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1.
Chromosomal rearrangements can result from crossing over during ectopic homologous recombination between dispersed repetitive DNA. We have previously shown that meiotic ectopic recombination between artificially dispersed ade6 heteroalleles in the fission yeast Schizosaccharomyces pombe frequently results in chromosomal rearrangements. The same recombination substrates have been studied in mitotic recombination. Ectopic recombination rates in haploids were approximately 1-4 x 10(-6) recombinants per cell generation, similar to allelic recombination rates in diploids. In contrast, ectopic recombination rates in heterozygous diploids were 2.5-70 times lower than allelic recombination or ectopic recombination in haploids. These results suggest that diploid-specific factors inhibit ectopic recombination. Very few crossovers occurred in ade6 mitotic recombination, either allelic or ectopic. Allelic intragenic recombination was associated with 2% crossing over, and ectopic recombination between multiple different pairing partners showed 1-7% crossing over. These results contrast sharply with the 35-65% crossovers associated with meiotic ade6 recombination and suggest either differential control of resolution of recombination intermediates or alternative pathways of recombination in mitosis and meiosis.  相似文献   

2.
F. Prado  A. Aguilera 《Genetics》1995,139(1):109-123
We have constructed novel DNA substrates (one inverted and three direct repeats) based on the same 0.6-kb repeat sequence to study deletions and inversions in Saccharomyces cerevisiae. Spontaneous deletions occur six to eight times more frequently than inversions, irrespective of the distance between the repeats. This difference can be explained by the observation that deletion events can be mediated by a recombination mechanism that can initiate within the intervening sequence of the repeats. Spontaneous and double-strand break (DSB) -induced deletions occur as RAD52-dependent and RAD52-independent events. Those deletion events initiated through a DSB in the unique intervening sequence require the Rad1/Rad10 endonuclease only if the break is distantly located from the flanking DNA repeats. We propose that deletions can occur as three types of recombination events: the conservative RAD52-dependent reciprocal exchange and the nonconservative events, one-ended invasion crossover, and single-strand annealing (SSA). We suggest that one-ended invasion is RAD52 dependent, whereas SSA is RAD52 independent. Whereas deletions, like inversions, occur through reciprocal exchange, deletions can also occur through SSA or one-ended invasion. We propose that the contribution of reciprocal exchange and one-ended invasion crossover vs. SSA events to overall spontaneous deletions is a feature specific for each repeat system, determined by the initiation event and the availability of the Rad52 protein. We discuss the role of the Rad1/Rad10 endonuclease on the initial steps of one-ended invasion crossover and SSA as a function of the location of the initiation event relative to the repeats. We also show that the frequency of recombination between repeats is the same independent of their location (whether on circular plasmids, linear minichromosomes, or natural chromosomes) and have similar RAD52 dependence.  相似文献   

3.
In this study, we examined homologous recombination in mammalian cells using a gene targeting assay in which the introduction of a double-strand-break (DSB) in the vector-borne region of homology to the chromosome resulted in targeted vector integration. The vector-borne DSB was flanked with small palindromic insertions that, when encompassed within heteroduplex DNA (hDNA) formed during targeted vector integration, were capable of avoiding the activity of the mismatch repair (MMR) system. When used in conjunction with an isolation procedure in which the product(s) of each targeted vector integration event were retained for molecular analysis, information about recombination mechanisms was obtained. The examination of marker segregation patterns in independent recombinants revealed the following, (i) hDNA tracts could form simultaneously on each side of the DSB and in both participating homologous regions. Clonal analysis of sectored recombinants revealed that, in the homologous repeats generated by the recombination event, vector-borne palindrome and chromosomal markers were linked in the expected way in each strand of the hDNA intermediate, (ii) hDNA tracts were subject to MMR processing that occurred on opposite sides of the DSB, and (iii) in the majority of recombinants, the vector-borne marker was replaced with the corresponding marker from the chromosome. Bidirectional hDNA formation and MMR processing of both sides of the DSB are consistent with the double-strand-break repair (DSBR) model of recombination.  相似文献   

