No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Drosophila ERCC1 Is Required for a Subset of MEI-9-Dependent Meiotic Crossovers
Abstract
Drosophila MEI-9 is the catalytic subunit of a DNA structure-specific endonuclease required for nucleotide excision repair (NER). The enzymatic activity of this endonuclease during NER requires the presence of a second, noncatalytic subunit called ERCC1. In addition to its role in NER, MEI-9 is required for the generation of most meiotic crossovers. To better understand the role of MEI-9 in crossover formation, we report here the characterization of the Drosophila Ercc1 gene. We created an Ercc1 mutant through homologous gene targeting. We find that Ercc1 mutants are identical to mei-9 mutants in sensitivity to DNA-damaging agents, but have a less severe reduction in the number of meiotic crossovers. MEI-9 protein levels are reduced in Ercc1 mutants; however, overexpression of MEI-9 is not sufficient to restore meiotic crossing over in Ercc1 mutants. We conclude that MEI-9 can generate some meiotic crossovers in an ERCC1-independent manner.
... Incision site mapping of the resulting products identifies two symmetrical cuts even in the presence of asymmetric lesions or substrates (Murchie and Lilley 1993;Jensch et al. 1989;Jensch and Kemper 1986;Birkenkamp and Kemper 1995). These observations demonstrate the highly specialized role of these enzymes for the dual, coordinated cleavage of target substrates, unlike the single incision event that occurs for flap endonucleases such as XPF-ERCC1 or XPG (Cox and Parry 1968;Fishman Lobell et al. 1992) SpRad16-Swi10 XPF NER, MTS (Carr et al. 1994;Schmidt et al. 1989) AtRAD1-RAD10 XPF NER, ICL, SSA (Gallego et al. 2000;Dubest et al. 2002) DmMEI-9-ERCCI XPF NER, ICL, Meiosis (Radford et al. 2005) HsXPF(ERCC4)-ERCC1 XPF NER, ICL (Biggerstaff et al. 1993;van Vuuren et al. 1993 (Andersen et al. 2009;Fekairi et al. 2009;Svendsen et al. 2009;Munoz et al. 2009) N/D not determined, NER nucleotide excision repair, SSA single-strand annealing, MTS mating-type switching, HR homologous recombination, RF replication fork support, ICL interstrand crosslink repair, rDNA ribosomal DNA maintenance, DSBR double-strand break repair (Guzder et al. 1995;Aboussekhra et al. 1995). Resolvases also generally display substrate specificity for HJs over other intact DNA substrates (Benson and West 1994;Whitby and Dixon 1998;Dickie et al. 1987). ...
... MEI-9 is undoubtedly the XPF homolog in flies, as mutants in this gene also display the UV and interstrand crosslink sensitivities expected for an NER defect (Table 1) (Radford et al. 2007;Yildiz et al. 2004;Sekelsky et al. 1995). Moreover, these DNA repair defects are identical to the mutant phenotypes of the ERCC1 homolog (Radford et al. 2005), suggesting that NER is also catalyzed in D. melanogaster by the MEI-9-ERCC1 heterodimeric nuclease, as in all other eukaryotes studied to date ( Fig. 3; Table 1). However, ERCC1 mutations cause a significantly milder defect in meiotic CO formation than MEI-9 mutants (Radford et al. 2005), suggesting that MEI-9 may participate with an alternate subunit for this function. ...
... Moreover, these DNA repair defects are identical to the mutant phenotypes of the ERCC1 homolog (Radford et al. 2005), suggesting that NER is also catalyzed in D. melanogaster by the MEI-9-ERCC1 heterodimeric nuclease, as in all other eukaryotes studied to date ( Fig. 3; Table 1). However, ERCC1 mutations cause a significantly milder defect in meiotic CO formation than MEI-9 mutants (Radford et al. 2005), suggesting that MEI-9 may participate with an alternate subunit for this function. Supporting this conjecture, MEI-9 also interacts with the fly Slx4 homolog, MUS312. ...
Homologous recombination is required for maintaining genomic integrity by functioning in high-fidelity repair of DNA double-strand breaks and other complex lesions, replication fork support, and meiotic chromosome segregation. Joint DNA molecules are key intermediates in recombination and their differential processing determines whether the genetic outcome is a crossover or non-crossover event. The Holliday model of recombination highlights the resolution of four-way DNA joint molecules, termed Holliday junctions, and the bacterial Holliday junction resolvase RuvC set the paradigm for the mechanism of crossover formation. In eukaryotes, much effort has been invested in identifying the eukaryotic equivalent of bacterial RuvC, leading to the discovery of a number of DNA endonucleases, including Mus81-Mms4/EME1, Slx1-Slx4/BTBD12/MUS312, XPF-ERCC1, and Yen1/GEN1. These nucleases exert different selectivity for various DNA joint molecules, including Holliday junctions. Their mutant phenotypes and distinct species-specific characteristics expose a surprisingly complex system of joint molecule processing. In an attempt to reconcile the biochemical and genetic data, we propose that nicked junctions constitute important in vivo recombination intermediates whose processing determines the efficiency and outcome (crossover/non-crossover) of homologous recombination.
... Second, all three of the identified exchange gene products interact in a yeast two-hybrid assay (Yildiz et al. 2002; Radford et al. 2005). On the basis of these findings, exchange class proteins have been proposed to be directly involved in the reaction that generates crossovers (Carpenter and Sandler 1974; Baker and Hall 1976). ...
... hdm mutants are sensitive to the DNA-damaging agent MMS: All previously described genes in the exchange class have a role in repairing somatic DNA damage. For example, mei-9 and Ercc1 mutants are hypersensitive to the DNA-damaging agent methyl methanesulfonate (MMS) and both are required for NER (Sekelsky et al. 1995; Radford et al. 2005). To test the possibility that HDM functions in somatic DNA repair, we measured the survival of hdm mutants after MMS treatment. ...
... To test the possibility that HDM functions in somatic DNA repair, we measured the survival of hdm mutants after MMS treatment. Sensitivity to MMS was assayed as described previously (Radford et al. 2005), with mutant and control flies laying eggs in vials for 2 days followed by addition of MMS on the third day. The relative survival rate following a treatment was measured as the ratio of mutant to wild-type flies. ...
Three Drosophila proteins, ERCC1, MUS312, and MEI-9, function in a complex proposed to resolve double-Holliday-junction intermediates into crossovers during meiosis. We report here the characterization of hold'em (hdm), whose protein product belongs to a single-strand-DNA-binding superfamily of proteins. Mutations in hdm result in reduced meiotic crossover formation and sensitivity to the DNA-damaging agent methyl methanesulfonate. Furthermore, HDM physically interacts with both MEI-9 and ERCC1 in a yeast two-hybrid assay. We conclude that HDM, MEI-9, MUS312, and ERCC1 form a complex that resolves meiotic recombination intermediates into crossovers.
... This crossfunctionality can be partly due to changes in complex composition that may modulate specificity of recognition and processing of a given intermediate. In D. melanogaster, where 90–95% of meiotic COs do not require MUS81 [10], COs are dependent on a protein complex consisting of MEI-9, an ortholog of the mammalian XPF and S. cerevisiae Rad1 nucleotide excision repair (NER) endonucleases , ERCC1, an XPF interaction partner, MUS312 and HDM, a member of a superfamily of proteins with ssDNA binding activity111213141516. However, ERCC1 and HDM are only required for a subset of CO events [12,13]. ...
... In D. melanogaster, where 90–95% of meiotic COs do not require MUS81 [10], COs are dependent on a protein complex consisting of MEI-9, an ortholog of the mammalian XPF and S. cerevisiae Rad1 nucleotide excision repair (NER) endonucleases , ERCC1, an XPF interaction partner, MUS312 and HDM, a member of a superfamily of proteins with ssDNA binding activity111213141516. However, ERCC1 and HDM are only required for a subset of CO events [12,13]. In S. cerevisiae, SLX4 was first identified in a synthetic-lethal screen for genes required for viability in the absence of SGS1, a member of the RecQ family of DNA helicases implicated in the human Bloom and Werner syndromes [17] . ...
Author Summary
Homologous recombination (HR) is a process that provides for the accurate and efficient repair of DNA double-strand breaks (DSBs) incurred by cells, thereby maintaining genomic integrity. Proper processing of HR intermediates is critical for biological processes ranging from replication fork restart to the accurate partitioning of chromosomes during meiotic cell divisions. This is further emphasized by the fact that impaired processing of HR intermediates in both mitotic and meiotic cells can result in tumorigenesis and congenital defects. Therefore, the identification of components involved in HR is essential to understand the molecular mechanism of HR. Here, we identify HIM-18/SLX-4 in C. elegans, a protein conserved from yeast to humans that interacts with the nucleases SLX-1 and XPF-1 and is required for DSB repair in the germline. Impaired HIM-18 function results in increased DNA damage sensitivity, the accumulation of recombination intermediates, decreased meiotic crossover frequencies, altered late meiotic chromosome remodeling, the formation of fragile connections between homologs, and an increased chromosome nondisjunction. Finally, HIM-18 is localized to both mitotic and meiotic nuclei in wild-type germlines. We propose that HIM-18 function is required during the processing of late HR intermediates resulting from replication fork collapse and meiotic DSBs.
... It was shown that mei-genes are functional both in meiotic recombination and in maintaining the integrity of somatic chromosomes (Gatti 1979;Koana et al. 2004). Mutations in these genes sharply reduce meiotic crossing-over and cause a significant increase in the frequency of chromosomal aberrations in the nerve ganglia cells of irradiated larvae (Gatti 1979;Radford et al. 2005). Consequently, a decrease in the activity of these genes can induce genetic instability not only in meiotic (mei-41), but also mitotic (mei-9, mei-41) cells of unirradiated offspring. ...
