RESUMEN
Although meiotic gene conversion has long been known to be accompanied by crossing-over, a direct test of the converse has not been possible. An experiment was designed to determine whether crossing-over is accompanied by gene conversion in Saccharomyces cerevisiae. Nine restriction site heterologies were introduced into a 9-kilobase chromosomal interval that exhibits 22 percent crossing-over. Of all the exchange events that occurred, at least 59 percent of meiotic crossovers are accompanied by gene conversion of one or more of the restriction site heterologies. The average gene conversion tract length was 1.5 kilobases. An unexpected result was that the introduction of as few as seven heterozygosities significantly altered the outcome of recombination events, reducing the frequency of crossovers by 50 percent and increasing the number of exceptional tetrads. This alteration results from a second recombination event induced by repair of heteroduplex DNA containing multiple mismatched base pairs.
Asunto(s)
Meiosis , Recombinación Genética , Saccharomyces cerevisiae/genética , Reparación del ADN , Replicación del ADN , Enzimas de Restricción del ADN/metabolismo , ADN de Hongos/análisis , HeterocigotoRESUMEN
In S. cerevisiae, mutations in genes that encode telomerase components, such as the genes EST1, EST2, EST3, and TLC1, result in the loss of telomerase activity in vivo. Two telomerase-independent mechanisms can overcome the resulting senescence. Type I survival is characterized by amplification of the subtelomeric Y' elements with a short telomere repeat tract at the terminus. Type II survivors arise through the abrupt addition of long tracts of telomere repeats. Both mechanisms are dependent on RAD52 and on either RAD50 or RAD51. We show here that the telomere elongation pathway in yeast (type II) is dependent on SGS1, the yeast homolog of the gene products of Werner's (WRN) and Bloom's (BLM) syndromes. Survival in the absence of SGS1 and EST2 is dependent upon RAD52 and RAD51 but not RAD50. We propose that the RecQ family helicases are required for processing a DNA structure specific to eroding telomeres.
Asunto(s)
ADN Helicasas/genética , Saccharomyces cerevisiae/genética , Telomerasa/metabolismo , Telómero , Supervivencia Celular/genética , ADN Helicasas/fisiología , Mutación , RecQ Helicasas , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiaeRESUMEN
Mismatch repair (MMR) proteins repair mispaired DNA bases and have an important role in maintaining the integrity of the genome [1]. Loss of MMR has been correlated with resistance to a variety of DNA-damaging agents, including many anticancer drugs [2]. How loss of MMR leads to resistance is not understood, but is proposed to be due to loss of futile MMR activity and/or replication stalling [3] [4]. We report that inactivation of MMR genes (MLH1, MLH2, MSH2, MSH3, MSH6, but not PMS1) in isogenic strains of Saccharomyces cerevisiae led to increased resistance to the anticancer drugs cisplatin, carboplatin and doxorubicin, but had no effect on sensitivity to ultraviolet C (UVC) radiation. Sensitivity to cisplatin and doxorubicin was increased in mlh1 mutant strains when the MLH1 gene was reintroduced, demonstrating a direct involvement of MMR proteins in sensitivity to these DNA-damaging agents. Inactivation of MLH1, MLH2 or MSH2 had no significant effect, however, on drug sensitivities in the rad52 or rad1 mutant strains that are defective in mitotic recombination and removing unpaired DNA single strands. We propose a model whereby MMR proteins - in addition to their role in DNA-damage recognition - decrease adduct tolerance during DNA replication by modulating the levels of recombination-dependent bypass. This hypothesis is supported by the finding that, in human ovarian tumour cells, loss of hMLH1 correlated with acquisition of cisplatin resistance and increased cisplatin-induced sister chromatid exchange, both of which were reversed by restoration of hMLH1 expression.
