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1.
Repair of DNA double-strand breaks can occur by either nonhomologous end joining or homologous recombination. Most nonhomologous end joining requires a specialized ligase, DNA ligase IV (Lig4). In Drosophila melanogaster, double-strand breaks created by excision of a P element are usually repaired by a homologous recombination pathway called synthesis-dependent strand annealing (SDSA). SDSA requires strand invasion mediated by DmRad51, the product of the spn-A gene. In spn-A mutants, repair proceeds through a nonconservative pathway involving the annealing of microhomologies found within the 17-nt overhangs produced by P excision. We report here that end joining of P-element breaks in the absence of DmRad51 does not require Drosophila LIG4. In wild-type flies, SDSA is sometimes incomplete, and repair is finished by an end-joining pathway that also appears to be independent of LIG4. Loss of LIG4 does not increase sensitivity to ionizing radiation in late-stage larvae, but lig4 spn-A double mutants do show heightened sensitivity relative to spn-A single mutants. Together, our results suggest that a LIG4-independent end-joining pathway is responsible for the majority of double-strand break repair in the absence of homologous recombination in flies. 相似文献
2.
The Drosophila melanogaster DmRAD54 Gene Plays a Crucial Role in Double-Strand Break Repair after P-Element Excision and Acts Synergistically with Ku70 in the Repair of X-Ray Damage 下载免费PDF全文
Rolf Kooistra Albert Pastink Jos B. M. Zonneveld Paul H. M. Lohman Jan C. J. Eeken 《Molecular and cellular biology》1999,19(9):6269-6275
The RAD54 gene has an essential role in the repair of double-strand breaks (DSBs) via homologous recombination in yeast as well as in higher eukaryotes. A Drosophila melanogaster strain deficient in the RAD54 homolog DmRAD54 is characterized by increased X-ray and methyl methanesulfonate (MMS) sensitivity. In addition, DmRAD54 is involved in the repair of DNA interstrand cross-links, as is shown here. However, whereas X-ray-induced loss-of-heterozygosity (LOH) events were completely absent in DmRAD54(-/-) flies, treatment with cross-linking agents or MMS resulted in only a slight reduction in LOH events in comparison with those in wild-type flies. To investigate the relative contributions of recombinational repair and nonhomologous end joining in DSB repair, a DmRad54(-/-)/DmKu70(-/-) double mutant was generated. Compared with both single mutants, a strong synergistic increase in X-ray sensitivity was observed in the double mutant. No similar increase in sensitivity was seen after treatment with MMS. Apparently, the two DSB repair pathways overlap much less in the repair of MMS-induced lesions than in that of X-ray-induced lesions. Excision of P transposable elements in Drosophila involves the formation of site-specific DSBs. In the absence of the DmRAD54 gene product, no male flies could be recovered after the excision of a single P element and the survival of females was reduced to 10% compared to that of wild-type flies. P-element excision involves the formation of two DSBs which have identical 3' overhangs of 17 nucleotides. The crucial role of homologous recombination in the repair of these DSBs may be related to the very specific nature of the breaks. 相似文献
3.
Dianov GL O'Neill P Goodhead DT 《BioEssays : news and reviews in molecular, cellular and developmental biology》2001,23(8):745-749
In addition to double- and single-strand DNA breaks and isolated base modifications, ionizing radiation induces clustered DNA damage, which contains two or more lesions closely spaced within about two helical turns on opposite DNA strands. Post-irradiation repair of single-base lesions is routinely performed by base excision repair and a DNA strand break is involved as an intermediate. Simultaneous processing of lesions on opposite DNA strands may generate double-strand DNA breaks and enhance nonhomologous end joining, which frequently results in the formation of deletions. Recent studies support the possibility that the mechanism of base excision repair contributes to genome stability by diminishing the formation of double-strand DNA breaks during processing of clustered lesions. 相似文献
4.
We have examined eight germline revertants generated by the excision of Tc1 from a site within the unc-22 gene of Caenorhabditis elegans. A rich variety of rearrangements accompanied Tc1 excision at this site, including transposon 'footprints', deletions of sequences flanking the insertion site and direct nontandem duplications of flanking DNA. With only modest modification the double-strand gap repair model for transposition, recently proposed by Engles and coworkers (Cell 62: 515-525 1990), can explain even the most complex of these rearrangements. In light of this model rearrangements of the target site accompanying transposition/excision may not be the end result of imprecise excision of the element. Instead, these rearrangements may be the result of imprecise repair of the double-strand gap by the host replication and repair machinery. Sequences surrounding an insertion site influence the fidelity of gap repair by this machinery. This may lead to a number of possible resolutions of a double-strand gap as documented here for a Tc1 site in unc-22. 相似文献
5.