The article is devoted to the study of the role of intracellular mechanisms in the formation of radiation-induced genetic instability and its transgenerational effect in cells of different tissues of the descendants of Drosophila melanogaster mutant strains whose parents were exposed to chronic radiation (0.42 and 3.5 mGy/h). The level of DNA damage (alkali-labile sites (ALS), single-strand (SSB) and double-strand (DSB) breaks) in cells of somatic (nerve ganglia, imaginal discs) and generative (testis) tissues from directly irradiated animals and their unirradiated offspring was evaluated. Confident transgenerational instability (on the level of ALSs and SSBs), observed only in somatic tissues and only at the higher dose rate, is characteristic for mus209 mutant strains defective in excision repair and, less often, for mus205 and mus210 mutant strains. The greatest manifestation of radiation-induced genetic instability was found in evaluating the DSBs. Dysfunction of the genes mus205, mus304, mei-9 and mei-41, which are responsible for postreplicative repair, excision repair, recombination and control of the cell cycle, affects transgenerational changes in the somatic tissues of the offspring of parents irradiated in both low and high dose rates. In germ cells, the key role in maintaining genetic stability under chronic irradiation is played by the non-recombination postreplication repair mus101 gene. We revealed the tissue specificity of the radiation-induced effects, transgenerational transmission and accumulation of DNA damage to descendants of chronically irradiated animals.
... We discovered that mild attenuation of DNA repair within skeletal muscle leads to systemic responses that limit age-related intestinal degeneration and extend lifespan. Using RNA interference (RNAi)-mediated targeting of the Mei-9-ERCC1 heterodimeric DNA repair nuclease (S1 Fig and [ 34,35]) specifically in adult thoracic skeletal muscle (using Act88FGal4, active in longitudinal indirect flight muscle but absent from other muscle found in the carcass and intestine [36]), we found that inhibiting DNA repair can dose-and age-dependently promote DNA damage, ultimately leading to muscle that presents hallmarks of tissue damage and premature aging (loss of proteostasis, Fig 1A and 1B). Attenuating Mei-9 alone (Act88FGal4>UAS-Mei-9 RNAi ) results in mild up-regulation of DNA damage and age-related increases in markers of muscle tissue damage/stress when compared thoracic muscle of young (10 d) Act88FG4>+(w 1118 ) controls, and flies with DNA repair attenuation specifically in thoracic muscle (mu-specific, Act88FG4>UAS-Mei-9 RNAi or Act88FG4>UAS-Mei-9 RNAi , UAS-ERCC1 RNAi ); assayed by phospho-H2aV immunostaining (red), counterstained with phalloidin ("Phall"; green, actin filaments) and DAPI (blue). ...
Author summary
Aging in multicellular organisms is characterized by a progressive decline in the proper function of organs. This deterioration of organ function is a risk factor for many diseases. However, it is unlikely that organs age in isolation, as damage in one organ can presumably impact aging of other organs through either beneficial or detrimental cross-talk. Our work attempts to explore this aspect of aging using fruit flies as a model system. We uncovered that damaged fly muscle can protect against aging in other organs, such as the intestine, through the secretion of a blood-borne factor named Diedel. This blood-borne factor presumably allows damaged organs to communicate with each other during aging. Related factors are also found in certain viruses, which have been hijacked from insect genomes to promote viral spreading during infection. Using this information, we found that viral Diedel inhibits death of infected cells, allowing viruses to spread. Similarly, host (insect) Diedel also blocks cell death in organs during aging, thus limiting deterioration of organ function and extending the organism’s lifespan.
... This is followed by an ingenious generation of a linearized targeting DNA in vivo by using sequence specific FLP recombinase to excise the integrated 'donor' as an episomal circle that then is linearized by the rare restriction enzyme I-SceI. While this method is extremely powerful it is time and labor-intensive, also the rates of insertion with homologous recombination can vary greatly and could prove to be particularly challenging for constructs with low targeting efficiency (Manoli et al., 2005;Radford et al., 2005;Jones et al., 2007). ...
Transcriptional repressors bind cis-regulatory elements of target genes in a sequence specific manner. To antagonize transcription, repressors primarily function by recruiting accessory proteins, co-repressors, which in turn largely function by modifying chromatin structure. Although a repressor could function by recruiting just a single co-repressor, many recruit more than one, with Brinker (Brk) from Drosophila recruiting the co-repressors, CtBP and Groucho (Gro), in addition to possessing a third repression domain, 3R. Previous studies indicated that Gro is sufficient for Brk to repress target genes in the wing imaginal disc, questioning why it should need to recruit CtBP, a ’short-range’ co-repressor compared to Gro that can function over longer distances. To resolve this I have used genomic engineering to generate a series of endogenous brk mutants that are unable to recruit Gro, CtBP and/or have the 3R domain deleted. Analysis of these mutants reveals that while the recruitment of Gro is necessary and is almost sufficient for Brk to make a morphologically wild-type fly, it is insufficient during oogenesis where Brk must utilize CtBP and 3R to pattern the egg-shell appropriately. Gro insufficiency during oogenesis can be explained by its downregulation in Brk-expressing cells through phosphorylation downstream of EGFR signaling, thus making it unavailable for Brk which must then resort to CtBP and/or 3R for repressive activity. The present study dissects the mechanism of activity of a transcription factor and its co-repressors and is the first to do so in multicellular eukaryotes in a physiologically relevant manner; additionally its findings provide a better understanding of why transcription factors in general may utilize more than one co-repressor.
... Thus, we created a null allele of nobo by a conventional knock-out technique 24,25 . In our nobo knock-out allele (nobo KO ), an almost entire coding sequence region of nobo was replaced with a mini-white gene targeting cassette (Fig. 2a). ...
In insects, the precise timing of moulting and metamorphosis is strictly guided by ecdysteroids that are synthesised from dietary cholesterol in the prothoracic gland (PG). In the past decade, several ecdysteroidogenic enzymes, some of which are encoded by the Halloween genes, have been identified and characterised. Here, we report a novel Halloween gene, noppera-bo (nobo), that encodes a member of the glutathione S-transferase family. nobo was identified as a gene that is predominantly expressed in the PG of the fruit fly Drosophila melanogaster. We generated a nobo knock-out mutant, which displayed embryonic lethality and a naked cuticle structure. These phenotypes are typical for Halloween mutants showing embryonic ecdysteroid deficiency. In addition, the PG-specific nobo knock-down larvae displayed an arrested phenotype and reduced 20-hydroxyecdysone (20E) titres. Importantly, both embryonic and larval phenotypes were rescued by the administration of 20E or cholesterol. We also confirm that PG cells in nobo loss-of-function larvae abnormally accumulate cholesterol. Considering that cholesterol is the most upstream material for ecdysteroid biosynthesis in the PG, our results raise the possibility that nobo plays a crucial role in regulating the behaviour of cholesterol in steroid biosynthesis in insects.
... To detect germline targeting events, these progeny are crossed to an appropriate stock and their offspring are screened for those that appear to have a new insertion of the targeting DNA. Stocks generated from different independent integration events are tested to identify any in which the targeting DNA has inserted into the target locus by homologous recombination, which in some cases is a small minority of the events (e.g., Radford et al. 2005). In the original "ends-in" method, homologous recombination results in a tandem duplication. ...
> In this commentary, K. Nicole Crown and Jeff Sekelsky examine new methods for gene targeting in Drosophila . These methods, which allow for rapid replacement of large regions adjacent to existing transgenic insertions, are described in two articles published in this month's GENETICS: [“Long-
... By combining these two methods, the efficiency of ends-out gene targeting could be greatly enhanced. Background mutations arising during HR based gene targeting have been reported (Rong et al., 2002; Greenberg et al., 2003; Lankenau et al., 2003; Radford et al., 2005); however, it is not routinely taken into account when using this technique. In a recent targeting case (O'Keefe et al., 2007), the Drosophila gene Wwox was knocked out by ends-in gene targeting strategy, and the resulted homozygous mutants (Wwox 1 ) were viable and fertile but showed an increased IR (ionizing radiation) sensitivity. ...
Functional biological research has benefited tremendously by analyses of the phenotypes of mutant organisms which can be generated
through targeted mutation of genes. In Drosophila, compared with random mutagenesis methods gene targeting has gained its popularity because it can introduce any desired mutation
into a gene of interest. However, applications of gene targeting have been limited because the targeting efficiency varies
with different genes, and the time and labor of targeting procedure are intensive. Nevertheless, improvement of gene targeting
and development of its variant technologies have received much attention of scientists. Here we review recent progress that
has been made in expanding the applications of gene targeting, which include the ΦC31 integration system and zinc-finger nucleases
induced gene targeting, and new strategies that generate more efficient and reliable gene targeting.
Keywordsgene targeting-ends-in-ends-out-ΦC31 integration system-zinc-finger nucleases (ZFNs)-homologous recombination (HR)-
Drosophila melanogaster
... The following double-balanced recombinant stocks were created for these studies: UAS-miR-11; UAS-dE2F1, UAS-dDP/ TM6B and UAS-p35; UAS-dE2F1, UAS-dDP/TM6B. Construction of miR-11 mutant donor transgene A donor construct was built in pBluescript and then subcloned into pTargetB (Radford et al. 2005). A 5.2-kb left arm fragment was generated by PCR with the primers ATCGGGTACCGCT TCTCTTTTGCGTTTGTTGC, and ATCGGGATCCCACGCG GGTCACAGAGAAAG, and was cloned into KpnI and BamHI of pBluescript. ...
The E2F family of transcription factors regulates the expression of both genes associated with cell proliferation and genes that regulate cell death. The net outcome is dependent on cellular context and tissue environment. The mir-11 gene is located in the last intron of the Drosophila E2F1 homolog gene dE2f1, and its expression parallels that of dE2f1. Here, we investigated the role of miR-11 and found that miR-11 specifically modulated the proapoptotic function of its host gene, dE2f1. A mir-11 mutant was highly sensitive to dE2F1-dependent, DNA damage-induced apoptosis. Consistently, coexpression of miR-11 in transgenic animals suppressed dE2F1-induced apoptosis in multiple tissues, while exerting no effect on dE2F1-driven cell proliferation. Importantly, miR-11 repressed the expression of the proapoptotic genes reaper (rpr) and head involution defective (hid), which are directly regulated by dE2F1 upon DNA damage. In addition to rpr and hid, we identified a novel set of cell death genes that was also directly regulated by dE2F1 and miR-11. Thus, our data support a model in which the coexpression of miR-11 limits the proapoptotic function of its host gene, dE2f1, upon DNA damage by directly modulating a dE2F1-dependent apoptotic transcriptional program.
