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Genetics, Vol. 170, 221-235, May 2005, Copyright © 2005
doi:10.1534/genetics.104.034538
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* Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia
Developmental Biology Center, University of California, Irvine, California 92697
1 Corresponding author: Institute of Gene Biology, Russian Academy of Sciences, 34/5 Vavilov St., Moscow 119334, Russia.
E-mail: georgiev_p{at}mail.ru
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Thus, a potential candidate that may play a role in chromosome end protection is the heterodimeric Ku protein, which is a sequence-independent DNA end-binding protein that plays various roles at different kinds of DNA ends (JACKSON 2002; BERTUCH and LUNDBLAD 2003b). Crystal structure of human Ku bound to DNA (WALKER et al. 2001) showed that the Ku70 and Ku80 subunits interlock to form a ring-shaped molecule with a large central cavity for binding DNA and globular domains on the outside of the structure, which are potentially available to interact with other factors. At broken DNA ends, Ku promotes DNA repair as part of the nonhomologous end-joining (NHEJ) pathway. Ku is also a component of telomeres in yeast, mammals (SALDANHA et al. 2003), and plants (GALLEGO et al. 2003), where an essential role of the Ku70/Ku80 heterodimer in telomere metabolism has been well demonstrated. Ku, however, has variable effects on telomere length in different species. Saccharomyces cerevisiae and Schizosaccharomyces pombe cells deficient in Ku70 exhibit shortened but stable telomeres (BOULTON and JACKSON 1996; PORTER et al. 1996; BAUMANN and CECH 2000). By contrast, telomeres are longer than wild type in Arabidopsis thaliana Ku70 (RIHA and SHIPPEN 2003) and Ku80 mutants (GALLEGO et al. 2003), and both short and longer telomeres have been reported for Ku80 mutant mice (SAMPER et al. 2000; D'ADDA DI FAGAGNA et al. 2001). It is noteworthy, however, that Ku-deficient mammalian cells display telomeric fusions that are independent of the length of the telomeric repeats (BAILEY et al. 1999; SAMPER et al. 2000).
Recent results have revealed a complex role for Ku proteins in telomere metabolism in yeast. In Ku-defective strains, the structure of single-stranded termini is altered by nucleolytic activity (GRAVEL et al. 1998; POLOTNIANKA et al. 1998; MARINGELE and LYDALL 2002). Yeast strains that lack Ku also have substantially shortened telomeres in part because of enhanced nuclease action (BERTUCH and LUNDBLAD 2003a) and in part due to impaired regulation of telomerase (PETERSON et al. 2001; STELLWAGEN et al. 2003). Ku interacts with specific regions of yeast telomerase RNA to facilitate telomerase action at telomeres and different regions of Ku80 are required for its functions at telomeres and in ligation of broken ends (BERTUCH and LUNDBLAD 2003a; STELLWAGEN et al. 2003). In mammalian cells, Ku may associate with hTERT, and this interaction may function to regulate the access of telomerase to telomeric DNA ends (CHAI et al. 2002).
Ku homologs have also been identified in Drosophila. DmKu70 has been cloned independently as a gene encoding yolk protein factor 1, Ypf1b (JACOBY and WENSINK 1994, 1996), and as a gene that encodes a protein that binds to the inverted repeats of P elements, Irbp (BEALL et al. 1994). Thus, Ku70 appears to play a role in recognizing and/or healing of double-strand breaks at the donor site of an excised P element, a function that would correspond to its role in NHEJ in yeast and mammals. The gene maps to cytological position 86E4 (FlyBase, FBgn0011774). Irbp RNA is most abundant in ovaries and early embryos (BEALL et al. 1994; JACOBY and WENSINK 1994). It was reported earlier that Ku70 corresponds to the mutagen-sensitive gene mus309 (BEALL and RIO 1996), but subsequent studies revealed that mus309 is the Drosophila homolog of the human Bloom syndrome gene and encodes a helicase of the RECQ family (KUSANO et al. 2001). However, the mutagen sensitivity and female sterility of mus309 could be partially rescued by Ku70 transgenes, suggesting functional interaction between these two genes involved in DNA break repair (BEALL and RIO 1996; KUSANO et al. 2001). Less is known about the Drosophila Ku80 gene (cytological position 35D4, FlyBase FBgn0041627), although it has been cloned and sequenced by G. B. Gloor (GenBank no. AF23772).