Asunto(s)
Disparidad de Par Base , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Resistencia a Antineoplásicos/genética , Endonucleasas/fisiología , Proteínas de Neoplasias/genética , Antineoplásicos/farmacología , Carboplatino/farmacología , Cisplatino/farmacología , Enzimas Reparadoras del ADN , Doxorrubicina/farmacología , Farmacorresistencia Microbiana , Mutación , Proteína Recombinante y Reparadora de ADN Rad52 , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efectos de la radiación , Proteínas de Saccharomyces cerevisiae , Células Tumorales Cultivadas/efectos de los fármacos , Rayos UltravioletaRESUMEN
Expanded trinucleotide repeats underlie a growing number of human diseases. The human FMR1 (CGG)(n) array can exhibit genetic instability characterized by progressive expansion over several generations leading to gene silencing and the development of the fragile X syndrome. While expansion is dependent upon the length of uninterrupted (CGG)(n), instability occurs in a limited germ line and early developmental window, suggesting that lineage-specific expression of other factors determines the cellular environment permissive for expansion. To identify these factors, we have established normal- and premutation-length human FMR1 (CGG)(n) arrays in the yeast Saccharomyces cerevisiae and assessed the frequency of length changes greater than 5 triplets in cells deficient in various DNA repair and replication functions. In contrast to previous studies with Escherichia coli, we observed a low frequency of orientation-dependent large expansions in arrays carrying long uninterrupted (CGG)(n) arrays in a wild-type background. This frequency was unaffected by deletion of several DNA mismatch repair genes or deletion of the EXO1 and DIN7 genes and was not enhanced through meiosis in a wild-type background. Array contraction occurred in an orientation-dependent manner in most mutant backgrounds, but loss of the Sgs1p resulted in a generalized increase in array stability in both orientations. In contrast, FMR1 arrays had a 10-fold-elevated frequency of expansion in a rad27 background, providing evidence for a role in lagging-strand Okazaki fragment processing in (CGG)(n) triplet repeat expansion.
Asunto(s)
Reparación del ADN , ADN Recombinante/metabolismo , Exodesoxirribonucleasas , Síndrome del Cromosoma X Frágil/genética , Proteínas Fúngicas/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas de Unión al ARN , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Repeticiones de Trinucleótidos , Tirosina 3-Monooxigenasa , Proteínas 14-3-3 , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , ADN Helicasas/deficiencia , ADN Helicasas/genética , ADN Helicasas/fisiología , Reparación del ADN/genética , ADN de Hongos/metabolismo , ADN Recombinante/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Proteínas Fúngicas/genética , Humanos , Modelos Genéticos , Conformación de Ácido Nucleico , Proteínas Quinasas/deficiencia , Proteínas Quinasas/genética , Proteínas Quinasas/fisiología , Proteínas/genética , Proteínas/fisiología , RecQ Helicasas , Saccharomyces cerevisiae/metabolismo , Expansión de Repetición de TrinucleótidoRESUMEN
Efficient genetic recombination requires near-perfect homology between participating molecules. Sequence divergence reduces the frequency of recombination, a process that is dependent on the activity of the mismatch repair system. The effects of chromosomal divergence in diploids of Saccharomyces cerevisiae in which one copy of chromosome III is derived from a closely related species, Saccharomyces paradoxus, have been examined. Meiotic recombination between the diverged chromosomes is decreased by 25-fold. Spore viability is reduced with an observable increase in the number of tetrads with only two or three viable spores. Asci with only two viable spores are disomic for chromosome III, consistent with meiosis I nondisjunction of the homeologs. Asci with three viable spores are highly enriched for recombinants relative to tetrads with four viable spores. In 96% of the class with three viable spores, only one spore possesses a recombinant chromosome III, suggesting that the recombination process itself contributes to meiotic death. This phenomenon is dependent on the activities of the mismatch repair genes PMS1 and MSH2. A model of mismatch-stimulated chromosome loss is proposed to account for this observation. As expected, crossing over is increased in pms1 and msh2 mutants. Furthermore, genetic exchange in pms1 msh2 double mutants is affected to a greater extent than in either mutant alone, suggesting that the two proteins act independently to inhibit homeologous recombination. All mismatch repair-deficient strains exhibited reductions in the rate of chromosome III nondisjunction.
Asunto(s)
Proteínas Portadoras , Cromosomas Fúngicos , Reparación del ADN , Recombinación Genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Mapeo Cromosómico , Intercambio Genético , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Diploidia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Marcadores Genéticos , Meiosis , Modelos Genéticos , Proteínas MutL , Proteína 2 Homóloga a MutS , No Disyunción Genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/fisiología , Esporas FúngicasRESUMEN
We have measured gene conversion tract length in strains of the yeast Saccharomyces cerevisiae containing three to six restriction site heterozygosities in a 9-kb interval. Tetrads containing a conversion were identified genetically by nonmendelian segregation of a marker in the middle of the interval. Gene conversions accompanied by a crossover have a tract length of 1.4 kb +/- 0.7 kb, which is indistinguishable from a tract length of 1.6 +/- 0.8 for conversions without an associated exchange. Among tetrads identified first as having a crossover in the interval, the average gene conversion tracts were apparently significantly shorter (0.71 +/- 1). We provide evidence that this apparent difference is due to the method of measuring conversion tracts and does not reflect a real difference in tract length. We also provide evidence that the number and position of restriction site markers alters the apparent distribution of the conversion tracts. More than ninety percent of the conversion tracts spanning three or more sites were continuous.