... Drosophila mei-9 mutants exhibit increased sensitivity to UV irradiation compared to wildtype flies ( Fig. 1A; (Radford et al., 2005)). When UV-irradiated at the late second instar larval stage (60 hrs after egg laying), larvae homozygous or hemizygous for the loss of function allele mei-9 a eclose at lower rates than wild-type (OreR) control larvae (Fig. 1A). ...
Metazoans adapt to changing environmental conditions and to harmful challenges by attenuating growth and metabolic activities systemically. Recent studies in mice and flies indicate that endocrine signaling interactions between insulin/IGF signaling (IIS) and innate immune signaling pathways are critical for this adaptation, yet the temporal and spatial hierarchy of these signaling events remains elusive. Here, we identify and characterize a program of signaling interactions that regulates the systemic response of the Drosophila larva to localized DNA damage. We provide evidence that epidermal DNA damage induces an innate immune response that is kept in check by systemic repression of IIS activity. IIS repression induces NFκB/Relish signaling in the fat body, which is required for recovery of IIS activity in a second phase of the systemic response to DNA damage. This systemic response to localized DNA damage thus coordinates growth and metabolic activities across tissues, ensuring growth homeostasis and survival of the animal.
... It is tempting to speculate that AtSHOC1 might fulfil the XPF catalytic function in a complex with AtPTD. This may be similar to the role of Mei-9 and ERCC1 in Drosophila meiotic CO formation ( Sekelsky et al., 1995;Radford et al., 2005). Interestingly, the numbers of late recombination nodules shown by electron microscopy were not statistically significantly different in Atptd (c. ...
Meiosis is a central feature of sexual reproduction. Studies in plants have made and continue to make an important contribution to fundamental research aimed at the understanding of this complex process. Moreover, homologous recombination during meiosis provides the basis for plant breeders to create new varieties of crops. The increasing global demand for food, combined with the challenges from climate change, will require sustained efforts in crop improvement. An understanding of the factors that control meiotic recombination has the potential to make an important contribution to this challenge by providing the breeder with the means to make fuller use of the genetic variability that is available within crop species. Cytogenetic studies in plants have provided considerable insights into chromosome organization and behaviour during meiosis. More recently, studies, predominantly in Arabidopsis thaliana, are providing important insights into the genes and proteins that are required for crossover formation during plant meiosis. As a result, substantial progress in the understanding of the molecular mechanisms that underpin meiosis in plants has begun to emerge. This article summarizes current progress in the understanding of meiotic recombination and its control in Arabidopsis. We also assess the relationship between meiotic recombination in Arabidopsis and other eukaryotes, highlighting areas of close similarity and apparent differences.
... Drosophila stocks and crosses were maintained on a standard medium at 25uC. The following mutant alleles were used unless otherwise noted-ord 10 [20], c(2)M EP , pch2 EY01788a (pch2 EY ), c(3)G 68 [18], hdm g7 , mei-218 1 , rec 1 and rec 2 [49], Ercc1 X [30] , mei-9 a , spn-A 1 , spn- B Bu , sir2 17 [38] ...
Author Summary
Meiosis is a specialized cell division in which diploid organisms form haploid gametes for sexual reproduction. This is accomplished by a single round of replication followed by two consecutive divisions. At the first meiotic division, the segregation of homologous chromosomes in most organisms is dependent upon genetic recombination, or crossing over. Crossing over must therefore be regulated to ensure that every pair of homologous chromosomes receives at least one reciprocal exchange. Homologous chromosomes that do not receive a crossover frequently undergo missegregation, yielding gametes that do not contain the normal chromosome number, conditions frequently associated in humans with infertility and birth defects. The pch2 gene is widely conserved and in Drosophila melanogaster is required for a meiosis-specific checkpoint that delays progression when crossover formation is defective. However, the underlying process that the checkpoint is monitoring remains unclear. Here we show that defects in axis components and homolog alignment are sufficient to induce checkpoint activity and increase crossing over across the genome. Based on these observations, we hypothesize that the checkpoint may monitor the integrity of chromosome axes and function to promote an optimal number of crossovers during meiosis.
... Studies using Drosophila also identified a conserved ICL repair protein, MUS312, which appears to play an important role in the repair of DSBs created during crosslink processing. As in mammals, flies with mutations in either mei-9 (the XPF homolog) or ercc1 are sensitive to nitrogen mustard [Yildiz et al., 2004; Radford et al., 2005]. Several years ago, a third protein that interacts with MEI-9 and ERCC1, named MUS312, was identified [Yildiz et al., 2002]. ...
DNA interstrand crosslinks (ICLs) are complex lesions that covalently link both strands of the DNA double helix and impede essential cellular processes such as DNA replication and transcription. Recent studies suggest that multiple repair pathways are involved in their removal. Elegant genetic analysis has demonstrated that at least three distinct sets of pathways cooperate in the repair and/or bypass of ICLs in budding yeast. Although the mechanisms of ICL repair in mammals appear similar to those in yeast, important differences have been documented. In addition, mammalian crosslink repair requires other repair factors, such as the Fanconi anemia proteins, whose functions are poorly understood. Because many of these proteins are conserved in simpler metazoans, nonmammalian models have become attractive systems for studying the function(s) of key crosslink repair factors. This review discusses the contributions that various model organisms have made to the field of ICL repair. Specifically, it highlights how studies performed with C. elegans, Drosophila, Xenopus, and the social amoeba Dictyostelium serve to complement those from bacteria, yeast, and mammals. Together, these investigations have revealed that although the underlying themes of ICL repair are largely conserved, the complement of DNA repair proteins utilized and the ways in which each of the proteins is used can vary substantially between different organisms.
... In meiosis, however, Mei-9 also associates with a novel partner, Mus312 ( Yildiz et al. 2002). Surprisingly, ercc1 mutants are not as CO-defective as either mei-9 or mus312, suggesting that Mei-9 may partner with alternative subunits in different contexts ( Radford et al. 2005). Although it is still relatively unclear how ERCC1 contributes to the enzymatic activity or structural selectivity of the XPF-ERCC1 complex, ERCC1 binds ssDNA and may define substrate recognition and protein interaction properties of the complex ( Tsodikov et al. 2005). ...
Meiotic recombination ensures accurate chromosome segregation during the first meiotic division and
provides a mechanism to increase genetic heterogeneity among the meiotic products. Unlike homologous
recombination in somatic (vegetative) cells, where sister chromatid interactions prevail and crossover formation
is avoided, meiotic recombination is targeted to involve homologs, resulting in crossovers to connect the
homologs before anaphase of the first meiotic division. The mechanisms responsible for homolog choice and
crossover control are poorly understood, but likely involve meiosis-specific recombination proteins, as
well as meiosis-specific chromosome organization and architecture. Much progress has been made to identify
and biochemically characterize many of the proteins acting during meiotic recombination. This review will
focus on the proteins that generate and process heteroduplex DNA, as well as those that process DNA junctions
during meiotic recombination, with particular attention to how recombination activities promote crossover
resolution between homologs.
... Yeast two-hybrid analysis was performed as described previously (Radford et al., 2005). ...
DNA recombination and repair pathways require structure-specific endonucleases to process DNA structures that include forks, flaps, and Holliday junctions. Previously, we determined that the Drosophila MEI-9-ERCC1 endonuclease interacts with the MUS312 protein to produce meiotic crossovers, and that MUS312 has a MEI-9-independent role in interstrand crosslink (ICL) repair. The importance of MUS312 to pathways crucial for maintaining genomic stability in Drosophila prompted us to search for orthologs in other organisms. Based on sequence, expression pattern, conserved protein-protein interactions, and ICL repair function, we determined that the mammalian ortholog of MUS312 is BTBD12. Orthology between these proteins and S. cerevisiae Slx4 helped identify a conserved interaction with a second structure-specific endonuclease, SLX1. Genetic and biochemical evidence described here and in related papers suggest that MUS312 and BTBD12 direct Holliday junction resolution by at least two distinct endonucleases in different recombination and repair contexts.
... Thus, the early steps in meiotic recombination appear to be similar between Drosophila and S. cerevisiae. In contrast, later stages of crossover production are different, since most crossovers in Drosophila require the XPF/Rad1 ortholog MEI-9 [10,11], its binding partner ERCC1 [12], and several novel proteins, including MUS-312 [13] and MEI-218 [14]. In addition, it is not known whether noncrossovers in Drosophila are derived from a DHJ intermediate or SDSA, although SDSA is the most common pathway for repair of mitotic DSBs generated by transposable element excision151617. ...
Crossovers ensure the accurate segregation of homologous chromosomes from one another during meiosis. Here, we describe the identity and function of the Drosophila melanogaster gene recombination defective (rec), which is required for most meiotic crossing over. We show that rec encodes a member of the mini-chromosome maintenance (MCM) protein family. Six MCM proteins (MCM2-7) are essential for DNA replication and are found in all eukaryotes. REC is the Drosophila ortholog of the recently identified seventh member of this family, MCM8. Our phylogenetic analysis reveals the existence of yet another family member, MCM9, and shows that MCM8 and MCM9 arose early in eukaryotic evolution, though one or both have been lost in multiple eukaryotic lineages. Drosophila has lost MCM9 but retained MCM8, represented by REC. We used genetic and molecular methods to study the function of REC in meiotic recombination. Epistasis experiments suggest that REC acts after the Rad51 ortholog SPN-A but before the endonuclease MEI-9. Although crossovers are reduced by 95% in rec mutants, the frequency of noncrossover gene conversion is significantly increased. Interestingly, gene conversion tracts in rec mutants are about half the length of tracts in wild-type flies. To account for these phenotypes, we propose that REC facilitates repair synthesis during meiotic recombination. In the absence of REC, synthesis does not proceed far enough to allow formation of an intermediate that can give rise to crossovers, and recombination proceeds via synthesis-dependent strand annealing to generate only noncrossover products.
... Gene targeting. Disruption of Obp57d and Obp57e was carried out by the ends-out method using the vectors provided by Dr. Sekelsky [26]. A hsp70-white marker gene was excised from pBS-70w with SphI and XhoI and subcloned into the SmaI site of pBSII after blunting to obtain pBSII-70w. ...
