Recombination-induced CAG trinucleotide repeat expansions in yeast involve the MRE11–RAD50–XRS2 complex
2000; Springer Nature; Volume: 19; Issue: 10 Linguagem: Inglês
10.1093/emboj/19.10.2381
ISSN1460-2075
AutoresGuy‐Franck Richard, Geoffrey M. Goellner, Cynthia T. McMurray, James E. Haber,
Tópico(s)Fungal and yeast genetics research
ResumoArticle15 May 2000free access Recombination-induced CAG trinucleotide repeat expansions in yeast involve the MRE11–RAD50–XRS2 complex Guy-Franck Richard Guy-Franck Richard Rosenstiel Center and Department of Biology, MS029, Brandeis University, Waltham, MA, 02454-9110 USA Present address: Unité de Génétique Moléculaire des Levures, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, Cedex 15, France Search for more papers by this author Geoffrey M. Goellner Geoffrey M. Goellner Molecular Neurosciences Program, Mayo Clinic and Foundation, Rochester, MN, 55905 USA Search for more papers by this author Cynthia T. McMurray Cynthia T. McMurray Molecular Neurosciences Program, Mayo Clinic and Foundation, Rochester, MN, 55905 USA Department of Pharmacology, Mayo Clinic and Foundation, Rochester, MN, 55905 USA Search for more papers by this author James E. Haber Corresponding Author James E. Haber Rosenstiel Center and Department of Biology, MS029, Brandeis University, Waltham, MA, 02454-9110 USA Search for more papers by this author Guy-Franck Richard Guy-Franck Richard Rosenstiel Center and Department of Biology, MS029, Brandeis University, Waltham, MA, 02454-9110 USA Present address: Unité de Génétique Moléculaire des Levures, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, Cedex 15, France Search for more papers by this author Geoffrey M. Goellner Geoffrey M. Goellner Molecular Neurosciences Program, Mayo Clinic and Foundation, Rochester, MN, 55905 USA Search for more papers by this author Cynthia T. McMurray Cynthia T. McMurray Molecular Neurosciences Program, Mayo Clinic and Foundation, Rochester, MN, 55905 USA Department of Pharmacology, Mayo Clinic and Foundation, Rochester, MN, 55905 USA Search for more papers by this author James E. Haber Corresponding Author James E. Haber Rosenstiel Center and Department of Biology, MS029, Brandeis University, Waltham, MA, 02454-9110 USA Search for more papers by this author Author Information Guy-Franck Richard1,2, Geoffrey M. Goellner3, Cynthia T. McMurray3,4 and James E. Haber 1 1Rosenstiel Center and Department of Biology, MS029, Brandeis University, Waltham, MA, 02454-9110 USA 2Present address: Unité de Génétique Moléculaire des Levures, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, Cedex 15, France 3Molecular Neurosciences Program, Mayo Clinic and Foundation, Rochester, MN, 55905 USA 4Department of Pharmacology, Mayo Clinic and Foundation, Rochester, MN, 55905 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:2381-2390https://doi.org/10.1093/emboj/19.10.2381 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Recombination induced by double-strand breaks (DSBs) in yeast leads to a higher proportion of expansions to contractions than does replication-associated tract length changes. Expansions are apparently dependent on the property of the repeat array to form hairpins, since DSB repair of a CAA87 repeat induces only contractions of the repeat sequence. DSB-repair efficiency is reduced by 40% when DNA synthesis must traverse a CAG98 array, as compared with a CAA87 array. These data indicate that repair- associated DNA synthesis is inhibited by secondary structures formed by CAG98 and that these structures promote repeat expansions during DSB repair. Overexpression of Mre11p or Rad50p suppresses the inhibition of DSB repair by CAG98 and significantly increases the average size of expansions found at the recipient locus. Both effects are dependent on the integrity of the Mre11p–Rad50p–Xrs2p complex. The Mre11 complex thus appears to be directly involved in removing CAG or CTG hairpins that arise frequently during DNA synthesis accompanying gene conversion of these trinucleotide repeats. Introduction A growing number of human diseases involving trinucleotide repeat expansions have been discovered (Ashley and Warren, 1995; Reddy and Housman, 1997; Richards and Sutherland, 1997). This class of