Given the sequence-independent end-binding features of Ku and its demonstrated functions at yeast and mammalian telomeres, we initiated studies to ask if Ku might play a detectable role at Drosophila telomeres. As Ku deficiencies in yeast, plants, and mammals cause changes in telomere length, we asked if similar effects could be detected in Drosophila. The Drosophila equivalent of telomerase-telomere interaction might be thought of as the interaction of HeT-A and TART RNA intermediates with the chromosome ends to initiate reverse transcription of these elements. We therefore investigated the role of the Ku70 and Ku80 proteins in telomeric retrotransposon transposition frequency and studied their effect on chromosome elongation by terminal conversion. For these studies we used truncated but capped X chromosomes with breaks in the yellow (y) gene, which have been used in the past to investigate the dynamic events that occur at terminally deficient chromosomes (BIESSMANN and MASON 1988; BIESSMANN et al. 1990a,b, 1992a; MIKHAILOVSKY et al. 1999; KAHN et al. 2000). The y gene, which is required for larval and adult cuticle pigmentation (WALTER et al. 1991), is suitable for these studies, because the presence of specific enhancers or the addition of promoter activity brought in by the HeT-A element (DANILEVSKAYA et al. 1997; GOLUBOVSKY et al. 2001; MASON et al. 2003) can be monitored as distinct cuticle pigmentation changes. Enhancers controlling y expression in the wings and body cuticle are located in the upstream region of the gene, whereas the enhancer controlling expression in bristles resides within the large intron (GEYER and CORCES 1987; BIESSMANN and MASON 1988; MARTIN et al. 1989).
Here we report that partial inactivation of either the Ku70 or the Ku80 gene in heterozygous deficiencies significantly increased the frequency of HeT-A/TART additions to the ends of terminally truncated chromosomes. A decrease of the Ku80 or Ku70 concentration also strongly enhanced terminal gene conversion. In addition, we found that the stability of the terminally deficient chromosomes is sensitive to Ku70 dose. Taken together, these results suggest that Ku proteins are involved in controlling accessibility of the chromosome ends and consequently play a role in length regulation of Drosophila telomeres.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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To study events at a chromosome end, we used terminal-deficiency (TD) chromosomes, with the break in the y gene region designated as yTD. The yTD chromosomes with a y2-like phenotype (wild-type pigmentation in bristles and lack of pigmentation in the body and wings) were designated y2TD. The yTD chromosomes with darker wing and body pigmentation (yr-like phenotype) were designated yrTD. The origin of the y alleles was described previously (MIKHAILOVSKY et al. 1999; KAHN et al. 2000; MELNIKOVA and GEORGIEV 2002). Other genetic markers are described in LINDSLEY and ZIMM (1992) and in FlyBase (http://flybase.bio.indiana.edu). The y ac chromosome has a deletion of the yellow and achaete genes but not of any vital genes and thus allowed us to examine the behavior of the y gene on the homolog in the absence of other y sequences. The y1 allele contained a single-base-pair change in the start codon (GEYER et al. 1990). As a result, the y1 allele has an intact regulatory region but a nonproductive coding region and therefore causes a null phenotype: lack of pigmentation in all parts of the cuticle. The y phenotype was determined visually by the extent of pigmentation in different tissues of adult flies in 3- to 5-day-old females developing at 25°.
Molecular methods:
For Southern blot hybridization, DNA from adult flies was isolated using a published protocol (ASHBURNER 1989). Treatment of DNA with restriction endonucleases, blotting, fixation, and hybridization with radioactive probes were performed as described in the protocols for Hybond-N+ nylon membrane (Amersham, Arlington Heights, IL) and in the laboratory manual (SAMBROOK et al. 1989). Probes were labeled by random priming of gel-isolated fragments after appropriate restriction endonuclease digestion of plasmid subclones.
The junctions between newly transposed mobile elements and the DNA terminus were determined by DNA amplification with two oligonucleotide primers from the y gene and the HeT-A element, respectively. PCR reactions with genomic DNA were done using Taq polymerase from Fermentas AB (Vilnius, Lithuania). Reactions of 20 µl usually contained 100 ng of genomic DNA, 50 µM each of the dNTPs, 1x PCR buffer (supplied by the manufacturer), 2.5 mM MgCl2, 10 ng of each primer, and 1 unit Taq polymerase. PCR reactions were done in a Mastercycler personal (Eppendorf, Madison, WI) at an annealing temperature of 2°–3° below the melting temperature of the primers used, with 1 min synthesis at 72° for 25 cycles. Nucleotide map positions for the primers are given in parentheses in accordance with the y sequences (GEYER et al. 1986) and the 3'-UTR of the HeT-A element (BIESSMANN et al. 1992b). Primers in the y gene were y1, 5'-CCTGGAACATTGCAC-3' (3053–3039); y2, 5'-AAGACGGCGTCACCAAGGTGATC-3' (3101–3078); and y3, 5'-ACTTCCACTTACCATCACGCCAG-3' (3293–3271). Primers in the HeT-A element were h1, 5'-TGTTGCAAGTGGCGCGCATCC-3' (456–434) and h2, 5'-GGTGCTTCCGTACTTCTGGCGG-3' (359–338). The primer in TART was t1, 5'-CGAAACGCAACAACAAAATGG-3'; (1124–1144, TARTC).