4.
A. Nussbaum  M. Shalit    A. Cohen 《Genetics》1992,130(1):37-49
To test the double-strand break (DSB) repair model in recombination by the RecE pathway of Escherichia coli, we constructed chimeric phages that allow restriction-mediated release of linear plasmid substrates of the bioluminescence recombination assay in infected EcoRI+ cells. Kinetics of DSB repair and expression of recombination products were followed by Southern hybridization and by the bioluminescence recombination assay, respectively. Plasmid recombinants were analyzed with restriction endonucleases. Our results indicate that a DSB can induce more than one type of RecE-mediated recombination. A DSB within the homology induced intermolecular recombination that followed the rules of the DSB repair model: (1) Recombination was enhanced by in vivo restriction. (2) Repair of the break depended on homologous sequences on the resident plasmid. (3) Break-repair was frequently associated with conversion of alleles that were cis to the break. (4) Conversion frequency decreased as the distance from the break increased. (5) Some clones contained a mixture of plasmid recombinants as expected by replication of a heteroduplex in the primary recombinant. The rules of the DSB repair model were not followed when recombination was induced by a DSB outside the homology. Both the cut and the uncut substrates were recipients in conversion events. Recombination events were associated with deletions that spanned the break site, but these deletions did not reach the homology. We propose that a break outside the homology may stimulate a RecE-mediated recombination pathway that does not involve direct participation of DNA ends in the homologous pairing reaction.  相似文献   

5.
Genetic recombination of Xenopus laevis 5 S DNA in bacteria   总被引:2,自引:0,他引:2  
The behavior in genetic recombination of Xenopus laevis 5 S DNA has been examined, with particular emphasis on the role of 15-base-pair tandem repeats in the A + T-rich spacer. Fragments of 5 S DNA were introduced into Escherichia coli cells as inserts in the recombination vectors, lambda rva and lambda rvb. Intermolecular recombinants were selected in which, because of properties of the phage vectors, the crossover event must have occurred within the 5 S DNA inserts. Inserts from individual recombinants have been characterized in detail. The effects of varying the number (n) of 15-base-pair repeats and the recombination capabilities of the phage and host have been investigated. In these crosses, unequal crossovers can occur, yielding inserts different in size from the parental inserts. When the number of 15-mers is large (n = 12 or 20), most of the unequal crossovers have occurred within the 15-mers, resulting in an altered n value, although other homologies within the 5 S DNA sequence can also support unequal events. Increasing n in the parental inserts modestly increases the overall frequency of recombination and the percentage of altered inserts. We conclude that, in a bacterial setting, the 15-base-pair repeats stimulate recombination only slightly by allowing alternative registers for heteroduplex formation. The degree of stimulation observed is less than predicted by one simple model.  相似文献   

6.
Summary Hydroxyurea (HU) inhibits the premeiotic DNA replication and the meiotic events that follow, namely readiness, recombination commitment, haploidisation, sporulation commitment and ascus formation. Short incubations with HU (2–4 hrs) during the premeiotic replication (i.e. starting between 3 and 6.5 hrs in sporulation medium) allow the resumption of the replication at a normal rate following the removal of the drug. The other meiotic events are similarly delayed by the approximate length of the treatment. In these experiments, intragenic recombination in ade2 reached a higher level than in the controls (x1.3–2.0 in one pair of heteroalleles and x3.0–4.0 in another pair). The recombination response to short HU treatments was not observed for a pair of heteroalleles in ade2 that normally shows a high level of meiotic recombination (750 per 106 cells), nor was the response observed in a pair of heteroalleles in lys2. HU treatments have almost no effect on sporulating cells from 8 hrs onwards. At 7–7.5 hrs the meiotic cells are very sensitive to the drug and even short treatments cause cell death and massive DNA degradation.  相似文献   

7.
Li J  Baker MD 《Genetics》2000,154(3):1281-1289
We examined mechanisms of mammalian homologous recombination using a gene targeting assay in which the vector-borne region of homology to the chromosome bore small palindrome insertions that frequently escape mismatch repair when encompassed within heteroduplex DNA (hDNA). Our assay permitted the product(s) of each independent recombination event to be recovered for molecular analysis. The results revealed the following: (i) vector-borne double-strand break (DSB) processing usually did not yield a large double-strand gap (DSG); (ii) in 43% of the recombinants, the results were consistent with crossover at or near the DSB; and (iii) in the remaining recombinants, hDNA was an intermediate. The sectored (mixed) genotypes observed in 38% of the recombinants provided direct evidence for involvement of hDNA, while indirect evidence was obtained from the patterns of mismatch repair (MMR). Individual hDNA tracts were either long or short and asymmetric or symmetric on the one side of the DSB examined. Clonal analysis of the sectored recombinants revealed how vector-borne and chromosomal markers were linked in each strand of individual hDNA intermediates. As expected, vector-borne and chromosomal markers usually resided on opposite strands. However, in one recombinant, they were linked on the same strand. The results are discussed with particular reference to the double-strand-break repair (DSBR) model of recombination.  相似文献   