microsatellites has the property of undergoing rapid and sometimes massive expansion of their repeat number, during germline transmission or early embryogenesis. One striking feature of all expansion-associated microsatellites is their propensity to form secondary structures. CAG/CTG trinucleotide repeats have been shown to form stable hairpins in vitro (Gacy et al., 1995; Mitas et al., 1995b; Yu and Mitas, 1995), as do CCG/CGG trinucleotide repeats (Gacy et al., 1995; Mitas et al., 1995a; Yu et al., 1997). GAA repeats involved in Friedreich's ataxia (Campuzano et al., 1996) were shown to form triple helices in vitro (Gacy et al., 1998; Sakamoto et al., 1999). The ability to form stable secondary structures is thought to play a role during the expansion process, perhaps by slowing down or stalling replication (Samadashwily et al., 1997). Replication pausing at trinucleotide repeats has been shown in vitro for CCG (Usdin and Woodford, 1995) and GAA (Gacy et al., 1998), and for CTG and CCG in vivo (Samadashwily et al., 1997). The mechanism by which trinucleotide secondary structures mediate expansions is not well understood. Recent data suggest that several mechanisms may be needed to give rise to the full spectrum of large expansions observed in human disease (reviewed by Wells, 1996; McMurray, 1999; Richard et al., 1999a). In model systems, trinucleotide repeats can undergo tract length changes during replication, as shown in both Escherichia coli (Kang et al., 1995) and Saccharomyces cerevisiae (Maurer et al., 1996; Freudenreich et al., 1997; Miret et al., 1998; Schweitzer and Livingston, 1998). However, in wild-type strains, starting with CAG templates up to 100 repeats, the vast majority of tract length changes are deletions (Maurer et al., 1996; Freudenreich et al., 1997; Miret et al., 1998; Schweitzer and Livingston, 1998). In contrast, in mutant strains carrying mutations in proteins involved in DNA replication or repair, expansions are frequently seen. In S.cerevisiae, the absence of the Rad27/FEN-1 nuclease that processes Okazaki fragments during DNA replication causes increases in the size of CAG repeats (Freudenreich et al., 1998; Schweitzer and Livingston, 1998; Spiro et al., 1999). Rad27/FEN-1 is also implicated in long-patch excision repair (Gary et al., 1999). In E.coli, CAG repeats were dramatically expanded in a strain carrying a mutation in the SbcC gene (Sarkar et al., 1998). The SbcC–SbcD protein complex exhibits 3′–5′ double-strand DNA exonucleolytic and single-strand DNA endonucleolytic activities in vitro (Connelly and Leach, 1996). In addition, the SbcC–SbcD complex cleaves DNA hairpins in vitro (Connelly et al., 1998). The SbcC–SbcD nuclease is highly conserved through evolution, from bacteria to humans. Yeast and mammalian homologues of SbcC and SbcD, the Rad50 and Mre11 proteins, respectively, exhibit in vitro biochemical activities similar to their bacterial counterparts (Paull and Gellert, 1998; Usui et al., 1998). However, RAD50 and MRE11 are involved in many different recombinational processes (reviewed by Haber, 1998), and their possible roles in trinucleotide expansions in eukaryotic cells remain to be elucidated. Although CAG expansions certainly occur with a low frequency during replication, it is important to characterize microsatellite length changes that arise during recombination, especially given the association between repeat expansions in humans with transmission of the germline. Recently, Jakupciak and Wells (1999) have shown that recombination occurred between two DNA molecules carrying CAG/CTG repeats in E.coli. These recombination events led to contractions and expansions of the repeats, were RecA- and RecBC-dependent, and were accompanied by cross-overs. In addition, Freudenreich et al. (1998) showed that long CAG/CTG repeats were fragile sites in yeast during vegetative growth, and Jankowski et al. (2000) showed that meiotic double-strand breaks (DSBs) are formed within CAG/CTG repeats in yeast. In order to understand by which mechanism(s) triplet repeats may be expanded in eukaryotic cells, it is thus relevant to study trinucleotide repeat instability during DSB repair. We have developed a system to assess the instability of repeated sequences