The products of amplification were fractionated by electrophoresis in 1.5% agarose gels in TAE, cloned in pBluescript (Stratagene, La Jolla, CA), and sequenced using the Amersham sequence kit (Amersham, Arlington Heights, IL).
The 12-kb clone covering the Ku80 gene with its regulatory region was cloned by PCR (BD Advantage 2 PCR enzyme system) with the primers Ku80-1, 5'-GCGGGCGCACAGGCGACACCAGTA-3'; Ku80-2, 5'-GCCCCACAGGGGTCCTGCTACCCT-3'; Ku80-3, 5'-GTGGACCTCCCCGGAAATCCACCC-3'; and Ku80-4, 5'-CCACGGGCCGATTCGTGCGCCACC-3'. PCR reactions with genomic DNA in 50 µl usually contained 100 ng of genomic DNA, 100 µM each of the dNTPs, 1x Advantage 2 PCR buffer, 20 ng of each primer, PCR-grade water, and 1x Advantage 2 polymerase mix. PCR reactions were done at an annealing temperature of 2° below the melting temperature of the primers used, with 5–8 min synthesis at 68° for 35 cycles. The products of amplification were fractionated by electrophoresis in 0.8% agarose gels in TAE and the expected 12-kb DNA fragment was cloned in CaSpeR4. This P{Ku80+} construct, together with a P element with defective inverted repeats providing transposase, P25.7wc (KARESS and RUBIN 1984), was injected into y ac w1118; If/CyO preblastoderm embryos as described (RUBIN and SPRADLING 1982). G0 flies were crossed with y ac w1118 flies, and transgenic progeny were identified by eye color. The transformants were examined by Southern blot hybridization to check for transposon integrity and copy number. Chromosome localization of the P{Ku80+} transgene insertion in CyO was determined by crossing the transformants to y ac w1118.
RNA extraction and Northern hybridization were done as described in DANILEVSKAYA et al. (1999). The HeT-A probe was derived from element 23Zn-1 (GenBank accession no. U06920), open reading frame nucleotides 1746–4421.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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HeT-A and TART attachment to a broken chromosome end:
First, we tested whether a decrease of functional Ku70 or Ku80 concentration could change the frequency of HeT-A/TART attachments to a broken chromosomal terminus. As there are no available mutations in the Ku80 gene, we tested two deficiencies, Df(2L)r1 (referred to here as DfKu801) and Df(2L)TE35BC-24 (referred to here as DfKu802) that uncover the Ku80 gene. In the initial experiments with the Ku70 gene, we used the large deficiency, Df(3R)M-Kx1 [referred to here as Df(Ku701)] that uncovers the Ku70 (Irbp) gene. Later we tested Df(3R)Ku70thoR1 (referred to here as DfKu70R1), which has a deletion of the Ku70 (Irbp) gene (KUSANO et al. 2001).
To study the frequency of HeT-A and TART attachments to broken chromosome ends, we used a terminally truncated chromosome, yTD, with breaks in the upstream y gene region. Breaks located between –1200 and –140 bp upstream of the y transcription start result in a y2-like phenotype with yellow-colored aristae, y2(A–) (Figure 1A). Addition of either HeT-A- or TART-carrying promoter activity (DANILEVSKAYA et al. 1997) restores arista pigmentation, which allowed us to monitor new transpositions of these retroelements to the terminal sequences.
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Both tested Ku70 deficiencies had a much lower effect on elongation frequency than the Ku80 deficiencies (Table 1). Flies carrying DfKu701 and a yTD chromosome had reduced viability that complicated the analysis. For this reason most results were obtained with the small DfKu70R1 deficiency that includes only the Ku70 gene. For three generations, deficiencies of Ku70 did not cause an increase of elongation, but beginning in the fourth generation, elongation increased to a frequency of 9.0%, which is comparable to DfKu80.