Despite its morphological similarity to the other species in the Drosophila melanogaster species complex, D. sechellia has evolved distinct physiological and behavioral adaptations to its host plant Morinda citrifolia, commonly known as Tahitian Noni. The odor of the ripe fruit of M. citrifolia originates from hexanoic and octanoic acid. D. sechellia is attracted to these two fatty acids, whereas the other species in the complex are repelled. Here, using interspecies hybrids between D. melanogaster deficiency mutants and D. sechellia, we showed that the Odorant-binding protein 57e (Obp57e) gene is involved in the behavioral difference between the species. D. melanogaster knock-out flies for Obp57e and Obp57d showed altered behavioral responses to hexanoic acid and octanoic acid. Furthermore, the introduction of Obp57d and Obp57e from D. simulans and D. sechellia shifted the oviposition site preference of D. melanogaster Obp57d/e(KO) flies to that of the original species, confirming the contribution of these genes to D. sechellia's specialization to M. citrifolia. Our finding of the genes involved in host-plant determination may lead to further understanding of mechanisms underlying taste perception, evolution of plant-herbivore interactions, and speciation.
Sexually reproducing eukaryotes use recombination between homologous chromosomes to promote chromosome segregation during meiosis. Meiotic recombination is almost universally conserved in its broad strokes, but specific molecular details often differ considerably between taxa, and the proteins that constitute the recombination machinery show substantial sequence variability. The extent of this variation is becoming increasingly clear because of recent increases in genomic resources and advances in protein structure prediction. We discuss the tension between functional conservation and rapid evolutionary change with a focus on the proteins that are required for the formation and repair of meiotic DNA double-strand breaks. We highlight phylogenetic relationships on different time scales and propose that this remarkable evolutionary plasticity is a fundamental property of meiotic recombination that shapes our understanding of molecular mechanisms in reproductive biology.
In this chapter, we examine landmark discoveries made in mitotic and meiotic recombination using Drosophila as a model organism. Mitotic recombination is detrimental to cell viability, causing loss of heterozygosity and genome rearrangements, and unrepaired double-strand breaks (DSBs) can result in cell death. To avoid these outcomes, cells utilize multiple pathways to ensure that mitotic DSBs are repaired into non-crossover products. Here we discuss the major mitotic DSB repair pathways homologous recombination repair (especially synthesis-dependent strand annealing) and end joining. In contrast to mitotic cells, crossovers are beneficial during meiosis. Meiotic recombination is important to ensure proper chromosome segregation during gametogenesis and defects in crossover formation result in aneuploidy and sterility. Accordingly, we discuss key regulatory mechanisms of meiotic recombination that promote repair of meiotic DSBs as crossovers and distribution of crossovers along chromosomes.
Proper number and placement of meiotic crossovers is vital to chromosome segregation, with failures in normal crossover distribution often resulting in aneuploidy and infertility. Meiotic crossovers are formed via homologous repair of programmed double-strand breaks (DSBs). Although DSBs occur throughout the genome, crossover placement is intricately patterned, as observed first in early genetic studies by Muller and Sturtevant. Three types of patterning events have been identified. Interference, first described by Sturtevant in 1915, is a phenomenon in which crossovers on the same chromosome do not occur near one another. Assurance, initially identified by Owen in 1949, describes the phenomenon in which a minimum of one crossover is formed per chromosome pair. Suppression, first observed by Beadle in 1932, dictates that crossovers do not occur in regions surrounding the centromere and telomeres. The mechanisms behind crossover patterning remain largely unknown, and key players appear to act at all scales, from the DNA level to inter-chromosome interactions. There is also considerable overlap between the known players that drive each patterning phenomenon. In this review we discuss the history of studies of crossover patterning, developments in methods used in the field, and our current understanding of the interplay between patterning phenomena.
The XPF-ERCC1 complex, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair, and homologous recombination. XPF-ERCC1 incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here, we have examined the role of the XPF-ERCC1 complex in the model bryophyte Physcomitrella patens which exhibits uniquely high gene targeting frequencies. We undertook targeted knockout of the Physcomitrella ERCC1 and XPF genes. Mutant analysis shows that the endonuclease complex is essential for resistance to UV-B and to the alkylating agent MMS, and contributes to the maintenance of genome integrity but is also involved in gene targeting in this model plant. Using different constructs we determine whether the function of the XPF-ERCC1 endonuclease complex in gene targeting was removal of 3′ non-homologous termini, similar to SSA, or processing of looped-out heteroduplex intermediates. Interestingly, our data suggest a role of the endonuclease in both pathways and have implications for the mechanism of targeted gene replacement in plants and its specificities compared to yeast and mammalian cells.
Crossover formation as a result of meiotic recombination is vital for proper segregation of homologous chromosomes at the end of meiosis I. In most organisms, crossovers are generated through two crossover pathways: Class I and Class II. Meiosis-specific protein complexes ensure accurate crossover placement and formation by promoting and inhibiting the formation of crossovers in both crossover pathways. In Drosophila , Class I crossovers are promoted and Class II crossovers are prevented by a complex that contains MCM (mini-chromosome maintenance) and MCM-like proteins, REC (ortholog of Mcm8), MEI-217, and MEI-218, collectively called the mei-MCM complex. However, little is known about how the mei-MCMs function within the Class I and II crossover pathways. In this study, we perform genetic analysis to understand how specific regions and motifs of REC and MEI-218 contribute to crossover formation and distribution. We see that while the N-terminus of MEI-218 is dispensable for crossover formation, REC's conserved AAA ATPase motifs exhibit differential requirements for Class I and Class II crossover formation. REC-dependent ATP hydrolysis, but not ATP binding, is required for promoting the formation of Class I, MEI-9 dependent crossovers. Conversely, the ability for REC to both bind and hydrolyze ATP is required for REC's Class II anti-crossover role, yet to varying degrees, suggesting that REC forms multiple complexes that require different REC-dependent ATP binding functions. These results provide genetic insight into the mechanism in which mei-MCMs promote Class I crossovers and inhibit Class II crossovers.
Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which may function as a mitochondrial iron sensor since it is inactivated by iron; (iii) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (iv) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle.
Formation of an oocyte involves a specialized cell division termed meiosis. In meiotic prophase I (the initial stage of meiosis), chromosomes undergo elaborate events to ensure the proper segregation of their chromosomes into gametes. These events include processes leading to the formation of a crossover that, along with sister chromatid cohesion, forms the physical link between homologous chromosomes. Crossovers are formed as an outcome of recombination. This process initiates with programmed double-strand breaks that are repaired through the use of homologous chromosomes as a repair template. The accurate repair to form crossovers takes place in the context of the synaptonemal complex, a protein complex that links homologous chromosomes in meiotic prophase I. To allow proper execution of meiotic prophase I events, signaling processes connect different steps in recombination and synapsis. The events occurring in meiotic prophase I are a prerequisite for proper chromosome segregation in the meiotic divisions. When these processes go awry, chromosomes missegregate. These meiotic errors are thought to increase with aging and may contribute to the increase in aneuploidy observed in advanced maternal age female oocytes.
In this chapter, we examine landmark discoveries made in mitotic and meiotic recombination using Drosophila as a model organism. Mitotic recombination is detrimental to cell viability, causing loss of heterozygosity and genome rearrangements, and unrepaired double-strand breaks (DSBs) can result in cell death. To avoid these outcomes, cells utilize multiple pathways to ensure that mitotic DSBs are repaired into noncrossover products. Here we discuss the major mitotic DSB-repair pathways homologous recombination repair (especially, synthesis-dependent strand annealing) and end joining. In contrast to mitotic cells, crossovers are beneficial during meiosis. Meiotic recombination is important to ensure proper chromosome segregation during gametogenesis and defects in crossover formation result in aneuploidy and sterility. Accordingly, we discuss key regulatory mechanisms of meiotic recombination that promote repair of meiotic DSBs as crossovers. Lastly, we examine a novel model for meiotic recombination in Drosophila.
DNA repair declines with age and correlates with longevity in many animal species. In this study, we 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.
Meiosis is essential for sexual reproduction and recombination is a critical step required for normal meiosis. Understanding the underlying molecular mechanisms that regulate recombination is important for medical, agricultural and ecological reasons. Readily available molecular and cytological tools make Arabidopsis an excellent system to study meiosis. Here we review recent developments in molecular genetic analyses on meiotic recombination. These include studies on plant homologs of yeast and animal genes, as well as novel genes that were first identified in plants. The characterizations of these genes have demonstrated essential functions from the initiation of recombination by double-strand breaks to repair of such breaks, from the formation of double-Holliday junctions to possible resolution of these junctions, both of which are critical for crossover formation. The recent advances have ushered a new era in plant meiosis, in which the combination of genetics, genomics, and molecular cytology can uncover important gene functions.
Drosophila is an attractive model system in which powerful tools
in genetics and cytology can be used to identify and characterize the genes required for meiotic recombination.
This article reviews recent developments in understanding how pairing and synapsis proceed in the absence
of double-strand breaks (DSBs), the relationship of DSB formation to synapsis, how crossovers are determined
and formed, and the role that chromosome structure, including specialized sites, plays in regulating DSB
formation and repair.
The BLM helicase has been shown to maintain genome stability by preventing accumulation of aberrant recombination intermediates. We show here that the Saccharomyces cerevisiae BLM ortholog, Sgs1, plays an integral role in normal meiotic recombination, beyond its documented activity limiting aberrant recombination intermediates. In wild-type meiosis, temporally and mechanistically distinct pathways produce crossover and noncrossover recombinants. Crossovers form late in meiosis I prophase, by polo kinase-triggered resolution of Holliday junction (HJ) intermediates. Noncrossovers form earlier, via processes that do not involve stable HJ intermediates. In contrast, sgs1 mutants abolish early noncrossover formation. Instead, both noncrossovers and crossovers form by late HJ intermediate resolution, using an alternate pathway requiring the overlapping activities of Mus81-Mms4, Yen1, and Slx1-Slx4, nucleases with minor roles in wild-type meiosis. We conclude that Sgs1 is a primary regulator of recombination pathway choice during meiosis and suggest a similar function in the mitotic cell cycle.