arising during gene conversion in S.cerevisiae. A DSB is created on a chromosome, such that the ends of the DSB are homologous to donor sequences located on a plasmid. The donor also contains a set of repeated sequences that DNA polymerases associated with repair synthesis must traverse (Figure 1A). Using this system, when the donor contained eight 375-bp repeats, Pâques et al. (1998) found that almost 50% of the gene conversions exhibited expansions or contractions (from one to 13 copies) and virtually all the tract length changes were found in the recipient locus. These results were interpreted in terms of a synthesis-dependent strand annealing (SDSA) mechanism of gene conversion in which the two newly synthesized DNA strands are displaced from the intact template and are both found in the recipient locus (reviewed by Pâques and Haber, 1999). A similar set of experiments were performed by Richard et al. (1999b), studying the fate of short CAG repeats (<40 repeats). In that study, ∼20% of DSB-induced gene conversions led to contraction of the repeat, again virtually all in the recipient locus. Figure 1.Survival of yeast cells after DSB induction. (A) Experimental system. The HO recognition site was inserted on chromosome III, inside the LEU2 gene. Cells were transformed with a centromeric plasmid containing the LEU2 gene. Different inserts were cloned inside the plasmid-borne LEU2 gene. After HO induction, the DSB was repaired by gene conversion using the plasmid-borne LEU2 template. Analysis of recombinants after DSB repair was carried out with or without the plasmid template, in order to distinguish between plasmid-borne and chromosome-borne repeats. For each repeat tract studied, sequence of the Watson (top) strand is shown. (B and C) Frequencies of survival after DSB repair. Yeast cells were plated in parallel on glucose and galactose plates. After 4–5 days at 30°C, colonies were scored on both plates. The frequency of survival was calculated as the ratio of the number of colonies growing on galactose plates over the number of colonies growing on glucose plates. The average frequencies and the standard errors were calculated from a set of 2–4 independent experiments. (B) Survival of a wild-type strain containing different templates after HO induction. (C) Survival of different mutant strains containing either a CAG98 or CAA87. Download figure Download PowerPoint In the present work, we examined HO-induced gene conversions of longer CAG triplet repeats, and tested the effects of the Mre11p–Rad50p–Xrs2p protein complex on trinucleotide tract length changes, during gene conversion-associated DNA synthesis. We show that gene conversion involving the copying of a CAG98 array produces both expansions and contractions, whereas only deletions were found during replication. Moreover, CAG sequences, but not CAA sequences, impede the efficiency of DNA synthesis during gene conversion. This sequence-specific effect can be suppressed by overexpression of the Mre11 and Rad50 proteins. This suggests that the Mre11p–Rad50p–Xrs2p complex is responsible for the removal of hairpin structures that arise during repair-associated DNA synthesis. We conclude that DNA synthesis during recombinational repair of DSBs can be an important source of expansions of CAG repeats. Results An HO endonuclease recognition site was inserted into the LEU2 gene of chromosome III, in strain YFP17. Homology to the HO cleavage site at MAT, HML and HMR was deleted (Pâques et al., 1998). Expression of the HO endonuclease gene was controlled by a galactose-inducible promoter (Sandell and Zakian, 1993). A DSB is created within the LEU2 gene of chromosome III, such that sequences near the ends of the DSB are homologous to donor sequences located on a plasmid (Figure 1A). Repair of the DSB requires the presence of the homologous sequences on the plasmid. Thus, when HO was expressed in the absence of the donor plasmid, 99.7% of cells died, as they were unable to repair the DSB. However, when a plasmid containing an intact LEU2 gene was introduced to provide homology to the HO-cleaved LEU2, ∼22% of cells were able to repair the DSB using the plasmid sequence as a template (Pâques et al., 1998, and Figure 1B). We then supplied different