Asunto(s)
Intercambio Genético , Conversión Génica , Meiosis , Mapeo Cromosómico , Marcadores Genéticos , Plásmidos , Recombinación Genética , Saccharomyces cerevisiae/genéticaRESUMEN
Each telomere in a single strain (S288C) of Saccharomyces cerevisiae was marked with a URA3 containing integrating vector having telomeric TG1-3 sequences. Efficiency of integrative transformation was enhanced by creating single random double-strand breaks in the integrating vector using DNAseI in the presence of Mn2+ ions. A total of 327 transformants were screened by CHEF gels of intact chromosomal DNA. Transformants with homology to the vector at particular chromosomal bands were then screened by Southern analysis with several restriction enzymes to confirm telomeric locations. CHEF gels of NotI and/or SfiI digests were also analyzed to determine left or right arm locations. In some cases allelism of marked telomeres was determined genetically. Transformation was performed by lithium acetate and electroporation with varying results. Electroporation resulted in 50% (75/150) of the integrants at the internal URA3 location rather than telomeres. There were also two rearrangements involving URA3 and the telomere of another chromosome. Lithium acetate transformation resulted in fewer integrants at the internal URA3 location (5/84) and no rearrangements. All telomeres were marked with approximately the same efficiency ranging from 0 to 11 hits in the first 240 transformants. These marked telomeres can be used to complete the physical maps of chromosomes in which the telomere regions are absent. The marked telomeres can be cloned with the appropriate restriction enzymes, thus completing the cloning of individual chromosomes for sequencing projects. The analysis of these clones will lead to a better understanding of telomere region biology. The methodology can also be applied to telomeres of other organisms once they are cloned as telomeric YACs.
Asunto(s)
Cromosomas Fúngicos/genética , Clonación Molecular/métodos , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Telómero/genética , Marcadores Genéticos/genética , Vectores Genéticos/genética , Transformación GenéticaRESUMEN
We have examined meiotic recombination between two defined leu2 heteroalleles present at the normal LEU2 locus and in leu2-containing plasmids inserted at four other genomic locations. In diploids where the two leu2 markers were present at allelic locations on parental homologs, the frequency of Leu2+ spores varied 38-fold, in a location-dependent manner. These results indicate that recombination in a genetic interval can be modulated by sequences at least 2.7 kb outside that interval. Leu2+ meiotic segregants were also recovered from diploids where LEU2 was marked with one heteroallele, and the other leu2 heteroallele was inserted at another genomic location. These products of ectopic interactions, between dispersed copies of leu2 sharing only 2.2 kb of homology, were recovered at a frequency comparable to that observed in corresponding allelic crosses. This high frequency of ectopic meiotic recombination was observed in crosses where both recombining partners could potentially pair with sequences at an allelic position. In addition, a significant fraction (22-50%) of these ectopic recombinants were associated with crossing over of flanking sequences.
Asunto(s)
Intercambio Genético , Conversión Génica , Genes Fúngicos , Saccharomyces cerevisiae/genética , Alelos , Genotipo , Meiosis , Saccharomyces cerevisiae/citología , Homología de Secuencia de Ácido NucleicoRESUMEN
We have developed a method by which the extent of physical exchange of DNA molecules can be determined throughout meiosis in the yeast Saccharomyces cerevisiae. We have used this technique to analyze the effect of five meiosis-defective mutations (rad6, rad50, rad52, rad57 and spo11) on the physical exchange of DNA molecules. In the same experiments, we have also measured other meiotic parameters, such as premeiotic DNA synthesis, commitment to intragenic recombination, haploidization, ascus formation, and viability. rad50 and spo11 diploids make an undetectable amount of physically recombined DNA and less than 1% of wild-type levels of viable intragenic recombinants. In contrast, diploids homozygous for rad52, rad6 or rad57 all yield significant amounts of novel restriction fragments which arise by recombination. rad57 diploids make nearly wild-type levels of the recombined restriction fragments, although they produce less than 10% of the wild-type levels of viable intragenic recombinants. rad52 strains are also capable of a significant (33%) amount of exchange of DNA molecules, but make less than 1% of wild-type levels of viable intragenic recombinants. rad6 diploids are also capable of undergoing a high level of exchange, as measured by the appearance of the recombined restriction fragment. In addition, rad6 diploids show an unusual allele- or locus-specific variability in the level of viable intragenic recombinants produced. Although rad6 diploids produce no viable spores, they are able to complete a significant amount of haploidization upon return to vegetative growth conditions.