To ask whether HeT-A and TART attachments had occurred in the yTD(A+) derivatives, 56 randomly selected females displaying a y2(A+) phenotype were crossed individually with y ac; If/CyO or with y ac; TM6/MKRS males as shown in Figure 2. The BamHI site in y located 
700–800 bp from the break of the original terminal deficiency was cut for the analysis of new DNA attachments to the ends of the deficient chromosomes (Figure 1B). We found that all selected yTD derivatives carried additions of new DNA sequences, which were of variable size. To identify the nature of the attached DNA fragments, the junctions between terminal y sequences and new DNA attachments were amplified by PCR and sequenced. The PCR primers were located in the y gene and in the conserved regions from the 3'-ends of HeT-A and TART, respectively. Of 21 independent yTD derivatives with terminal additions, 18 HeT-A and 3 TART attachments were identified (data not shown).
To reveal any possible DNA attachments that could not be selected by phenotype, we also examined all progeny without phenotypic changes obtained from individual yTD/yac females carrying either a Ku70 or a Ku80 deficiency (Figure 1C). Several new DNA additions that did not change the phenotype of the yTD alleles were detected. All of these new terminal extensions occurred in the progeny of flies carrying Ku80 deficiencies. We tried without success to clone the ends of these new additions by amplification of DNA between primers in the y gene and the 3'-end of HeT-A or TART. Since the primer in the 3'-UTR of HeT-A is located 400 bp upstream of the oligo(A) tail of the element, this part of the element should be present in these longer terminal elongation events if they are HeT-A. Perhaps these attachments are unusual versions of HeT-A and TART elements or unrelated DNA fragments. Thus, a low concentration of Ku80 causes an even higher frequency of new DNA additions.
Both Ku80 deficiencies resulted in a similar frequency of HeT-A/TART additions. The different origins of the Ku80 deficiencies diminishes the possibility that the observed 100-fold increase in terminal transpositions was due to a linked but unrelated mutation. To further test this possibility, we cloned the 12-kb fragment containing the Ku80 gene with its flanking regulatory regions into the CaSPeR4 plasmid (P{Ku80+}). One transformant was obtained that carried an insertion of the P{Ku80+} transposon on the CyO chromosome. To ask whether the P{Ku80+} transgene complements DfKu80, yTD/y ac, DfKu802/CyO females were crossed with y ac w/Y; If/CyO, P{Ku80+} males. yTD/y ac w; DfKu802/CyO, P{Ku80+} offspring were crossed to y ac w/Y; If/CyO, P{Ku80+} males. The frequency of HeT-A and TART attachments was monitored in three subsequent generations in y2TD/y ac w; DfKu802/CyO, P{Ku80+} females. In the presence of the transgene, the frequency of HeT-A/TART attachments was reduced to near wild-type levels (Table 1). We conclude that the deletion of the Ku80 gene in the deficiencies was responsible for the observed 100-fold increase in the frequency of HeT-A and TART attachments.
Erosion of terminal deficiencies:
Terminal chromosomal deficiencies lose 70–75 bp/generation (BIESSMANN and MASON 1988; BIESSMANN et al. 1990a, 1992a; LEVIS et al. 1993). To study the rate of terminal DNA loss in the presence of Ku70 (Irbp) and Ku80 deficiencies, we sampled DNA from yTD/yac; DfKu80/CyO and yTD/ac; Df(Ku70)/TM6 flies and control yTD/y ac; If/CyO; TM6/MKRS flies over six consecutive generations. In every generation, the size of the terminal fragments was measured using Southern blot analysis (Figure 1D). At the beginning of the experiment the chromosome ends were located at –800 bp and, after five generations, at –500 bp. In the presence of the Ku80 mutant and in the control, the chromosomes lost DNA sequences from the end at a rate of 70–80 bp/generation, indicating that the stability of the terminal chromosomal deficiencies is not sensitive to the concentration of the Ku80 protein. yTD/yac DfKu70R1 chromosomes exhibited a similar overall terminal loss, but starting in the second generation, y1-like females appeared among the progeny of DfKu70R1 mothers with a frequency 10–20%. The appearance of y1-like flies suggests occasional deletion of a larger DNA fragment than the expected 70–80 bp/generation. The progeny of 17 y1-like females were examined by Southern blot analysis (Figure 1E); all of them had termini at +230 relative to the y transcription start site, deleting the TATA promoter. Thus, the stability of the TD chromosomes may be sensitive to the Ku70 concentration.