templates in which the cells survive by a gene conversion event in which the repair DNA polymerases must traverse an ∼300 bp insertion in the template (Figure 1A). When the template contained 300 bp of phage λ DNA, 21.3% of the cells survived (Figure 1B); similarly a donor containing CAA87 gave 22.0% survival, very similar to results when there was no insertion in the template. However, when the donor carried CAG98, survival dropped to 13.8%. We conclude that CAG repeats, but not CAA repeats, reduce efficient gene conversion. This is consistent with the idea that CAG repeats, but not CAA repeats, are capable of forming hairpin structures that impede new DNA synthesis during repair (Samadashwily et al., 1997; McMurray, 1999). We have performed several experiments to confirm that CAA/GTT repeats form no hairpins. In contrast to CAG, CTG, CGG and GAC, DNA-melting analysis shows that repeating GTT has no melting transition consistent with the absence of structure (data not shown). Additionally, GTT25 sequences on polyacrylamide gels migrate at a position that is consistent only with an unstructured strand and not with a hairpin. The lack of structure has also been confirmed functionally. Spiro et al. (1999) showed that repeating GTT has a 1000-fold lower expansion rate relative to hairpin-forming sequences such as CTG or GAC. The low mutation rate of GTT was identical in frequency to random sequences with no secondary structure. In addition, Moore et al., (1999) showed that heteroduplex DNA containing CAG or CTG repeats is not efficiently repaired in vivo, whereas CAA loops are repaired. These results strongly suggest that CAA repeats do not form the same structures in vivo as CAG or CTG repeats. If secondary structures in CAG98 cause the reduction in survival, then proteins involved in cleaving or unwinding these secondary structures might increase the efficiency of successfully traversing these sequences and thus increase survival. We, therefore, evaluated cell survival under conditions when MRE11 was overexpressed. MRE11, fused to the ADH1 promoter, was carried on a centromere-containing plasmid in an mre11Δ strain. MRE11 overexpression suppressed the CAG-dependent recombination defect, so that survival occurred at the same level as for the other 300 bp inserts (Figure 1C). MRE11 overexpression did not affect repair of the CAA template. This suppression of CAG-specific inhibition of repair was dependent on having an intact Mre11–Rad50–Xrs2 complex, because a rad50Δ mre11Δ double mutant strain carrying the overexpression MRE11 plasmid had the same low survival as rad50Δ or rad50Δ mre11Δ (Figure 1C). Similarly, overexpressing RAD50 suppressed the defect in gene conversion of CAG repeats, but this suppression disappeared in a mre11Δ rad50Δ strain (Figure 1C). Thus overexpression of two members of the Mre11–Rad50–Xrs2 complex suppressed the CAG-specific reduction in successful recombination, presumably by removing a secondary structure in DNA (presumably a hairpin) that would impair DNA synthesis during DSB repair. There is also an effect of deleting the genes encoding the Mre11–Rad50–Xrs2 complex. Deletions of mre11Δ, rad50Δ or xrs2Δ, as well as a triple mutant (rad50Δ mre11Δ xrs2Δ) reduced the efficiency of gene conversion with the CAG98 template, relative to wild type (Figure 1C). However, this effect is not CAG-specific, as we found a similar reduction in strains carrying LEU2 or LEU2::λ300 templates (data not shown). If MRE11 is required to remove hairpins, then expression of a defective mre11 mutant should prevent the suppression of the recombination defect in cells using the CAG template. The Mre11-3 protein carries a leucine substitution at a histidine residue conserved in all phosphoesterases of the SbcD/Mre11 family (Bressan et al., 1998). The mre11-3 (H125L, D126V) mutant is similar to mre11-H125N, which has been shown to be defective in in vitro nuclease assays (Moreau et al., 1999). Curiously, both mre11-3 and mre11-H125N mutants have proven to be nearly wild-type in many mitotic phenotypes that are affected by an mre11Δ mutation, including spontaneous mitotic recombination, radiation sensitivity, mating-type switching, 5′ to 3′ resection of a DSB, non-homologous end-joining and telomere