End-joining of blunt DNA double-strand breaks in mammalian fibroblasts is precise and requires DNA-PK and XRCC4 总被引:3,自引:0,他引:3
DNA double-strand break repair by non-homologous end-joining (NHEJ) is generally considered to be an imprecise repair pathway. In order to study repair of a blunt, 5' phosphorylated break in the DNA of mammalian fibroblasts, we used the E. coli cut-and-paste type transposon Tn5. We found that the Tn5 transposase can mediate transposon excision in Chinese hamster cell lines. Interestingly, a blunt 5' phosphorylated break could efficiently be repaired without loss of nucleotides in wild type fibroblasts. Catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) deficiency reduced the efficiency of joining four-fold without reducing precision, whereas both efficiency and accuracy of joining were affected in Ku80 or XRCC4 mutant cell lines. These results show that both the DNA-PK and the XRCC4/ligase IV complexes are required for NHEJ and that other, more error-prone, repair processes cannot efficiently substitute for joining of blunt breaks produced in living cells. Interestingly, the severity of the end-joining defect differs between the various mutants, which may explain the difference in the severity of the phenotypes, which have been observed in the corresponding mouse models. 相似文献
6.
The transposases of DNA transposable elements catalyze the excision of the element from the host genome, but are not involved in the repair of the resulting double-strand break. To elucidate the role of various host DNA repair and damage response proteins in the repair of the hairpin-ended double strand breaks (DSBs) generated during excision of the maize Ac element in Arabidopsis thaliana, we deep-sequenced hundreds of thousands of somatic excision products from a variety of repair- or response-defective mutants. We find that each of these repair/response defects negatively affects the preservation of the ends, resulting in an enhanced frequency of deletions, insertions, and inversions at the excision site. The spectra of the resulting repair products demonstrate, not unexpectedly, that the canonical nonhomologous end joining (NHEJ) proteins DNA ligase IV and KU70 play an important role in the repair of the lesion generated by Ac excision. Our data also indicate that auxiliary NHEJ repair proteins such as DNA ligase VI and DNA polymerase lambda are routinely involved in the repair of these lesions. Roles for the damage response kinases ATM and ATR in the repair of transposition-induced DSBs are also discussed. 相似文献
7.
Lig4 and rad54 are required for repair of DNA double-strand breaks induced by P-element excision in Drosophila 下载免费PDF全文
Romeijn RJ Gorski MM van Schie MA Noordermeer JN Mullenders LH Ferro W Pastink A 《Genetics》2005,169(2):795-806
Site-specific double-strand breaks (DSBs) were generated in the white gene located on the X chromosome of Drosophila by excision of the w(hd) P-element. To investigate the role of nonhomologous end joining (NHEJ) and homologous recombination (HR) in the repair of these breaks, the w(hd) P-element was mobilized in flies carrying mutant alleles of either lig4 or rad54. The survival of both lig4- and rad54-deficient males was reduced to 25% in comparison to the wild type, indicating that both NHEJ and HR are involved in the repair P-induced gaps in males. Survival of lig4-deficient females was not affected at all, implying that HR using the homologous chromosome as a template can partially compensate for the impaired NHEJ pathway. In rad54 mutant females survival was reduced to 70% after w(hd) excision. PCR analysis indicated that the undamaged homologous chromosome may compensate for the potential loss of the broken chromosome in rad54 mutant females after excision. Molecular analysis of the repair junctions revealed microhomology (2-8 bp)-dependent DSB repair in most products. In the absence of Lig4, the 8-bp target site duplication is used more frequently for repair. Our data indicate the presence of efficient alternative end-joining mechanisms, which partly depend on the presence of microhomology but do not require Lig4. 相似文献
8.