In contrast to Ku80 deficiencies, deficiencies of Ku70 (Irbp) reduced viability of flies carrying TD chromosomes. In the presence of DfKu70R1, the viability of yTD/y ac females was reduced twofold compared with y ac/y ac females. To test any cooperation of Ku70 and Ku80 in protecting the ends of terminally deficient chromosomes, we tried to combine deficiencies for Ku70 and Ku80 with the yTD chromosome. However, among the progeny of the cross of yTD/y ac; DfKu802/CyO females with y ac/Y; TM6/DfKu70R1 males, we did not obtain any yTD/y ac females bearing both the Ku70 and the Ku80 deficiencies. Moreover, we obtained only two weak yTD/y ac females bearing DfKu70R1 among 134 yTD females. We conclude that some genetic factor on the DfKu70R1 chromosome may be contributing to reduced viability in the presence of the Ku80 and yTD chromosomes. However, in the absence of a duplication for Irbp, it is currently not possible to distinguish between a direct effect of the reduction in Ku70 concentration and genetic background effects.
Terminal gene conversion when a template for DNA synthesis is located on the homologous chromosome:
In the previous experiments we were unable to monitor the frequency of terminal gene conversion, because the homologous y ac chromosome carried a deletion of the y locus. We therefore also used as a homolog the y1 w chromosome, which carries the y1 allele that has an intact regulatory region but a nonproductive coding region yielding a y null phenotype.
The same two terminal deficiencies as before were used, yTD-700 and yTD-800 (Figure 3A). The addition of at least the body enhancer (at –1600 bp) to the ends of the deficient chromosomes via gene conversion would partially restore y expression in the body, giving rise to yr revertants. Further addition of y sequences increases the extent of pigmentation of the body cuticle and wing blades (MIKHAILOVSKY et al. 1999), allowing the observation of conversion tracks >800–900 bp. As in the first set of experiments, alternative elongation by HeT-A or TART transposition can be scored by the restoration of pigmentation in the aristae.
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In the case of DfKu70R1, we examined 3990 yTD/y w; DfKu70R1/TM6 flies during the F2–5 generations. Again, the frequency of new events was strongly increased at the fourth generation from the initial cross. In total we found 151 independent yr-like females (3.8%) and 16 y2(A+) females (0.4%), which were characterized on Southern blots with probes 1 and 2 as described above (Figure 3C). Thus, DfKu80 increased the frequency of terminal gene conversion 260-fold over control levels, while DfKu70 had a less pronounced effect, but still showed an 80-fold elevation over controls.
Terminal gene conversion when a template for DNA synthesis is located on the same chromosome:
Finally, we studied the effect of Ku70(Irbp) and Ku80 deficiencies on gene conversion in the presence of a template for gene conversion on the same terminally truncated chromosome. For this, derivatives of yTD2h2, which contain a terminally truncated X chromosome with duplicated y genes, including 875 bp of upstream y sequences up to the chromosomal end, were used (Figure 4A, top). In addition to this y duplication, a gypsy retrotransposon is inserted between the y enhancers and promoter at the –700-bp position. These yTD2h2 flies displayed a y2-like phenotype because the gypsy insulator blocks the interaction between the wing and body enhancers and the promoter (GEYER et al. 1986; GAUSE et al. 1998).
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Using Southern blot analysis, two yTD2h2/y ac; If/CyO; TM6/MKRS stocks were selected (Figure 4B) in which the termini of TD chromosomes were located at –900 or –1100 bp relative to the y transcription start site of the distal y copy (see Figure 4A). To activate y expression in the body and wings by acquiring a second gypsy insulator, the minimal size of the terminal DNA elongation by gene conversion must be 400–600 bp (see Figure 4A, middle). yTD2h2/y ac; DfKu70R1/TM6 and DfKu802/CyO females were constructed as described in Figure 2 and crossed individually to y ac/Y; DfKu70R1/TM6 or DfKu802/CyO males for four subsequent generations. yTD2h2/y ac; If/CyO; or yTD2h2/y ac; TM6/MKRS flies obtained in these crosses were used as an internal control. Only 3 (0.1%) yr-like females were found among 3423 females from the control, while both DfKu802 and DfKu70R1 induced terminal gene conversion with high frequency. Among 2464 yTD2h2/y w; DfKu802/CyO females scored during F2–5 generations, we found 563 (23%) yr-like females, which is 230-fold over the control. In the case of DfKu70R1, 94 (3.8%) yr-like females were found among 2445 yTD2h2/y ac; DfKu70R1/TM6 females during F3–5 generations, a 38-fold increase above the control.