Flow cytometry is a powerful technique that allows the researcher to measure fluorescence emissions on a per-cell basis, at multiple wavelengths, in populations of thousands of cells. In this chapter, we outline the use of flow cytometry for the analysis of cells from Drosophila's imaginal discs, which are developing epithelial organs that give rise to, but not exclusively, the wings, eyes, and legs of the adult. A variety of classical and transgenic genetic methods can be used to mark cells (e.g., mutant, or overexpressing a gene, or in a particular compartment) in these organs with green fluorescent protein (GFP), which is readily detected by flow cytometry. After dissecting an organ out of the animal and dissociating it into single cells, a flow cytometer can be used to assay the size, DNA content, and other parameters in GFP-marked experimental cells as well as GFP-negative control cells from the same sample. Specific marked cell populations can also be physically sorted, and then used in diverse biochemical assays. This chapter includes protocols for isolation and dissociation of larval imaginal discs and pupal appendages for flow cytometry, and as well as for flow cytometric acquisition and analysis. In addition, we present protocols for performing flow cytometry on fixed or live-cultured Drosophila S2 cells.
The purification of native protein complexes requires the availability of sufficient amounts of starting material. Drosophila melanogaster embryos have proven to be a rich source for nuclear protein complexes. Here we describe establishment and maintenance of a fly facility for the production of large amounts of embryos, protocols for the production of nuclear extracts, and a scheme for the chromatographic purification of a nuclear multisubunit protein complex.
Drosophila melanogaster is one of the best characterized model systems for genetic analysis. Protein biochemical methods have lagged behind for quite some time but meanwhile have reached a state where protein interaction networks can be elucidated at a similar speed and accuracy as genetic interactions. Therefore, Drosophila now offers the advantages of both genetic and biochemical approaches.
Here, we present a basic method for the purification of the endogenous Par-6/aPKC protein complex, which plays a central role in orchestrating asymmetric cell divisions in the developing nervous system of Drosophila. The procedure can be subdivided into the following steps: acquisition of sufficient starting material, complex stabilization by crosslinking (optional), purification of the protein complex by immunoprecipitation, separation of the isolated material on a polyacrylamide gel, sample preparation for mass spectrometry, and sample analysis. The protocol can easily be adapted to different affinity-tagged or endogenous protein complexes of interest.
To understand Drosophila development and other genetically controlled processes, it is often desirable to identify differences in gene expression levels. An experimental approach to investigate these processes is to catalog the transcriptome by hybridization of mRNA to DNA microbar-rays. In these experiments mRNA-derived hybridization probes are produced and hybridized to an array of DNA spots on a solid support. The labeled cDNAs of the complex hybridization probe will bind to their complementary sequences and provide quantification of the relative concentration of the corresponding transcript in the starting material. However, such approaches are often limited by the scarcity of the experimental sample because standard methods of probe preparation require microgram quantities of mRNA template. Linear RNA amplification can alleviate such limitations to support the generation of microarray hybridization probes from a few 100 pg of mRNA. These smaller quantities can be isolated from a few 100 cells. Here, we present a linear amplification protocol designed to preserve both the relative abundance of transcripts as well as their sequence complexity.
The regular appearance and the repetitive nature of the Drosophila eye, consisting of several hundred identical multicellular units, the ommatidia, has long served as an invaluable experimental system to study cell-cell interactions, inductive signaling events, cell proliferation, programmed cell death, cell differentiation, cell organization, and planar cell polarity among others. Importantly, the eye is dispensable for viability and fertility of the fly and thus, it can easily be manipulated, making it an ideal target for genetic screens. This chapter described an essential technique in the analysis of different genotypes in the adult fly eye, and allows detailed analyses with single cell resolution.
Drosophila pupal (P) wing development entails a series of dynamic developmental events, such as epithelial and glial morphogenesis, that are of outstanding interest to cell biologists. Here, we first describe how to prepare P and prepupal (PP) wings for immunofluorescence microscopy. This protocol has been optimized to visualize wing epithelial architecture, such as polarized cortical domains of planar cell polarity proteins. We then provide a protocol to prepare pupae for whole mount live imaging of P wings. This procedure has allowed us to live-image glial cell migration and proliferation along wing sensory nerves.
With the live imaging of embryos, the dynamics of developmental processes, such as tissue remodeling, cell morphogenesis, and, campus protein dynamics can be observed and quantified. This has greatly improved the mechanistic understanding of biological processes. Here we describe how embryos can be prepared for imaging mainly, but not only, fluorescent proteins and probes. This chapter is a users' guide that addresses the following aspects of fluorescent embryo imaging: (1) How to handle and prepare embryos for live microscopy. (2) What microscopic setups are available for embryo imaging and what should they be used for. (3) How to practically use fluorescent imaging setups depending on the experimental context: large-scale imaging of multiple embryos, high-resolution four-dimensional imaging of single embryos, studies of protein dynamics, and so on. (4) Finally, we focus on pitfalls and how to overcome a variety of possible problems encountered during live imaging.
The Drosophila embryonic ventral epidermis has served as a unique tissue for the genetic analysis of patterning. Two types of epidermal cells are easily distinguished: those that secrete short, thick hair-like structures called denticles and cells that only secrete smooth cuticle. Denticle-secreting cells form segmentally repeated belts. Within each belt, six types of denticles can be recognized according to size, shape, and orientation (types 1-6). They are arranged in a stereotypical manner within each denticle belt. This pattern results from the spatially organized activation of several signaling pathways during embryogenesis. Cuticle patterns therefore provide a sensitive readout of signaling activity and other patterning mechanisms. Here, I describe methods of preparation and analysis of cuticles from 1st instar larvae as well as from 3rd instar larvae. In addition, a protocol to simultaneously analyze cuticles and beta-galactosidase activity of embryos expressing lacZ reporter genes is presented.
We present detailed protocols for two methods of gene targeting in Drosophila. The first, ends-out targeting, is identical in concept to gene replacement techniques used routinely in mammalian and yeast cells. In Drosophila, the targeted gene is replaced by the marker gene white
+ (although options exist to generate unmarked targeted alleles). This approach is simple in both the molecular cloning and the genetic manipulations. Ends-out will likely serve most investigators’ purposes to generate simple gene deletions or reporter gene “knock-ins.”
The second method, ends-in targeting, targets a wild-type gene with an engineered mutated copy and generates a duplication structure at the target locus. This duplication can subsequently be reduced to one copy, removing the wild-type gene and leaving only the introduced mutation. Although more complicated in the cloning and genetic manipulations (see
Note 1), this approach has the benefit that the mutations may be introduced with no other remnant of the targeting procedure. This “surgical” approach will appeal to investigators who desire minimal perturbation to the genome, such as single nucleotide mutation.
Although both approaches appear to be approximately equally efficient (see
Note 2), each method has separate strengths and drawbacks. The choice of which approach is best depends on the researcher’s goal.
RNA interference (RNAi) has become a widely used tool to analyze biological functions in vivo and in vitro. With the availability of an increasing number of Drosophila cell lines, a variety of different processes can be studied ranging from cell cycle control defects to signaling pathway activities and changes in cell morphology. Owing to the ease of RNAi in Drosophila cells, this experimental system has become a preferred method to screen for novel cellular factors, before their in depth analysis. We here describe the experimental procedures for RNAi experiments in cultured Drosophila cells, starting from the design of long double-stranded RNAs, their synthesis by in vitro transcription and application for cell-based RNAi experiments from low to high-throughput formats. Finally, we show how phenotype analysis can be performed using cell-based assays for luminescence or flow cytometric analysis as examples.
Mobile elements were first used as a mutagenesis tool that introduces a molecular tag in the genes of interest. This facilitated subsequent molecular cloning and eventually promoted molecular analysis of a large number of fly genes. Soon after, P-elements were modified to detect genes not only based on a mutant phenotype but rather through revealing RNA or protein expression patterns (enhancer trap, gene trap). Owing to the typically imprecise mobilization of the P-elements these enhancer trap or gene trap insertions also provided means to generate (excision) mutants. Whereas the excision mutants are valuable deletions they are induced in a random fashion and the exact breakpoints have to be determined following molecular analysis. More recently, the introduction of recombination targets (flipase recombination targets) into P-elements has provided the ability to generate precise chromosomal deletions between preselected sites. Here we will summarize the current genetic approaches to generate different type of insertional and deletion mutations using specifically designed P-elements.
The development of a technique to stably integrate exogenous DNA into the germline of Drosophila melanogaster marked a milestone in the ability to study gene function in the fly.
On the molecular level germline transformation mainly relies on a particular transposable element, the D. melanogaster P-element. Based on certain features of the P-element, vectors have been designed for diverse applications like gene disruption, chromosome engineering, gene tagging, and inducible gene expression/repression. Despite the fact that an increasing number of other transposons have been utilized for germline transformation of Drosophila most transformation vectors are still P-element based.
Technically, microinjection serves as the method of choice to physically introduce transgenes into preblastoderm Drosophila embryos. Besides an appropriate technical equipment including suitable microcapillaries in conjunction with a micromanipulator, a microinjector, and a microscope, proper handling of the Drosophila embryos before and after microinjection is the key step to the generation of transgenic flies. Pioneer work in Drosophila also served as a general guideline for the transformation of other insect species including those with medical and agricultural importance.
The fruit fly Drosophila has played a central role in the development of biology during the 20th century. First chosen as a convenient organism to test evolutionary theories soon became the central element in an elaborate, fruitful, and insightful research program dealing with the nature and function of the gene. Through the activities of TH Morgan and his students, Drosophila did more than any other organism to lay down the foundations of genetics as a discipline and a tool for biology. In the last third of the century, a judicious blend of classical genetics and molecular biology focused on some mutants affecting the pattern of the Drosophila larva and the adult, and unlocked the molecular mechanisms of development. Surprisingly, many of the genes identified in this exercise turned to be conserved across organisms. This observation provided a vista of universality at a fundamental level of biological activity. At the dawn of the 21st century, Drosophila continues to be center stage in the development of biology and to open new ways of seeing cells and to understand the construction and the functioning of organisms.