Asunto(s)
Meiosis , Mutación , Recombinación Genética , Saccharomyces cerevisiae/genética , Enzimas de Restricción del ADN , Haploidia , Hibridación de Ácido Nucleico , Plásmidos , Saccharomyces cerevisiae/citologíaRESUMEN
Estimates of the rate and frequency distribution of deleterious effects were obtained for the first time by direct scoring and characterization of individual mutations. This was achieved by applying tetrad analysis to a large number of yeast clones. The genomic rate of spontaneous mutation deleterious to a basic fitness-related trait, that of growth rate, was U = 1.1 x 10(-3) per diploid cell division. Extrapolated to the fruit fly and humans, the per generation rate would be 0.074 and 0.92, respectively. This is likely to be an underestimate because single mutations with selection coefficients s < 0.01 could not be detected. The distribution of s > or = 0.01 was studied both for spontaneous and induced mutations. The latter were induced by ethyl methanesulfonate (EMS) or resulted from defective mismatch repair. Lethal changes accounted for approximately 30-40% of the scored mutations. The mean s of nonlethal mutations was fairly high, but most frequently its value was between 0.01 and 0.05. Although the rate and distribution of very small effects could not be determined, the joint share of such mutations in decreasing average fitness was probably no larger than approximately 1%.
Asunto(s)
Mutación , Saccharomyces cerevisiae/genética , Metanosulfonato de Etilo/farmacología , Mutágenos/farmacología , Fenotipo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Two yeast minisatellite alleles were cloned and inserted into a genetically defined interval in Saccharomyces cerevisiae. Analysis of flanking markers in combination with sequencing allowed the determination of the meiotic events that produced minisatellites with altered lengths. Tetrad analysis revealed that gene conversions, deletions, or complex combinations of both were involved in producing minisatellite variants. Similar changes were obtained following selection for nearby gene conversions or crossovers among random spores. The largest class of events involving the minisatellite was a 3:1 segregation of parental-size alleles, a class that would have been missed in all previous studies of minisatellites. Comparison of the sequences of the parental and novel alleles revealed that DNA must have been removed from the recipient array while a newly synthesized copy of donor array sequences was inserted. The length of inserted sequences did not appear to be constrained by the length of DNA that was removed. In cases where one or both sides of the insertion could be determined, the insertion endpoints were consistent with the suggestion that the event was mediated by alignment of homologous stretches of donor/recipient DNA.
Asunto(s)
ADN de Hongos/genética , Conversión Génica , Meiosis/genética , Repeticiones de Minisatélite , Saccharomyces cerevisiae/genética , Alelos , Secuencia de Bases , Cartilla de ADN/genética , Reordenamiento Génico , Variación Genética , Esporas Fúngicas/genéticaRESUMEN
The presence of multiple heterologies in a 9-kilobase (kb) interval results in a decrease in meiotic crossovers from 26.0% to 10.1%. There is also an increase from 3.5% to 11.1% in gene conversions and ectopic recombinations between the flanking homologous MAT loci. The hypothesis that mismatch repair of heteroduplex DNA containing several heterologies would lead to a second round of recombination has now been tested by examining the effect of a mutation that reduces mismatch correction. The repair-defective pms1-1 allele restores the pattern of recombination to nearly that seen in congenic diploids without the heterologies. Mismatch repair-induced recombination causes a significant increase in MAT conversions and ectopic recombination events with as few as two heterozygosities separated by 0.3-0.7 kb, but not when the mismatches are separated by greater than 1 kb. The frequency of these events depends on both the number and position of the heterozygosities relative to the flanking homologous MAT loci used to detect the events. The creation of recombinogenic lesions by mismatch repair in yeast could be analogous to the creation of recombinogenic lesions in dam- Escherichia coli. We suggest that the repair of heteroduplex DNA containing multiple mismatches may produce chromosomal rearrangements and gamete inviability when naturally polymorphic chromosomes undergo meiotic recombination.
