila oogenesis regulates the translation of Gurken through Chk2/Mnk. Curr. Biol. 12, 1645-1651. PubMed ID: 12361566
Abdu, U., Klovstad, M., Butin-Israeli, V., Bakhrat, A. and Schüpbach T. (2007). An essential role for Drosophila hus1 in somatic and meiotic DNA damage responses. J. Cell Sci. 120(Pt 6): 1042-9. PubMed ID: 17327271
Bao, S., Lu, T., Wang, X., Zheng, H., Wang, L. E., Wei, Q., Hittelman, W. N. and Li, L. (2004). Disruption of the Rad9/Rad1/Hus1 (9-1-1) complex leads to checkpoint signaling and replication defects. Oncogene 23: 5586-5593. PubMed ID: 15184880
Bermudez, V. P., Lindsey-Boltz, L. A., Cesare, A. J., Maniwa, Y., Griffith, J. D., Hurwitz, J. and Sancar, A. (2003). Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro. Proc. Natl. Acad. Sci. 100: 1633-1638. PubMed ID: 12578958
Byun, T. S., Pacek, M., Yee, M. C., Walter, J. C., and Cimprich, K. A. (2005). Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint. Genes Dev. 19: 1040-1052. PubMed ID: 15833913
Delacroix, S., Wagner, J. M., Kobayashi, M., Yamamoto, K. and Karnitz, L. M. (2007). The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1. Genes Dev. 21(12): 1472-7. PubMed ID: 17575048
Ellison, V. and Stillman, B. (2003). Biochemical characterization of DNA damage checkpoint complexes: Clamp loader and clamp complexes with specificity for 5' recessed DNA. PLoS Biol. 1: 231-243. PubMed ID: 14624239
Enoch, T., Carr, A. M. and Nurse, P. (1992). Fission yeast genes involved in coupling mitosis to completion of DNA replication. Genes Dev. 6: 2035-2046. PubMed ID: 1427071
Ghabrial, A. and Schüpbach, T. (1999). Activation of a meiotic checkpoint regulates translation of Gurken during Drosophila oogenesis. Nat. Cell Biol. 1: 354-357. PubMed ID: 10559962
Hofmann, E. R., Milstein, S., Boulton, S. J., Ye, M., Hofmann, J. J., Stergiou, L., Gartner, A., Vidal, M. and Hengartner, M. O. (2002). Caenorhabditis elegans HUS1 is a DNA damage checkpoint protein required for genome stability and EGL-1-mediated apoptosis. Curr. Biol. 12: 1908-1918. PubMed ID: 12445383
Hong, E. J. and Roeder, G. S. (2002). A role for Ddc1 in signaling meiotic double-strand breaks at the pachytene checkpoint. Genes Dev. 16: 363-376. PubMed ID: 12445383
Joyce, E. F. and McKim, K. S. (2009). Drosophila PCH2 is required for a pachytene checkpoint that monitors double-strand-break-independent events leading to meiotic crossover formation. Genetics 181: 39-51. PubMed ID: 18957704
Klovstad, M., Abdu, U. and Schüpbach, T. (2008). Drosophila brca2 is required for mitotic and meiotic DNA repair and efficient activation of the meiotic recombination checkpoint, PLoS Genet. 4: e31. PubMed ID: 18266476
Kostrub, C. F., Knudsen, K., Subramani, S. and Enoch, T. (1998). Hus1p, a conserved fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response to DNA damage. EMBO J. 17: 2055-2066. PubMed ID: 9524127
Lee, J., Kumagai, A., and Dunphy, W. G. (2003). Claspin, a Chk1-regulatory protein, monitors DNA replication on chromatin independently of RPA, ATR, and Rad17. Mol. Cell 11: 329-340. PubMed ID: 12620222
Melo, J. and Toczyski, D. (2002). A unified view of the DNA-damage checkpoint. Curr. Opin. Cell Biol. 14: 237-245. PubMed ID: 11891124
Pandita, R. K. et al. (2006). Mammalian rad9 plays a role in telomere stability, S- and G2-phase-specific cell survival, and homologous recombinational repair. Mol. Cell. Biol. 26: 1850-1864. PubMed ID: 16479004
Peretz, G., Arie, L. G., Bakhrat, A. and Abdu, U. (2009). The Drosophila hus1 gene is required for homologous recombination repair during meiosis. Mech. Dev. 126(8-9): 677-86. PubMed ID: 19501158
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date revised: 10 April 2010
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