Proteins belonging to the XPF/MUS81 family play important roles in the repair of DNA lesions caused by UV-light or DNA cross-linking agents. Most eukaryotes have four family members that assemble into two distinct heterodimeric complexes, XPF-ERCC1 and MUS81-EME1. Each complex contains one catalytic and one noncatalytic subunit and exhibits endonuclease activity with a variety of 3'-flap or fork DNA structures. The catalytic subunits share a characteristic core containing an excision repair cross complementation group 4 (ERCC4) nuclease domain and a tandem helix-hairpin-helix (HhH)(2) domain. Diverged domains are present in the noncatalytic subunits and may be required for substrate targeting. Vertebrates possess two additional family members, FANCM and Fanconi anemia-associated protein 24 kDa (FAAP24), which possess inactive nuclease domains. Instead, FANCM contains a functional Superfamily 2 (SF2) helicase domain that is required for DNA translocation. Determining how these enzymes recognize specific DNA substrates and promote key repair reactions is an important challenge for the future.
The generation and analysis of mutants is central to studies of gene function in model organisms. Methods for random mutagenesis in Drosophila melanogaster have been available for many years, but an alternative approach--targeted mutagenesis using homologous recombination--has only recently been developed. This approach has the advantage of specificity, because genes of interest can be altered. One might expect with a gene-targeting approach that the frequency of background mutations would be minimal. Unfortunately, we have found that this is not the case. Although the possibility of background mutations arising during homologous-recombination-based gene targeting has been raised in the literature, it is not routinely taken into account when using this technique. Our experience suggests that it can be a considerable problem but that it has a relatively simple solution.
The mammalian ortholog of yeast Slx4, BTBD12, is an ATM substrate that functions as a scaffold for various DNA repair activities. Mutations of human BTBD12 have been reported in a new sub-type of Fanconi anemia patients. Recent studies have implicated the fly and worm orthologs, MUS312 and HIM-18, in the regulation of meiotic crossovers arising from double-strand break (DSB) initiating events and also in genome stability prior to meiosis. Using a Btbd12 mutant mouse, we analyzed the role of BTBD12 in mammalian gametogenesis. BTBD12 localizes to pre-meiotic spermatogonia and to meiotic spermatocytes in wildtype males. Btbd12 mutant mice have less than 15% normal spermatozoa and are subfertile. Loss of BTBD12 during embryogenesis results in impaired primordial germ cell proliferation and increased apoptosis, which reduces the spermatogonial pool in the early postnatal testis. During prophase I, DSBs initiate normally in Btbd12 mutant animals. However, DSB repair is delayed or impeded, resulting in persistent γH2AX and RAD51, and the choice of repair pathway may be altered, resulting in elevated MLH1/MLH3 focus numbers at pachynema. The result is an increase in apoptosis through prophase I and beyond. Unlike yeast Slx4, therefore, BTBD12 appears to function in meiotic prophase I, possibly during the recombination events that lead to the production of crossovers. In line with its expected regulation by ATM kinase, BTBD12 protein is reduced in the testis of Atm(-/-) males, and Btbd12 mutant mice exhibit increased genomic instability in the form of elevated blood cell micronucleus formation similar to that seen in Atm(-/-) males. Taken together, these data indicate that BTBD12 functions throughout gametogenesis to maintain genome stability, possibly by co-ordinating repair processes and/or by linking DNA repair events to the cell cycle via ATM.
DMRT (Doublesex and Mab-3 related transcription factor) proteins generally associated with sexual differentiation in many organisms share a common DNA binding domain and are often expressed in reproductive tissues. Aside from doublesex, which is a central factor in the regulation of sex determination, Drosophila possesses three different dmrt genes that are of unknown function. Because the association with sexual differentiation and reproduction is not universal and some DMRT proteins have been found to play other developmental roles we chose to further characterize one of these Drosophila genes. We carried out genetic analysis of dmrt93B, which was previously found to be expressed sex-specifically in the developing somatic gonad and to affect testis morphogenesis in RNAi knockdowns. In order to disrupt this gene, the GAL4 yeast transcriptional activator followed by a polyadenylation signal was inserted after the dmrt93B start codon and introduced into the genome by homologous recombination. Analysis of the knock-in mutation as well as a small deletion removing all dmrt93B sequence demonstrate that loss of function causes partial lethality at the late pupal stage. Surprisingly, these mutations have no significant effect on gonad formation or male fertility. Analysis of GAL4-driven GFP reporter expression indicates that the dmrt93B promoter activity is highly specific to neurons in the suboesophageal and proventricular ganglion in larva and adult of both sexes suggesting a possible role in digestive tract function. Using the Capillary Feeder (CAFÉ) assay to measure daily food intake we find that reduction in this gene’s function leads to an increase in food consumption. These results suggest dmrt93 plays an important role in the formation or maintenance of neurons that affect feeding and support the idea that dmrt genes may not be restricted to roles in sexual differentiation.
Recent studies of meiotic recombination in the budding yeast and the model plant Arabidopsis thaliana indicate that meiotic crossovers (COs) occur through two genetic pathways: the interference-sensitive pathway and the interference-insensitive pathway. However, few genes have been identified in either pathway. Here, we describe the identification of the PARTING DANCERS (PTD) gene, as a gene with an elevated expression level in meiocytes. Analysis of two independently generated transferred DNA insertional lines in PTD showed that the mutants had reduced fertility. Further cytological analysis of male meiosis in the ptd mutants revealed defects in meiosis, including reduced formation of chiasmata, the cytological appearance of COs. The residual chiasmata in the mutants were distributed randomly, indicating that the ptd mutants are defective for CO formation in the interference-sensitive pathway. In addition, transmission electron microscopic analysis of the mutants detected no obvious abnormality of synaptonemal complexes and apparently normal late recombination nodules at the pachytene stage, suggesting that the mutant's defects in bivalent formation were postsynaptic. Comparison to other genes with limited sequence similarity raises the possibility that PTD may present a previously unknown function conserved in divergent eukaryotic organisms.
Nucleotide excision repair (NER) is a genome caretaker mechanism responsible for removing helix-distorting DNA lesions, most notably ultraviolet photodimers. Inherited defects in NER result in profound photosensitivity and the cancer-prone syndrome xeroderma pigmentosum (XP) or two progeroid syndromes: Cockayne and trichothiodystrophy syndromes. The heterodimer ERCC1-XPF is one of two endonucleases required for NER. Mutations in XPF are associated with mild XP and rarely with progeria. Mutations in ERCC1 have not been reported. Here, we describe the first case of human inherited ERCC1 deficiency. Patient cells showed moderate hypersensitivity to ultraviolet rays and mitomycin C, yet the clinical features were very severe and, unexpectedly, were compatible with a diagnosis of cerebro-oculo-facio-skeletal syndrome. This discovery represents a novel complementation group of patients with defective NER. Further, the clinical severity, coupled with a relatively mild repair defect, suggests novel functions for ERCC1.
Author Summary
DNA is a fragile thread that often breaks. When it does, the cell must find a way to splice the broken ends back together in order to continue its cycle of replication. Cells possess an array of ways to rejoin broken DNA ends, each with advantages and disadvantages. Some are “quick and dirty,” sacrificing accuracy for robustness. They do the basic job of resealing the break but often result in random base changes at the site of the repair. At the other extreme are methods with greater fidelity but added restrictions, such as requiring chromosome replication. We used an experimental system to obtain highly accurate measurements of the relative usage of various repair methods in developing germ cells of fruit flies. The measurements were made in normal flies as well as those carrying mutations at each of 11 genes involved in DNA repair. Most previous studies of these genes focused on specific biochemical pathways. Our results looked at how the repair apparatus as a whole compensates for defects in individual components. The data point to a “decision circuit” for matching each break to a repair method and provide new insight into how our DNA repair machinery protects the genome's integrity.
Meiotic recombination and DNA repair are mediated by overlapping sets of genes. In the yeast Saccharomyces cereuisiae, many genes required to repair DNA doublestrand breaks are also required for meiotic recombination. In contrast, mutations in genes required for nucleotide excision repair (NER) have no detectable effects on meiotic recombination in S. cereuisiae. The Drosophila melanogaster mei-9 gene is unique among known recombination genes in that it is required for both meiotic recombination and NER. We have analyzed the mei-9 gene at the molecular level and found that it encodes a homologue of the S. cereuisiae excision repair protein Radl, the probable homologue of mammalian XPF/ERCC4. Hence, the predominant process of meiotic recombination in Drosophila proceeds through a pathway that is at least partially distinct from that of S. cereuisiae, in that it requires an NER protein. The biochemical properties of the Radl protein allow us to explain the observation that mei-9 mutants suppress reciprocal exchange without suppressing the frequency of gene conversion.
In Saccharomyces cerevisiae, the RAD1 and RAD10 genes are involved in DNA nucleotide excision repair (NER) and in a pathway of mitotic recombination that occurs between
direct repeat DNA sequences. In this paper, we show that purified Rad1 and Rad10 interact with a synthetic bubble structure
and incise the DNA at the 5′-side of the centrally unpaired region. When Rad1-Rad10 and purified XPG protein (the human homolog
of yeast Rad2 protein) were co-incubated with the DNA substrate, we observed incisions at both ends of the bubble. This reaction
mimics the dual incision step in nucleotide excision repair in vivo. In addition, the recent suggestion that Rad1 can act to resolve Holliday junctions (Habraken, Y., Sung, P., Prakash, L.,
and Prakash, S.(1994) Nature 371, 531-534), explaining the recombination defect observed in rad1 mutants, has been further investigated. However, using proteins purified in two different laboratories we were unable to
show any interaction between Rad1 and synthetic Holliday junctions. The role that Rad1-Rad10 plays in recombination is likely
to resemble its activity in NER by acting upon partially unpaired DNA intermediates such as those formed by recombination
mechanisms involving single-strand DNA annealing.
The RAD10 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of UV-damaged DNA. We show
that the RAD10 gene is also required for mitotic recombination. The rad10 delta mutation lowered the rate of intrachromosomal
recombination of a his3 duplication in which one his3 allele has a deletion at the 3' end and the other his3 allele has a
deletion at the 5' end (his3 delta 3' his3 delta 5'). The rate of formation of HIS3+ recombinants in the rad10 delta mutant
was not affected by the rad1 delta mutation but decreased synergistically in the presence of the rad10 delta mutation in combination
with the rad52 delta mutation. These observations indicate that the RAD1 and RAD10 genes function together in a mitotic recombination
pathway that is distinct from the RAD52 recombination pathway. The rad10 delta mutation also lowered the efficiency of integration
of linear DNA molecules and circular plasmids into homologous genomic sequences. We suggest that the RAD1 and RAD10 gene products
act in recombination after the formation of the recombinogenic substrate. The rad1 delta and rad10 delta mutations did not
affect meiotic intrachromosomal recombination of the his3 delta 3' his3 delta 5' duplication or mitotic and meiotic recombination
of ade2 heteroalleles located on homologous chromosomes.