P element excision generates a DNA double-strand break at the transposon donor site. Genetic studies have demonstrated a strong bias toward repair of P element-induced DNA breaks by homologous recombination with the sister chromatid, suggesting that P element excision occurs after DNA replication, in G2 of the cell cycle. We developed methods to arrest Drosophila tissue culture cells and assay P element excision in either G1- or G2-arrested cells. Dacapo or tribbles transgene expression arrests cells in either G2 or G2, respectively. RNA-mediated gene interference (RNAi) directed against cyclin E or cyclin A arrests cells in G1 or G2, respectively. P element excision occurs efficiently in both G1- and G2-arrested cells, suggesting that cell cycle regulation of P element transposase does not occur in our somatic cell system. DNA double-strand break repair occurs by two predominant mechanisms: homologous recombination (HR) and non-homologous end joining (NHEJ). HR is thought to be restricted to the post-replicative, G2, phase of the cell cycle, while NHEJ may occur throughout the cell cycle. Our results indicate that NHEJ repair of an extrachromasomal plasmid substrate occurs at least as efficiently in G2-arrested cells as in asynchronous cells or in G1-arrested cells. 相似文献
9.
10.
DNA double-strand breaks are repaired by multiple mechanisms that are roughly grouped into the categories of homology-directed repair and non-homologous end joining. End-joining repair can be further classified as either classical non-homologous end joining, which requires DNA ligase 4, or “alternative” end joining, which does not. Alternative end joining has been associated with genomic deletions and translocations, but its molecular mechanism(s) are largely uncharacterized. Here, we report that Drosophila melanogaster DNA polymerase theta (pol theta), encoded by the mus308 gene and previously implicated in DNA interstrand crosslink repair, plays a crucial role in DNA ligase 4-independent alternative end joining. In the absence of pol theta, end joining is impaired and residual repair often creates large deletions flanking the break site. Analysis of break repair junctions from flies with mus308 separation-of-function alleles suggests that pol theta promotes the use of long microhomologies during alternative end joining and increases the likelihood of complex insertion events. Our results establish pol theta as a key protein in alternative end joining in Drosophila and suggest a potential mechanistic link between alternative end joining and interstrand crosslink repair. 相似文献
11.
Evidence for multiple cycles of strand invasion during repair of double-strand gaps in Drosophila 总被引:1,自引:0,他引:1
DNA double-strand breaks (DSBs), a major source of genome instability, are often repaired through homologous recombination pathways. Models for these pathways have been proposed, but the precise mechanisms and the rules governing their use remain unclear. In Drosophila, the synthesis-dependent strand annealing (SDSA) model can explain most DSB repair. To investigate SDSA, we induced DSBs by excision of a P element from the male X chromosome, which produces a 14-kb gap relative to the sister chromatid. In wild-type males, repair synthesis tracts are usually long, resulting in frequent restoration of the P element. However, repair synthesis is often incomplete, resulting in internally deleted P elements. We examined the effects of mutations in spn-A, which encodes the Drosophila Rad51 ortholog. As expected, there is little or no repair synthesis in homozygous spn-A mutants after P excision. However, heterozygosity for spn-A mutations also resulted in dramatic reductions in the lengths of repair synthesis tracts. These findings support a model in which repair DNA synthesis is not highly processive. We discuss a model wherein repair of a double-strand gap requires multiple cycles of strand invasion, synthesis, and dissociation of the nascent strand. After dissociation, the nascent strand may anneal to a complementary single strand, reinvade a template to be extended by additional synthesis, or undergo end joining. This model can explain aborted SDSA repair events and the prevalence of internally deleted transposable elements in genomes. 相似文献
12.
We examined the influence that heterologous sequences of different sizes have on the frequency of double-strand-break repair by gene conversion in Drosophila melanogaster. We induced a double-strand break on one X chromosome in female flies by P-element excision. These flies contained heterologous insertions of various sizes located 238 bp from the break site in cis or in trans to the break, or both. We observed a significant decrease in double-strand-break repair with large heterologous insertions located either in cis or in trans to the break. Reestablishing the homology by including the same heterologous sequence in cis and in trans to the double-strand break restored the frequency of gene conversion to wild-type levels. In one instance, an allelic nonhomologous insertion completely abolished repair by homologous recombination. The results show that the repair of a double-strand break by gene conversion requires chromosome pairing in the local region of the double-strand break. 相似文献
13.