length (Bressan et al., 1998; Moreau et al., 1999; S.E.Lee, D.Bressan, J.H.J.Petrini and J.E.Haber, manuscript submitted). The mre11-3 mutant does not disrupt the physical interaction between Rad50p and Mre11-3p (Bressan et al., 1998). In the assay described here, we found that overexpression of Mre11-3p in an mre11Δ strain did not relieve the CAG-dependent inhibition of gene conversion, giving the same results as for the mre11Δ strain (Figure 1C). Hence this mutation, which shows no significant defect in a variety of other mitotic phenotypes, is defective in suppressing CAG-mediated inhibition of DSB repair. This implies that the nuclease activity of Mre11p seen in vitro may be important in hairpin removal, or possibly hairpin unwinding (see below). We also note that Bressan et al. (1998) showed that Mre11-3p was expressed at the same level as wild-type Mre11p, so that the absence of an effect in this system cannot be explained by the instability of this mutant protein. Recombination produces expansions of CAG98 If secondary structure inhibits gene conversion, it may also affect the fidelity of copying CAG repeats. Changes in the number of repeated sequences may arise by replication slippage and/or by the pairing of newly synthesized DNA strands, both of which can be influenced by secondary structures that impede repair-associated DNA synthesis and perhaps promote dissociation of DNA polymerases (Pâques et al., 1998). We evaluated changes in repeat length accompanying gene conversion by determining the size of the trinucleotide repeats in both the recipient array at the LEU2 chromosomal locus and in the plasmid template (see Materials and methods). The sensitivity of our measurements was calculated to be ± 8 triplets. Hence, we were not able to detect small changes in size of <8 triplets in the following experiments. Without HO induction, only 8% of the templates carried on the plasmid showed tract length changes of the repeat, and all of them were contractions. Thus replication in the absence of DSB repair exhibited the same frequency of rearrangements, with deletions greatly outnumbering expansions, that have been seen in previous studies in yeast analyzing tract length changes of CAG repeats of comparable sizes (Maurer et al., 1996; Schweitzer and Livingston, 1997). Moreover, the frequency of tract length changes in the template sequences remained constant, again yielding mostly contractions, after HO-induced recombination (Table I). Table 1. Spontaneous and induced trinucleotide repeats rearrangements, in wild-type and mutant strains Strain Genotype Template Template contractions a Gene conversions of the recipient Before HO induction After HO induction Without tractlength change With tractexpansion With tractcontraction Total GFY510 WT CAG98 8% 12% 49 (57%) 11 (13%) 26 (30%) 86 (100%) GFY549 WT CTG98 72% 6 (27%) 2 (9%) 14 (64%) 22 (100%) GFY529 WT CAA87 3% 43 (90%) 0 5 (10%) 48 (100%) GFY508 rad50 CAG98 11% 43 (67%) 9 (14%) 12 (19%) 64 (100%) GFY513 mre11 CAG98 6% 35 (65%) 10 (18%) 9 (17%) 54 (100%) GFY515 rad50 mre11 CAG98 2% 27 (53%) 10 (20%) 14 (27%) 51 (100%) GFY527 mre11 ADH1::MRE11 CAG98 29% 18% b 46 (66%) 5 (7%) 19 (27%) 70 (100%) GFY528 rad50 mre11 ADH1::MRE11 CAG98 12% 34 (67%) 9 (17%) 8 (16%) 51 (100%) WT: wild type. a Template rearrangements were scored either during vegetative growth of independent cultures in the absence of HO induction and/or as the number of rearranged templates following HO induction and recovery of the survivors. All template rearrangements were contractions. b For strain GFY527, 15% of the templates recovered after HO induction showed expansions. None of the templates analyzed before HO induction showed expansions. In contrast, induction of DSB repair by HO endonuclease resulted in a significant number of both contractions and expansions. All the tract length changes were found in the recipient locus, as predicted by SDSA models of gene conversion (Pâques et al., 1998; Pâques and Haber, 1999). Examples are shown in Figure 2. Only 57% of the cells completing gene conversion in the wild-type strain faithfully copied 98 CAG triplets from the template into the chromosome. The remaining 43% contained either contractions of the CAG repeat (30%) or expansions (13%; Table I). When the same analysis was performed on the strain containing CAA87 as template, only 10% showed tract length changes after DSB repair. All tract length changes were contractions, all in the recipient locus (Table I). This argues that the CAG trinucleotide expansions we observe are apparently dependent on their ability to form hairpins during DSB repair. Figure 2.Changes in CAG repeats during gene conversion. Following HO induction of gene conversion between a chromosomal leu2 locus and a URA3-containing plasmid containing leu2::(CAG98), individual colonies were isolated. Cells lacking the URA3-marked plasmid were selected on 5-FOA and DNA was analyzed by Southern blots. The examples shown are from wild-type cells. Lane 1: uninduced control cells; lane 4: non-homologous recombination event giving rise to partial deletion of the LEU2 locus; lanes 2, 5, 11: gene conversion that faithfully copies CAG98 into the chromosomal locus; lanes 3, 6, 7, 8, 12, 15: gene conversion that yields a deletion in the recipient; lane 14: gene conversion that leads to an expansion of CAG repeats in the recipient; lanes 9, 10, 13: gene conversion in which the recipient contained heteroduplex DNA, producing a colony with two different-sized CAG repeats of equal intensity. Download figure Download PowerPoint Since overexpression of MRE11 increased survival of strains carrying the CAG-containing template, we wished to know if higher levels of MRE11 would also affect the proportions or sizes of tract length changes. The proportions of tract length changes after DSB repair when MRE11 was overexpressed with the CAG98-containing donor (GFY527) were 27% contractions and 7% expansions, which is statistically indistinguishable by a contingency χ2 test from the 30% deletions and 13% expansions that we observed with the wild-type strain GFY510. In the rad50 derivative overexpressing MRE11 (GFY528), there were 16% deletions and 17% expansions (Table I). These numbers are not significantly different from an mre11 or a rad50 strain alone (Table I). However, the proportions of expansions and contractions among the wild-type strains (GFY510 and GFY527) are statistically significantly different from those strains that are mutant for rad50 and/or mre11 (GFY508, GFY513, GFY515 and GFY528), by a contingency χ2 test (p <0.025). The absence of mre11 or rad50 appears to nearly double the proportion of expansions, from the average wild-type value of 26% of tract length changes to 47%. We conclude that overexpression of MRE11 did not change the ratio of contractions over expansions recovered after DSB repair, but the absence of either Mre11p or Rad50p does increase the proportion of expansions. We also examined the stability of the template when MRE11 was overexpressed, in the absence of HO induction. Out of 17 independent clones, five were found to carry a contraction of the CAG98 repeat, two of them exhibiting a double band, suggesting that contraction occurred during the vegetative growth of the clone. This result indicates that MRE11 overexpression does increase the instability of the CAG98 array during growth, presumably during repair-associated DNA synthesis, but does not induce spontaneous expansions of the template. Although MRE11 overexpression does not change the proportion of expansions after DSB repair, compared with wild-type, it did affect the size of expansions. Compared with an average expansion size of 25 ± 3 triplets in the wild-type strain, overexpressing MRE11 increased this average to 58 ± 11 triplets (Figure 3). All expansions except one were significantly larger (p <0.05 using a Student's t-test) than in the wild-type strain. This important increase was suppressed in a rad50 strain, in which expansion sizes were comparable to wild-type (Figure 3). The sizes of contractions after gene conversion were not significantly different in the five strains studied (Figure 3). The interpretation of this result will be discussed later. Figure 3.Trinucleotide repeat size changes after gene conversion using CAG98 or CAA87 as templates. For each strain, the white rectangle indicates the range of contractions detected after DSB repair at the recipient locus on the chromosome. Sizes of the shortest and largest