8.
The site-specific recombinase Cre must employ control mechanisms to impose directionality on recombination. When two recombination sites (locus of crossing over in phage P1, loxP) are placed as direct repeats on the same DNA molecule, collision between loxP-bound Cre dimers leads to excision of intervening DNA. If two sites are placed as inverted repeats, the intervening segment is flipped around. Cre catalyzes these reactions in the absence of protein co-factors. Current models suggest that directionality is controlled at two steps in the recombination pathway: the juxtaposition of loxP sites and the single-strand-transfer reactions within the synaptic complex. Here, we show that in Escherichia coli strain 294-Cre, directionality for recombination is altered when the expression of Cre is increased. This leads to deletion instead of inversion on substrates carrying two loxP sites as inverted repeats. The nucleotide sequence composition of loxP sites remaining in aberrant products indicates that site alignment and/or DNA strand transfer in the in vivo Cre-loxP recombination pathway are not always tightly controlled.  相似文献   

9.
Plasmid-Mediated Induction of Recombination in Yeast   总被引:5,自引:2,他引:3       下载免费PDF全文
R. Silberman  M. Kupiec 《Genetics》1994,137(1):41-48
Diploid yeast cells heteroallelic at the HIS3 locus were transformed with a minichromosome (centromeric plasmid) carrying homology to the HIS3 region and containing the same two mutations as were present in the chromosomes. When a double-strand break (DSB) was introduced in the region of homology, an increase in the recombination frequency between heteroalleles (leading to His(+) cells) was observed, although the plasmid was unable to donate wild-type information. This induction of recombination was dependent on the presence of homology between the plasmid sequences and the chromosomes. We show evidence for the physical involvement of the plasmid in tripartite recombination events, and we propose models that can explain the interactions between the plasmid-borne and chromosomal-borne alleles. Our results suggest that the mitotic induction of recombination by DNA damage is due to localized initiation of recombination events, and not to a general induction of recombination enzymes in the cell.  相似文献   

10.
11.
Loss of heterozygosity (LOH) is a common genetic alteration in tumors and often extends several megabases to encompass multiple genetic loci or even whole chromosome arms. Based on marker and karyotype analysis of tumor samples, a significant fraction of LOH events appears to arise from mitotic recombination between homologous chromosomes, reminiscent of recombination during meiosis. As DNA double-strand breaks (DSBs) initiate meiotic recombination, a potential mechanism leading to LOH in mitotically dividing cells is DSB repair involving homologous chromosomes. We therefore sought to characterize the extent of LOH arising from DSB-induced recombination between homologous chromosomes in mammalian cells. To this end, a recombination reporter was introduced into a mouse embryonic stem cell line that has nonisogenic maternal and paternal chromosomes, as is the case in human populations, and then a DSB was introduced into one of the chromosomes. Recombinants involving alleles on homologous chromosomes were readily obtained at a frequency of 4.6 x 10(-5); however, this frequency was substantially lower than that of DSB repair by nonhomologous end joining or the inferred frequency of homologous repair involving sister chromatids. Strikingly, the majority of recombinants had LOH restricted to the site of the DSB, with a minor class of recombinants having LOH that extended to markers 6 kb from the DSB. Furthermore, we found no evidence of LOH extending to markers 1 centimorgan or more from the DSB. In addition, crossing over, which can lead to LOH of a whole chromosome arm, was not observed, implying that there are key differences between mitotic and meiotic recombination mechanisms. These results indicate that extensive LOH is normally suppressed during DSB-induced allelic recombination in dividing mammalian cells.  相似文献   