To demonstrate that the yr derivatives were actually generated by gene conversion and had a structure as shown in Figure 4A, middle, the progeny of individual yr females were used for DNA preparation. Southern blot analysis showed a correlation between the y phenotype and the size of the terminal DNA fragment in the yr-like derivatives (Figure 4C). In most cases BamHI-digested DNAs isolated from the progeny of individual yr-like females showed broad bands of 2–10 kb with some minor bands of larger size, suggesting that terminal conversions extend the size of the original 1- to 1.4-kb BamHI terminal fragment (see Figure 4B).
Recombination between direct repeats located at the end of a truncated chromosome:
We reported previously that yTD2h2/y ac derivative females displaying a yr-like phenotype generated y2-like females with a frequency 0.2% (MELNIKOVA and GEORGIEV 2002). The new y2-like chromosomes likely originated by deletion of the duplicated y and gypsy sequences through recombination between homologous sequences (see Figure 4A, bottom). To determine if the reduced concentration of Ku70 or Ku80 enhanced the recombination between direct repeats, we compared the occurrence of y2-like females from yr-like females bearing either DfKu70R1/TM6 or DfKu802/CyO.
Four yTDrh chromosomes that had ends between –4400 and –8000 (yTDrh-4400, yTDrh-5000, yTDrh-5400, yTDrh-8000) relative to the y transcription start site in the distal gypsy element were selected (Figure 4A, middle, and Figure 4C). Six independent y2-like derivatives were found among 4920 yr-like females carrying DfKu802/CyO (0.12%). By Southern blot analysis, all y2-like derivatives lacked the internal 13-kb band, indicating the deletion of the duplicated y and gypsy sequences (Figure 4D). This frequency is similar to published values (MELNIKOVA and GEORGIEV 2002). Thus, DfKu802 does not influence the frequency of recombination between direct terminal repeats.
Three yTDrh chromosomes that had ends between –2200 and –5100 (yTDrh-2200, yTDrh-3400, yTDrh-5100) relative to the y transcription start site were selected by Southern blot analysis (Figure 4E). Among 1860 yr-like female progeny of yTDrh/y ac; DfKu70R1/TM6 females with the end of the TD chromosome at –2200 bp (i.e., in the distal gypsy element), 27 (1.5%) y2-like derivatives were found. In contrast, in two yTDrh/y ac; DfKu70R1/TM6 stocks with the ends of the TD chromosomes at either –3400 or –5100 bp, we found only 5 (0.17%) y2-like derivatives among 2935 yr-like females. Together with the results of DfKu80, this suggests that a lower concentration of Ku proteins does not influence the frequency of recombination among direct terminal repeats.
Southern blot analysis suggests that most of the y2-like derivatives obtained among the progeny of yTDrh-2200/y ac; DfKu70R1/TM6 females had the end of the TD chromosome in the region between the promoter of the distal y gene and the hobo insertion as indicated by the 2-kb band (Figure 4E). Since these chromosomes still contained the internal 10.7-kb fragment present in the original starting chromosome yTDrh-2200 hybridizing to the proximal copy of probe 3 (Figure 4E), we infer that these y2 derivatives were generated by extensive DNA loss induced by low concentration of Ku70 rather than by homologous recombination among the gypsy elements.