contractions are indicated, respectively, right and left of the rectangle, along with the average contraction size and the standard error. Similarly, the gray rectangle indicates the range of expansions detected after DSB repair. Sizes of the shortest and largest expansions are indicated, left and right of the rectangle, respectively, along with the average expansion size and the standard error. The vertical black lines indicate the original size of the CAG98 or of the CAA87 repeats. The arrows represent the median value of each of the distributions. For CAG98 repeats, the changes in the array arising spontaneously on the donor plasmid sequence are also shown. Download figure Download PowerPoint As a control, we also overexpressed MRE11 in a strain containing CAA87 as the template. Out of 36 survivors analyzed, all of them were faithfully repaired, and no expansion was detected. A short contraction of the repeat was found on one template. We concluded that the effect of MRE11 overexpression on expansions was specific to CAG repeats, thus probably dependent on their ability to form secondary structures that we presume to be hairpins. Orientation-independence of recombination-induced CAG/CTG tract length changes Several authors reported that the frequency of CAG/CTG tract length changes during replication in yeast is orientation-dependent, relative to the direction of the replication fork (Maurer et al., 1996; Freudenreich et al., 1997; Miret et al., 1998). In our experimental system, strand invasion and repair-associated DNA synthesis of the CAG98 template can theoretically occur from both ends (Figure 4). However, if for some reason, invasion occurs preferentially from one end, the orientation of the repeat could affect tract length changes during gene conversion. To address this question, we flipped the CAG98 insert in the plasmid template. The resulting CTG98 template was transformed into yeast (GFY549, Table I). There was a much higher proportion of tract length changes in the template (72%)—again, all contractions—than with the template in the CAG98 orientation (12%). These results confirm previous studies showing that the stability of a CAG/CTG repeat carried on a centromeric plasmid in yeast is orientation-dependent (Maurer et al., 1996; Freudenreich et al., 1997; Miret et al., 1998). Figure 4.Possible roles of Mre11p during gene conversion of a CAG trinucleotide repeat. After HO induction and processing of the 3′ ends of the DSB (A), strand invasion may occur and DNA synthesis proceeds through the repeats (B). If secondary structures are formed on the template, DNA synthesis will be stopped (E). Unwinding of one or both strands may occur (C), allowing annealing between the two newly synthesized strands (D), or re-invasion of the template. Formation of a hairpin on one of the newly synthesized strands would give rise to a heteroduplex DNA molecule that is resistant to mismatch repair (D′). Formation of a hairpin on each of the newly synthesized strands may block re-invasion and annealing, leading to cell death (F). Mre11p may help to bypass hairpins and allow DNA synthesis to proceed further. Alternatively, Mre11p may help to unwind or process the hairpin(s) formed on the newly synthesized strand(s), favoring re-invasion. Download figure Download PowerPoint When HO was induced and the CTG98 sequences were used as a template for gene conversion, there was also a statistically significantly higher frequency of tract length changes at the recipient locus than with CAG98 (p <0.01) (Table I). For this analysis we only considered the 22 cases where the template size was not apparently changed prior to induction of HO. Despite the increase in the frequency of tract length changes, the proportion of events that were expansions was not significantly different using CTG98 than with CAG98. Thus, we observed recombination-dependent expansions and contractions with both orientations of the template. The higher proportion of instability in the CTG98 orientation suggests that the DSB repair process is not entirely symmetrical, because if recombination proceeded the same way from each end, the orientation should not have had any effect. It is possib
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