A complex, which consists of ERCC1 (38 kDa) and a 112-kDa protein, was purified from HeLa cells to homogeneity. This complex
complemented the nucleotide excision repair defects of rodent ERCC-1, ERCC-4, and human XP-F mutant cell-free extracts, indicating
that the 112-kDa protein is XPF/ERCC4 and providing direct biochemical evidence that XPF and ERCC4 are identical. The XPF/ERCC4-ERCC1
complex has an endonuclease activity with preference for single-stranded DNA and a single-stranded region of duplex DNA with
a “bubble” structure. This complex also nicks supercoiled DNA weakly, and this nicking activity is stimulated by human replication
protein A when the DNA contains UV damage.
HO endonuclease-induced double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae can be repaired by the process of
gap repair or, alternatively, by single-strand annealing if the site of the break is flanked by directly repeated homologous
sequences. We have shown previously (J. Fishman-Lobell and J. E. Haber, Science 258:480-484, 1992) that during the repair
of an HO-induced DSB, the excision repair gene RAD1 is needed to remove regions of nonhomology from the DSB ends. In this
report, we present evidence that among nine genes involved in nucleotide excision repair, only RAD1 and RAD10 are required
for removal of nonhomologous sequences from the DSB ends. rad1 delta and rad10 delta mutants displayed a 20-fold reduction
in the ability to execute both gap repair and single-strand annealing pathways of HO-induced recombination. Mutations in RAD2,
RAD3, and RAD14 reduced HO-induced recombination by about twofold. We also show that RAD7 and RAD16, which are required to
remove UV photodamage from the silent HML, locus, are not required for MAT switching with HML or HMR as a donor. Our results
provide a molecular basis for understanding the role of yeast nucleotide excision repair gene and their human homologs in
DSB-induced recombination and repair.
HO endonuclease-induced double-strand breaks (DSBs) within a direct duplication of Escherichia coli lacZ genes are repaired either by gene conversion or by single-strand annealing (SSA), with > 80% being SSA. Previously it was demonstrated that the RAD52 gene is required for DSB-induced SSA. In the present study, the effects of other genes belonging to the RAD52 epistasis group were analyzed. We show that RAD51, RAD54, RAD55, and RAD57 genes are not required for SSA irrespective of whether recombination occurred in plasmid or chromosomal DNA. In both plasmid and chromosomal constructs with homologous sequences in direct orientation, the proportion of SSA events over gene conversion was significantly elevated in the mutant strains. However, gene conversion was not affected when the two lacZ sequences were in inverted orientation. These results suggest that there is a competition between SSA and gene conversion processes that favors SSA in the absence of RAD51, RAD54, RAD55 and RAD57. Mutations in RAD50 and XRS2 genes do not prevent the completion, but markedly retard the kinetics, of DSB repair by both mechanisms in the lacZ direct repeat plasmid, a result resembling the effects of these genes during mating-type (MAT) switching.
Hermansky-Pudlak syndrome (HPS) is an often-fatal autosomal recessive disease in which albinism, bleeding, and lysosomal storage result from defects of diverse cytoplasmic organelles: melanosomes, platelet dense bodies, and lysosomes. HPS is the most common single-gene disorder in Puerto Rico, with an incidence of 1 in 1,800. We have identified the HPS gene by positional cloning, and found homozygous frameshifts in this gene in Puerto Rican, Swiss, Irish and Japanese HPS patients. The HPS polypeptide is a novel transmembrane protein that is likely to be a component of multiple cytoplasmic organelles and that is apparently crucial for their normal development and function. The different clinical phenotypes associated with the different HPS frameshifts we observed suggests that differentially truncated HPS polypeptides may have somewhat different consequences for subcellular function.
The Wilms tumor suppressor protein, WT1, is a transcription factor capable of activating or repressing transcription of various cellular genes. The mechanisms involved in regulating the transcriptional activities of WT1 are beginning to be unraveled. It appears that physical interactions of other cellular proteins (p53 and par-4) with WT1 can modulate the function of WT1. Here, we report the identification and cloning of a novel WT1-interacting protein termed Ciao 1, a member of the WD40 family of proteins. Ciao 1 specifically interacts with WT1 both in vitro and in vivo. This interaction alters the mobility of a WT1.DNA complex in gel shift assays, and results in a decrease in transcriptional activation mediated by WT1. Ciao 1 does not inhibit binding of WT1 to its consensus nucleotide sequence and does not affect the repression activity of WT1. Thus, Ciao 1 appears to specifically modulate the transactivation activity of WT1 and may function to regulate the physiological functions of WT1 in cell growth and differentiation.
We used a recently developed method to produce mutant alleles of five endogenous Drosophila genes, including the homolog of the p53 tumor suppressor. Transgenic expression of the FLP site-specific recombinase and the I-SceI endonuclease generates extrachromosomal linear DNA molecules in vivo. These molecules undergo homologous recombination with the corresponding chromosomal locus to generate targeted alterations of the host genome. The results address several questions about the general utility of this technique. We show that genes not near telomeres can be efficiently targeted; that no knowledge of the mutant phenotype is needed for targeting; and that insertional mutations and allelic substitutions can be easily produced.
Xeroderma pigmentosum (XP) is a human genetic disease which is caused by defects in nucleotide excision repair. Since this repair pathway is responsible for removing UV irradiation-induced damage to DNA, XP patients are hypersensitive to sunlight and are prone to develop skin cancer. Based on the underlying genetic defect, the disease can be divided into the seven complementation groups XPA through XPG. XPF, in association with ERCC1, constitutes a structure-specific endonuclease that makes an incision 5' to the photodamage. XPF-ERCC1 has also been implicated in both removal of interstrand DNA cross-links and homology-mediated recombination and in immunoglobulin class switch recombination (CSR). To study the function of XPF in vivo, we inactivated the XPF gene in mice. XPF-deficient mice showed a severe postnatal growth defect and died approximately 3 weeks after birth. Histological examination revealed that the liver of mutant animals contained abnormal cells with enlarged nuclei. Furthermore, embryonic fibroblasts defective in XPF are hypersensitive to UV irradiation and mitomycin C treatment. No defect in CSR was detected, suggesting that the nuclease is dispensable for this recombination process. These phenotypes are identical to those exhibited by the ERCC1-deficient mice, consistent with the functional association of the two proteins. The complex phenotype suggests that XPF-ERCC1 is involved in multiple DNA repair processes.
FlyBase (http://flybase.org) is the primary repository of genetic and molecular data of the insect family Drosophilidae. For the most extensively studied
species, Drosophila melanogaster, a wide range of data are presented in integrated formats. Data types include mutant phenotypes, molecular characterization
of mutant alleles and aberrations, cytological maps, wild-type expression patterns, anatomical images, transgenic constructs
and insertions, sequence-level gene models and molecular classification of gene product functions. There is a growing body
of data for other Drosophila species; this is expected to increase dramatically over the next year, with the completion of draft-quality genomic sequences
of an additional 11 Drosphila species.
MEI-9 is the Drosophila homolog of the human structure-specific DNA endonuclease XPF. Like XPF, MEI-9 functions in nucleotide excision repair and interstrand crosslink repair. MEI-9 is also required to generate meiotic crossovers, in a function thought to be associated with resolution of Holliday junction intermediates. We report here the identification of MUS312, a protein that physically interacts with MEI-9. We show that mutations in mus312 elicit a meiotic phenotype identical to that of mei-9 mutants. A missense mutation in mei-9 that disrupts the MEI-9-MUS312 interaction abolishes the meiotic function of mei-9 but does not affect the DNA repair functions of mei-9. We propose that MUS312 facilitates resolution of meiotic Holliday junction intermediates by MEI-9.
Drosophila mei-9 is essential for several DNA repair and recombination pathways, including nucleotide excision repair (NER), interstrand crosslink repair, and meiotic recombination. To better understand the role of MEI-9 in these processes, we characterized 10 unique mutant alleles of mei-9. These include a P-element insertion that disrupts repair functions but not the meiotic function; three nonsense mutations, one of which has nearly wild-type levels of protein; three missense mutations, one of which disrupts the meiotic function but not repair functions; two small in-frame deletions; and one frameshift.
Thirteen X-linked mutants have been isolated in Drosophila melanogaster which render male and homozygous female larvae sensitive to the mutagen methyl methanesulfonate. Their characterization and preliminary assignment to functional groups is described. Four of these mutants are alleles of mei-41 (Baker and Carpenter 1972). Like previously isolated alleles of this locus, these mutants reduce fertility and increase loss and nondisjunction of the X-chromosome in homozygous females. The remaining mutants have been tentatively assigned to six functional groups (two mutants to the mus(1)101 locus, two to mus(1)102 , two to mus(1)103, and one each to mus(1)104, mus(1)105 , and mus(1)106). Several of the complementation groups can be distinguished on the basis of nondisjunction and cross sensitivity to mutagens. Females homozygous for the mei-41, mus(1)101 and mus(1)102 mutants exhibit elevated levels of nondisjunction. Mutants belonging to complementation groups mei-41, mus(1)101, and mus(1)104 are sensitive to nitrogen mustard (HN2) in addition to their MMS sensitivity. Among these mutants there is currently a direct correlation between sensitivity to HN2, sensitivity to 2-acetylaminofluorene and a deficiency in post-replication repair ( Boyd and Setlow 1976). Only the mei-41 mutants are hypersensitive to UV radiation, although several of the mutants exhibit sensitivity to gamma-rays. Semidominance is observed in female larvae of the mei-41, mus(1)104, and mus(1)103 mutants after exposure to high concentrations of MMS. The properties of the mutants generally conform to a pattern which has been established for related mutants in yeast. Additional properties of these mutants are summarized in Table 9.