HeT-A transcription:
As mutations in the Su(var)2-5 gene encoding heterochromatin protein 1 (HP1) activate HeT-A transcription in the heterozygous condition (SAVITSKY et al. 2002; PERRINI et al. 2004), we tested whether the reduced concentration of Ku70 or Ku80 also has an effect on HeT-A transcription. We compared HeT-A transcript levels in DfKu70R1, DfKu801/CyO, DfKu802/CyO, TM6/MKRS, and If/CyO females obtained in the same crosses. As a control, we used Oregon, y1 w, and y ac lines (low level of HeT-A transcription) and Su(var)2-505/CyO (high level of HeT-A transcription). When RNA from flies was probed with sequences from any part of HeT-A, the major species of RNA detected was the sense-strand transcript of 6 kb (DANILEVSKAYA et al. 1994, 1999; GEORGE and PARDUE 2003). This is the expected size of a full-length transcript from the HeT-A element. In the DfKu70R1, DfKu801, and DfKu802 lines, the level of HeT-A transcript was the same as in the control lines, while in the Su(var)2-505 line it was 10 times more abundant (Figure 5). Thus, in contrast to the Su(var)2-5 mutation, reduced concentration of Ku70 or Ku80 did not effect HeT-A transcript levels.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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In all three elongation mechanisms tested, the Ku70 and Ku80 deficiencies had different effects on the TD chromosomes, and deficiencies for Ku80 always enhanced terminal DNA elongation more dramatically than deficiencies for Ku70. Deficiencies of Ku70 did not show an increase of HeT-A transposition until the fourth generation, perhaps because a decrease of Ku70 may have an accumulative effect during several generations due to the genetic background. Reduction of Ku70 showed a stronger effect on the stability of the chromosome terminus as evidenced by occasional deletions of longer DNA fragments. This accelerated terminal erosion may be caused by less efficient replication of the terminal sequences or by increased exonuclease action in Ku70 deficient flies Thus, apart from the protection of the telomeric ends by the cooperation of the Ku subunits in a heterodimer, Ku70 and Ku80 may have independent functions in Drosophila telomere metabolism. On the basis of the observation that Ku70 and Ku80 can translocate to the nucleus without forming a heterodimeric complex, independent functions for Ku70 and Ku80 have been suggested (KOIKE 2002). Alternatively, it is possible that uncontrolled genetic background effects may have contributed to the relatively minor differences seen between DfKu70 and DfKu80. We were unable to test the combined effect of DfKu70 and DfKu80 because the double deficiency for Ku70 and Ku80 in combination with the yTD chromosomes has a fivefold lower viability, and surviving females are frequently sterile.
One might suggest that Ku could interact with the RNA intermediates of the telomeric retrotransposons in Drosophila similarly to the binding of Ku to a certain region of the telomerase RNA template in yeast, which mediates telomerase activity or helps to recruit telomerase to the chromosome end (BERTUCH and LUNDBLAD 2003a; STELLWAGEN et al. 2003). However, this appears unlikely, because in that case, reduced Ku levels should result in reduced chromosome-end elongation events, while the opposite has been observed here. Although our genetic approach did not allow us to examine events at natural telomeres in the absence of Ku70 or Ku80, as has been done in yeast (BOULTON and JACKSON 1996; PORTER et al. 1996; BAUMANN and CECH 2000), the 100-fold elevated levels of HeT-A and TART transposition to a broken, but capped, chromosome end in Ku deficiency strains suggest easier accessibility of the end, predicting the accumulation of longer retrotransposon arrays at chromosome tips over time. This has been found for mutations in Su(var)205, the gene that encodes the telomere-binding protein, HP1 (EISSENBERG et al. 1990).
Ku has quite diverse effects on telomeres in different organisms, possibly reflecting its various interactions with the complex telomere elongation mechanism that comprises telomerase as well as the structure of the telomere itself. Ku mutations in S. cerevisiae severely reduce telomere elongation and result in shortened telomeric tracts with longer 3' single-strand overhangs (BOULTON and JACKSON 1996; PORTER et al. 1996; GRAVEL et al. 1998; POLOTNIANKA et al. 1998; MARINGELE and LYDALL 2002). In addition, loss of Ku function disrupts telomeric silencing (BOULTON and JACKSON 1998; LAROCHE et al. 1998; MISHRA and SHORE 1999) and causes amplification and rearrangement of subtelomeric sequences in the fission yeast S. pombe (BAUMANN and CECH 2000). In mammals, Ku deficiencies may cause shorter or longer telomeres. Telomere length is influenced by complex interactions, which, when compromised, result in telomere fusions that are independent of the length of the telomeric repeat arrays (BAILEY et al. 1999; SAMPER et al. 2000; D'ADDA DI FAGAGNA et al. 2001; ESPEJEL et al. 2002). However, in A. thaliana, lack of Ku70/80 causes dramatic deregulation of telomere length control, and telomere tracts grow to more than double the size of wild type. Apparently, Ku does not alter telomerase activity in plants and Ku-deficient plant cells do not show end-to-end chromosome fusions (BUNDOCK et al. 2002; RIHA et al. 2002; GALLEGO et al. 2003). The results in Drosophila demonstrating an increase in three different telomere elongation mechanisms are more in line with the observations in plants.