12.
Errors associated with the repair of DNA double-strand breaks (DSBs) include point mutations caused by misincorporation during repair DNA synthesis or novel junctions made by nonhomologous end joining (NHEJ). We previously demonstrated that DNA synthesis is approximately 100-fold more error prone when associated with DSB repair. Here we describe a genetic screen for mutants that affect the fidelity of DSB repair. The substrate consists of inverted repeats of the trp1 and CAN1 genes. Recombinational repair of a site-specific DSB within the repeat yields TRP1 recombinants. Errors in the repair process can be detected by the production of canavanine-resistant (can1) mutants among the TRP1 recombinants. In wild-type cells the recombinational repair process is efficient and fairly accurate. Errors resulting in can1 mutations occur in <1% of the TRP1 recombinants and most appear to be point mutations. We isolated several mutant strains with altered fidelity of recombination. Here we characterize one of these mutants that revealed an approximately 10-fold elevation in the frequency of can1 mutants among TRP1 recombinants. The gene was cloned by complementation of a coincident sporulation defect and proved to be an allele of SAE2/COM1. Physical analysis of the can1 mutants from sae2/com1 strains revealed that many were a novel class of chromosome rearrangement that could reflect break-induced replication (BIR) and NHEJ. Strains with either the mre11s-H125N or rad50s-K81I alleles had phenotypes in this assay that are similar to that of the sae2/com1Delta strain. Our data suggest that Sae2p/Com1p plays a role in ensuring that both ends of a DSB participate in a recombination event, thus avoiding BIR, possibly by regulating the nuclease activity of the Mre11p/Rad50p/Xrs2p complex.  相似文献   

13.
Homologous recombination at the bacterial transposon Tn7 donor site is stimulated 10-fold when Tn7 is activated to transpose at high frequency in RecD(-) Escherichia coli, where recombination is focused near the ends of double-chain breaks. This is observed as an increase in recombination between two lacZ heteroalleles when one copy of lacZ carries within it a Tn7 that is transposing at high frequency. This stimulation of recombination is dependent upon the presence of homology with the donor site, is independent of SOS induction, and is not due to a global stimulation of recombination. When stimulated by Tn7 transposition, the conversion events giving rise to Lac(+) recombinants occur preferentially at the site of Tn7, suggesting that transposition is stimulating gene conversion at the donor site. These results support the model that Tn7 transposition occurs by a ``cut and paste' mechanism, leaving a double-chain break at the donor site that is repaired by the host homologous recombination machinery; normally, repair would use homology in a sister chromosome to regenerate a copy of the transposon. This proposed series of events allows transposition that is nonreplicative, per se, to be effectively replicative.  相似文献   

14.
Inverted DNA repeats: a source of eukaryotic genomic instability.   总被引:17,自引:5,他引:12       下载免费PDF全文
While inverted DNA repeats are generally acknowledged to be an important source of genetic instability in prokaryotes, relatively little is known about their effects in eukaryotes. Using bacterial transposon Tn5 and its derivatives, we demonstrate that long inverted repeats also cause genetic instability leading to deletion in the yeast Saccharomyces cerevisiae. Furthermore, they induce homologous recombination. Replication plays a major role in the deletion formation. Deletions are stimulated by a mutation in the DNA polymerase delta gene (pol3). The majority of deletions result from imprecise excision between small (4- to 6-bp) repeats in a polar fashion, and they often generate quasipalindrome structures that subsequently may be highly unstable. Breakpoints are clustered near the ends of the long inverted repeats (< 150 bp). The repeats have both intra- and interchromosomal effects in that they also create hot spots for mitotic interchromosomal recombination. Intragenic recombination is 4 to 18 times more frequent for heteroalleles in which one of the two mutations is due to the insertion of a long inverted repeat, compared with other pairs of heteroalleles in which neither mutation has a long repeat. We propose that both deletion and recombination are the result of altered replication at the basal part of the stem formed by the inverted repeats.  相似文献   

15.
McCulloch RD  Read LR  Baker MD 《Genetics》2003,163(4):1439-1447
Analysis of the crossover products recovered following transformation of mammalian cells with a sequence insertion ("ends-in") gene-targeting vector revealed a novel class of recombinant. In this class of recombinants, a single vector copy has integrated into an ectopic genomic position, leaving the structure of the cognate chromosomal locus unaltered. Thus, in this respect, the recombinants resemble simple cases of random vector integration. However, the important difference is that the two paired 3' vector ends have acquired endogenous, chromosomal sequences flanking both sides of the vector-borne double-strand break (DSB). In some cases, copying was extensive, extending >16 kb into nonhomologous flanking DNA. The results suggest that mammalian homologous recombination events can involve strand invasion and DNA synthesis by both 3' ends of the DSB. These DNA interactions are a central, predicted feature of the DSBR model of recombination.  相似文献   