Many naturally occurring Drosophila strains (P strains) contain P transposable elements that are relatively stable within that strain but transpose at a high frequency when introduced by suitable genetic crosses into strains lacking P elements (M strains). Natural P strains of Drosophila may contain P elements at 40 to 50 sites on the genome, but the majority of these are usually defective elements, containing internal deletions that render them unable to supply their own transposase functions. They are, however, mobilizable by transposase provided in trans by the intact, autonomous elements. This chapter discusses the vectors for P-mediated transformation in Drosophila. P elements are used to introduce cloned DNA sequences into the genome of the fly by inserting the desired sequence within a cloned P element. Such an engineered P element act as a vector for the transposition of a desired sequence into the genome if coinjected into early embryos with P element sequences that provide the required transposase functions in trans. The components necessary for P-mediated transposition are then a P-vector, into which can be cloned the sequences to be introduced into the Drosophila genome and a helper plasmid containing an active P-transposase gene.
We have cloned the RAD10 gene of Saccharomyces cerevisiae and physically mapped it to a 1.0-kb DNA fragment. Strains containing disruptions of the RAD10 gene were found to show enhanced UV sensitivity compared with the previously characterized rad10-1 or rad10-2 mutants. The UV sensitivity of the disruption mutant is comparable to the highly UV sensitive rad1-19, rad2-delta, and rad3-2 mutants.
The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for both nucleotide excision repair and certain mitotic
recombination events. Here, model recombination and repair intermediates were used to show that Rad1-Rad10-mediated cleavage
occurs at duplex-single-strand junctions. Moreover, cleavage occurs only on the strand containing the 3' single-stranded tail.
Thus, both biochemical and genetic evidence indicate a role for the Rad1-Rad10 complex in the cleavage of specific recombination
intermediates. Furthermore, these data suggest that Rad1-Rad10 endonuclease incises DNA 5' to damaged bases during nucleotide
excision repair.
We describe here a family of P elements that we refer to as type I repressors. These elements are identified by their repressor functions and their lack of any deletion within the first two-thirds of the canonical P sequence. Elements belonging to this repressor class were isolated from P strains and were made in vitro. We found that type I repressor elements could strongly repress both a cytotype-dependent allele and P element mobility in somatic and germline tissues. These effects were very dependent on genomic position. Moreover, we observed that an element's ability to repress in one assay positively correlated with its ability to repress in either of the other two assays. The type I family of repressor elements includes both autonomous P elements and those lacking exon 3 of the P element. Fine structure deletion mapping showed that the minimal 3' boundary of a functional type I element lies between nucleotide position 1950 and 1956. None of 12 elements examined with more extreme deletions extending into exon 2 made repressor. We conclude that the type I repressors form a structurally distinct group that does not include more extensively deleted repressor elements such as the KP element described previously.
The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
The structure-specific ERCC1/XPF endonuclease complex that contains the ERCC1 and XPF subunits is implicated in the repair of two distinct types of lesions in DNA: nucleotide excision repair (NER) for ultraviolet-induced lesions and bulky chemical adducts; and recombination repair of the very genotoxic interstrand cross-links.
Here, we present a detailed analysis of two types of mice with mutations in ERCC1, one in which the gene is 'knocked out', and one in which the encoded protein contains a seven amino-acid carboxy-terminal truncation. In addition to the previously reported symptoms of severe runting, abnormalities of liver nuclei and greatly reduced lifespan (which appeared less severe in the truncation mutant), both types of ERCC1-mutant mouse exhibited an absence of subcutaneous fat, early onset of ferritin deposition in the spleen, kidney malfunction, gross abnormalities of ploidy and cytoplasmic invaginations in nuclei of liver and kidney, and compromised NER and cross-link repair. We also found that heterozygosity for ERCC1 mutations did not appear to provide a selective advantage for chemically induced tumorigenesis. An important clue to the cause of the very severe ERCC1-mutant phenotypes is our finding that ERCC1-mutant cells undergo premature replicative senescence, unlike cells from mice with a defect only in NER.
Our results strongly suggest that the accumulation in ERCC1-mutant mice of endogenously generated DNA interstrand cross-links, which are normally repaired by ERCC1-dependent recombination repair, underlies both the early onset of cell cycle arrest and polyploidy in the liver and kidney. Thus, our work provides an insight into the molecular basis of ageing and highlights the role of ERCC1 and interstrand DNA cross-links.
To study the nuclear organization and dynamics of nucleotide excision repair (NER), the endonuclease ERCC1/XPF (for excision
repair cross complementation group 1/xeroderma pigmentosum group F) was tagged with green fluorescent protein and its mobility
was monitored in living Chinese hamster ovary cells. In the absence of DNA damage, the complex moved freely through the nucleus,
with a diffusion coefficient (15 ± 5 square micrometers per second) consistent with its molecular size. Ultraviolet light–induced
DNA damage caused a transient dose-dependent immobilization of ERCC1/XPF, likely due to engagement of the complex in a single
repair event. After 4 minutes, the complex regained mobility. These results suggest (i) that NER operates by assembly of individual
NER factors at sites of DNA damage rather than by preassembly of holocomplexes and (ii) that ERCC1/XPF participates in repair
of DNA damage in a distributive fashion rather than by processive scanning of large genome segments.
Nucleotide excision repair (NER) is the primary pathway for the removal of ultraviolet light-induced damage and bulky adducts from DNA in eukaryotes. During NER, the helix is unwound around the damaged site, and incisions are made on the 5' and 3' sides, to release an oligonucleotide carrying the lesion. Repair synthesis can then proceed, using the intact strand as a template. The incisions flanking the lesion are catalyzed by different structure-specific endonucleases. The 5' incision is made by a heterodimer of XPF and ERCC1 (Rad1p-Rad10p in Saccharomyces cerevisiae), and the 3' incision is made by XPG (Rad2p in S. cerevisiae). We previously showed that the Drosophila XPF homologue is encoded by the meiotic recombination gene mei-9. We report here the identification of the genes encoding the XPG and ERCC1 homologues (XPG(Dm) and ERCC1(Dm)). XPG(Dm) is encoded by the mus201 gene; we found frameshift mutations predicted to produce truncated XPG(Dm) proteins in each of two mus201 alleles. These mutations cause defects in nucleotide excision repair and hypersensitivity to alkylating agents and ultraviolet light, but do not cause hypersensitivity to ionizing radiation and do not impair viability or fertility. ERCC1(Dm) interacts strongly in a yeast two-hybrid assay with MEI-9, indicative of the presumed requirement for these polypeptides to dimerize to form the functional endonuclease. The Drosophila Ercc1 gene maps to polytene region 51D1-2. The nucleotide excision repair gene mus210 maps nearby (51E-F) but is distinct from Ercc1.
Drosophila offers many advantages as an experimental organism. However, in comparison with yeast and mouse, two other widely used eukaryotic
model systems, Drosophila suffers from an inability to perform homologous recombination between introduced DNA and the corresponding chromosomal loci.
The ability to specifically modify the genomes of yeast and mouse provides a quick and easy way to generate or rescue mutations
in genes for which a DNA clone or sequence is available. A method is described that enables analogous manipulations of the
Drosophila genome. This technique may also be applicable to other organisms for which gene-targeting procedures do not yet exist.
We previously described a method for targeted homologous recombination at the yellow gene of Drosophila melanogaster. Because only a single gene was targeted, further work was required to show whether the method could be extended to become generally useful for gene modification in Drosophila. We have now used this method to produce a knockout of the autosomal pugilist gene by homologous recombination between the endogenous locus and a 2.5-kb DNA fragment. This was accomplished solely by tracking the altered genetic linkage of an arbitrary marker gene as the targeting DNA moved from chromosome X or 2 to chromosome 3. The results indicate that this method of homologous recombination is likely to be generally useful for Drosophila gene targeting.
In this review, we describe the pathway for generating meiotic crossovers in Drosophila melanogaster females and how these events ensure the segregation of homologous chromosomes. As appears to be common to meiosis in most organisms, recombination is initiated with a double-strand break (DSB). The interesting differences between organisms appear to be associated with what chromosomal events are required for DSBs to form. In Drosophila females, the synaptonemal complex is required for most DSB formation. The repair of these breaks requires several DSB repair genes, some of which are meiosis-specific, and defects at this stage can have effects downstream on oocyte development. This has been suggested to result from a checkpoint-like signaling between the oocyte nucleus and gene products regulating oogenesis. Crossovers result from genetically controlled modifications to the DSB repair pathway. Finally, segregation of chromosomes joined by a chiasma requires a bipolar spindle. At least two kinesin motor proteins are required for the assembly of this bipolar spindle, and while the meiotic spindle lacks traditional centrosomes, some centrosome components are found at the spindle poles.
Drosophila mei-9 is essential for several DNA repair and recombination pathways, including nucleotide excision repair (NER), interstrand crosslink repair, and meiotic recombination. To better understand the role of MEI-9 in these processes, we characterized 10 unique mutant alleles of mei-9. These include a P-element insertion that disrupts repair functions but not the meiotic function; three nonsense mutations, one of which has nearly wild-type levels of protein; three missense mutations, one of which disrupts the meiotic function but not repair functions; two small in-frame deletions; and one frameshift.
Cullin-dependent ubiquitin ligases regulate a variety of cellular and developmental processes by recruiting specific proteins for ubiquitin-mediated degradation. Cullin proteins form a scaffold for two functional modules: a catalytic module comprised of a small RING domain protein Roc1/Rbx1 and a ubiquitin-conjugating enzyme (E2), and a substrate recruitment module containing one or more proteins that bind to and bring the substrate in proximity to the catalytic module. Here, we present evidence that the three Drosophila Roc proteins are not functionally equivalent. Mutation of Roc1a causes lethality that cannot be rescued by expression of Roc1b or Roc2 by using the Roc1a promoter. Roc1a mutant cells hyperaccumulate Cubitus interruptus, a transcription factor that mediates Hedgehog signaling. This phenotype is not rescued by expression of Roc2 and only partially by expression of Roc1b. Targeted disruption of Roc1b causes male sterility that is partially rescued by expression of Roc1a by using the Roc1b promoter, but not by similar expression of Roc2. These data indicate that Roc proteins play nonredundant roles during development. Coimmunoprecipitation followed by Western or mass spectrometric analysis indicate that the three Roc proteins preferentially bind certain Cullins, providing a possible explanation for the distinct biological activities of each Drosophila Roc/Rbx.