The question arises whether Ku may be a component of the terminal capping complex in Drosophila. Failure to effectively protect the chromosome ends because of shortened or absent telomeric repeats in yeast or human cells or by mutations in genes encoding important telomere-binding proteins, e.g., TRF2, often results in telomere-telomere fusions (VAN STEENSEL et al. 1998). Using mutations that cause telomere-telomere attachments, several potential components of the terminal capping complex in Drosophila have been identified. One is HP1 (FANTI et al. 1998), which binds to natural chromosome ends and to broken chromosome ends, such as those generated in the y gene region (BIESSMANN and MASON 1988). This telomere binding is independent of HeT-A and TART elements or of the subtelomeric repeats (FANTI et al. 1998; SIRIACO et al. 2002). Another proposed telomere capping protein is the HP1/ORC2-associated protein, HOAP (SHAREEF et al. 2001). Similar to mutations in Su(var)205 encoding HP1, the mutant caravaggio (cav), which lacks full-length HOAP, also exhibits a telomere fusion phenotype (CENCI et al. 2003). Finally, the Drosophila homologs of the mre/rad50 complex and ATM kinase, which play important roles at yeast and mammalian telomeres, are also required for telomere capping in Drosophila (QUEIROZ-MACHADO et al. 2001; BI et al. 2004; CIAPPONI et al. 2004; OIKEMUS et al. 2004; SILVA et al. 2004). However, Ku may provide a function at telomeres that is distinct from that of the capping complex, because a Ku70 deficiency does not exhibit a telomere fusion phenotype in neuroblasts (M. GATTI, personal communication).
Our results also suggest that a lower concentration of Ku proteins does not influence the frequency of recombination between duplicated y genes at the chromosome end. As suggested previously (SAVITSKY et al. 2002), these tandem repeats may facilitate loop formation, similar to the t-loop in mammals. This structure may partially protect the end of DNA from the transposition of HeT-A and TART elements and at the same time facilitate DNA elongation by terminal DNA conversion. Reduction of either Ku or HP1 concentration leads to increase of terminal DNA elongation but has no effect on recombination among these duplicated y genes. An explanation may be that suppression of terminal DNA recombination is mediated by a stable protein complex that is not affected by the decrease of concentration of one of its components.
What might be the function of Ku at Drosophila telomeres and at broken chromosome ends? Ku possesses ATPase and DNA helicase activity and is the DNA-binding moiety of the high-molecular-weight DNA-dependent protein kinase (DNA-PK) in mammals, which phosphorylates DNA repair factors, p53, and other proteins (SMITH and JACKSON 1999). In addition to its role in NHEJ, DNA-PK also plays a role in telomere protection, as DNA-PK-deficient mice exhibit not only severe immunodeficiency and radiosensitivity, but also an increased frequency of telomere fusions (GOYTISOLO et al. 2001). Interestingly, a gene encoding the catalytic subunit of DNA-PK is absent from D. melanogaster and D. pseudoobscura, but is present in another dipteran species, the mosquito Anopheles gambiae (DORé et al. 2004). Given the unique telomere structure of Drosophila, it is conceivable that Ku might be associated with a different kinase subunit at Drosophila telomeres or that it may interact with other proteins, such as components of the terminal capping complex.
In this context it might be revealing to compare the effects on elongation of TD chromosomes of reduced doses of the two known telomeric capping proteins, HP1 and HOAP, with those of Ku when tested in the same three assay systems. We show here that all three terminal elongation pathways are elevated in heterozygous Ku deficiencies. The presence of a heterozygous DfKu80 increased the frequency of HeT-A/TART attachments by 100-fold, the frequency of terminal gene conversion with the homolog by 260-fold, and conversion with tandem sequences on the same chromosome by 230-fold over controls. Similarly, heterozygous Su(var)205 mutants increased the frequency of HeT-A/TART attachments by 100-fold, increased conversion with tandem sequences on the same chromosome by 400-fold, but surprisingly had no effect on the frequency of terminal gene conversion with the homolog (SAVITSKY et al. 2002). While reduction of the concentration of either protein, HP1 or Ku, increases the rate of HeT-A transpositions and terminal gene conversion, transposition increases may be achieved by different means. In Su(var)2-5 mutations, HeT-A transcript levels are strongly elevated, but they are at wild-type levels in Ku mutants. Thus, HP1 appears to be more important in controlling HeT-A transcript levels, which in turn regulate transposition rates, while Ku may control accessibility of the telomere and be required for protecting the terminus from DNA repair. In the same test system, the cav mutation (CENCI et al. 2003) has only a weak effect on HeT-A/TART attachments (M. SAVITSKY and P. GEORGIEV, unpublished result). On the basis of these observations it seems reasonable to propose that the chromosome end is least protected when Ku levels are reduced, suggesting a central role for Ku in recruiting and/or assembling the terminal capping complex at the Drosophila chromosome end.
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JACOBY, D. B., and P. C. WENS