16.
Z. Silberstein  M. Shalit    A. Cohen 《Genetics》1993,133(3):439-448
The RecE recombination pathway is active in Escherichia coli recB recC sbcA mutants. To isolate and characterize products and intermediates of RecE-mediated, break-induced, intramolecular recombination, we infected recB recC sbcA mutants, expressing EcoRI endonuclease, with chimeric λ phages that allow EcoRI-mediated release of cloned linear recombination substrates. Substrates with direct terminal repeats recombined to yield a circular product with one copy of the repeated sequence. Some recombinants were heteroallelic for the recombining markers. Markers distant to the break were recovered in the circular product at a higher frequency than markers close to the break. To examine the heteroduplex structures that may have yielded the heteroallelic recombinants, nonreplicative substrates were employed. Some of the nonreplicative recombination products contained heteroduplexes, with a strong bias for paired strands ending 3' at the break. This strand bias in heteroduplex formation is consistent with recombination models that postulate homologous pairing of protruding 3' single-stranded ends.  相似文献   

17.
The budding yeast INO80 complex has a role in remodeling chromatin structure, and the SWR1 complex replaces a H2A/H2B dimer with a variant dimer, H2A.Z (Htz1)/H2B. It has been reported that these chromatin remodeling complexes contain Arp4 (actin-related protein) and actin in common and are recruited to HO endonuclease-induced DNA double-strand break (DSB) site. Reportedly, Ino80 can evict nucleosomes surrounding a HO-induced DSB; however, it has no apparent role to play in the repair of HO-induced DSB. Here we show that an essential factor for INO80 chromatin remodeling activity, Arp8, is involved in damage-induced sister chromatid recombination and interchromosomal recombination between heteroalleles. In contrast, arp6 mutants are proficient for recombination, indicating that the SWR1 complex does not promote recombination. Our data suggest that the remodeling of chromatin by the INO80 complex facilitates efficient homologous recombination upon DNA damages.  相似文献   

18.
DNA double strand breaks (DSB) can be repaired either via a sequence independent joining of DNA ends or via homologous recombination. We established a detection system in Drosophila melanogaster to investigate the impact of sequence constraints on the usage of the homology based DSB repair via single strand annealing (SSA), which leads to recombination between direct repeats with concomitant loss of one repeat copy. First of all, we find the SSA frequency to be inversely proportional to the spacer length between the repeats, for spacers up to 2.4 kb in length. We further show that SSA between divergent repeats (homeologous SSA) is suppressed in cell cultures and in vivo in a sensitive manner, recognizing sequence divergences smaller than 0.5%. Finally, we demonstrate that the suppression of homeologous SSA depends on the Bloom helicase (Blm), encoded by the Drosophila gene mus309. Suppression of homeologous recombination is a novel function of Blm in ensuring genomic integrity, not described to date in mammalian systems. Unexpectedly, distinct from its function in Saccharomyces cerevisiae, the mismatch repair factor Msh2 encoded by spel1 does not suppress homeologous SSA in Drosophila.  相似文献   

19.
Preferred crossover sites on polyomavirus DNA.   总被引:5,自引:3,他引:2       下载免费PDF全文
RmI is a hybrid replicon consisting of polyomavirus (Py) and mouse sequences that yields unit-length polyomavirus DNA via recombination between two directly repeated viral sequences of 182 base pairs (S repeats). To define the contribution of the S repeats in this intramolecular recombination, we derived from RmI a series of replicons containing the original S repeats as well as additional direct viral repeats which were 1 to 2 kilobases in length (L repeats). After mouse 3T6 cells were transfected with these constructs, recombination products that displayed the physical properties of homologous recombinants were detected. The structures of these recombinants indicated that whereas repeat length influences the likelihood of recombination, crossover occurs preferentially near the S repeats, provided that one of them is proximal to the viral origin of replication. This finding suggests that recombination near the S repeats depends on a process initiated near the viral origin of replication.  相似文献   

20.
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.  相似文献   

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