Removal of the Bloom Syndrome DNA Helicase Extends the Utility of Imprecise Transposon Excision for Making Null Mutations in Drosophila 下载免费PDF全文
Transposable elements are frequently used in Drosophila melanogaster for imprecise excision screens to delete genes of interest. However, these screens are highly variable in the number and size of deletions that are recovered. Here, we show that conducting excision screens in mus309 mutant flies that lack DmBlm, the Drosophila ortholog of the Bloom syndrome protein, increases the percentage and overall size of flanking deletions recovered after excision of either P or Minos elements.TRANSPOSABLE elements have a rich history as mutagenesis tools in Drosophila melanogaster (reviewed in Ryder and Russell 2003). Initially, researchers focused their efforts on the use of nonautonomous P-element transposons for gene disruption (Cooley et al. 1988). However, P elements have insertion biases, preferring to transpose into euchromatic regions, the 5′ regions of genes (Tsubota et al. 1985; Kelley et al. 1987), and to target sequence motifs similar to the octamer GGCCAGAC (O''Hare and Rubin 1983). These biases make it unlikely that full genome saturation will be reached using P-element mutagenesis. Therefore, mutational systems that utilize transposable elements with different insertion biases have been developed. These include Hobo (Smith et al. 1993); the lepidopteran-derived piggyBac element, which inserts at TTAA sites (Hacker et al. 2003; Horn et al. 2003); and Minos, a Tc-1/mariner-like element originally isolated from Drosophila hydei that inserts at TA dinucleotides (Franz and Savakis 1991; Loukeris et al. 1995). Using a combination of these transposons, the Drosophila Gene Disruption Project has generated inserts in ∼60% of the 14,850 annotated genes (Spradling et al. 1999; Bellen et al. 2004).In spite of the growing number of transposon insertions in the Drosophila genome, many are inserted in regions that do not completely abolish gene function, such as 5′-UTRs and introns. This can make it difficult to discern the true null phenotypes of genes. Furthermore, there still exist a sizable number of genes for which no transposon insertions are available. To address these issues, many transposons have been constructed with additional characteristics, such as FRT sites, that make generation of molecularly defined deletions by site-specific recombination relatively straightforward (Parks et al. 2004; Thibault et al. 2004; Ryder et al. 2007). However, until saturation of the genome with these designer transposons is achieved, their utility in creating single-gene deletions remains limited.A more general approach for generating single-gene deletions that has proven successful is the use of P elements in imprecise excision screens. Excision of a P element creates a DNA double-strand break with 17 nucleotide noncomplementary ends (Beall and Rio 1997). If the ends of the break are degraded prior to repair, a deletion of DNA flanking the original insertion site is created (reviewed in Hummel and Klambt 2008). On average, the frequency of flanking deletions recovered from imprecise excision screens is ∼1%. However, this frequency varies tremendously by locus and depends on a multitude of factors that are not well understood, including chromatin structure and local sequence context. Therefore, generation of suitable deletion mutants frequently involves screening many hundreds of independent lines.An alternative method that uses P elements to generate deletions involves screening for events associated with male recombination. These events, which probably arise through a hybrid element insertion mechanism, generate one-sided deletions of sizes ranging from several base pairs to several kilobases (Preston and Engels 1996). This method, although powerful, involves screening a large number of flies and requires two sequential screens to generate bidirectional deletions.P-element-induced double-strand breaks are preferentially repaired through homologous recombination using a sister chromatid or a homologous chromosome as a template (Engels et al. 1990). Previously, we and others have demonstrated that the Drosophila Bloom protein ortholog (DmBlm), a RecQ DNA helicase encoded by mus309, is involved in homology-directed repair of these breaks (Beall and Rio 1996; McVey et al. 2004a). In the absence of DmBlm, repair of a P-element-induced break on a plasmid or at a chromosomal locus frequently results in a large, flanking deletion. Several groups have applied this observation to imprecise excision screens using P elements and have successfully recovered multiple deletions (Astrom et al. 2003; Johansson et al. 2007; Y. Rong, unpublished data). However, a direct comparison between imprecise excision screens carried out in wild-type vs. mus309 mutant backgrounds has not been published, and little is known regarding the use of this technique with other types of transposable elements. In this study, we used three different transposons to test the hypothesis that the use of a mus309 mutant background in imprecise excision screens would result in a greater yield of deletions and that these deletions would be larger than those recovered from a wild-type background.Insertion Wild-type: % sterility mus309: % sterility Wild-type: % of males producing excision mus309: % of males producing excision Trf4-1EY14679 16.5 (97)a 15.4 (136) 32.1 (81) 62.6 (115) mus205EY20083 33.3 (108) 31.8 (88) 100 (72) 71.6 (60) Pvf1MB01242 10.4 (113) 9.9 (191) 71.2 (101) 69.7 (172) dpMB00453 40.2 (87) 49.2 (61) 73.0 (52) 87.1 (31) TequilaMB00537
11.3 (53)
14.2 (70)
83.0 (47)
70.0 (60)