Asunto(s)
Reparación del ADN , ADN de Hongos/metabolismo , Genes Fúngicos , Recombinación Genética , Saccharomyces cerevisiae/genética , Alelos , Expresión Génica , Genotipo , Mutación , Ácidos Nucleicos Heterodúplex/metabolismoRESUMEN
The Saccharomyces cerevisiae SGS1 gene is homologous to Escherichia coli RecQ and the human BLM and WRN proteins that are defective in the cancer-prone disorder Bloom's syndrome and the premature aging disorder Werner's syndrome, respectively. While recQ mutants are deficient in conjugational recombination and DNA repair, Bloom's syndrome cell lines show hyperrecombination. Bloom's and Werner's syndrome cell lines both exhibit chromosomal instability, sgs1 delta strains show mitotic hyperrecombination, as do Bloom's cells. This was manifested as an increase in the frequency of interchromosomal homologous recombination, intrachromosomal excision recombination, and ectopic recombination. Hyperrecombination was partially independent of both RAD52 and RAD1. Meiotic recombination was not increased in sgs1 delta mutants, although meiosis I chromosome missegregation has been shown to be elevated sgs1 delta suppresses the slow growth of a top3 delta strain lacking topoisomerase III. Although there was an increase in subtelomeric Y' instability in sgs1 delta strains due to hyperrecombination, no evidence was found for an increase in the instability of terminal telomeric sequences in a top3 delta or a sgs1 delta strain. This contrasts with the telomere maintenance defects of Werner's patients. We conclude that the SGS1 gene product is involved in the maintenance of genome stability in S. cerevisiae.
Asunto(s)
ADN Helicasas/genética , Genoma Fúngico , Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/genética , Alelos , Síndrome de Bloom/genética , Cromosomas Fúngicos , ADN-Topoisomerasas de Tipo I/genética , Diploidia , Humanos , Mitosis , Fenotipo , RecQ Helicasas , Recombinación Genética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae , Esporas Fúngicas , Telómero , Síndrome de Werner/genéticaRESUMEN
Mismatch repair proteins are a highly diverse group of proteins that interact with numerous DNA structures during DNA repair and replication. Here we review data for the role of Msh4, Msh5, Mlh1, Mlh3 and Exo1 in crossing over. Based on the paradigm of interactions developed from studies of mismatch repair, we propose models for the mechanism of crossover implementation by Msh4/Msh5 and Mlh1/Mlh3.
Asunto(s)
Disparidad de Par Base/genética , Reparación del ADN/fisiología , Meiosis/fisiología , Proteínas/metabolismo , Recombinación Genética/fisiología , Animales , Reparación del ADN/genética , Enzimas Reparadoras del ADN , Exodesoxirribonucleasas/metabolismo , Humanos , Meiosis/genética , Recombinación Genética/genéticaRESUMEN
BACKGROUND AND AIMS: Mismatch repair proteins play important roles during meiotic recombination in the budding yeast Saccharomyces cerevisiae and most eukaryotic organisms studied to date. To study the functions of the mismatch repair protein Mlh2p in meiosis, we constructed mlh2Delta strains and measured rates of crossing over, gene conversion, post-meiotic segregation and spore viability. We also analysed mlh1Delta, mlh3Delta, msh4Delta, msh5Delta, exo1Delta and mus81Delta mutant strains singularly and in various combinations. RESULTS: Loss of MLH2 resulted in a small but significant decrease in spore viability and a significant increase in gene conversion frequencies but had no apparent effect on crossing over. Deletion of MLH2 in mlh3Delta, msh4Delta or msh5Delta strains resulted in significant proportion of the "lost" crossovers found in single deletion strains being regained in some genetic intervals. We and others propose that there are at least two pathways to generate crossovers in yeast (Ross-Macdonald and Roeder, 1994; Zalevsky et al., 1999; Khazanehdari and Borts, 2000; Novak et al., 2001; de los Santos et al., 2003). Most recombination intermediates are processed by the "major", Msh4-dependent pathway, which requires the activity of Mlh1p/Mlh3p/Msh4p/Msh5p as well as a number of other proteins. The minor pathway(s) utilizes Mms4p/Mus81p. We suggest that the absence of Mlh2p allows some crossovers from the MSH4 pathway to traverse the MUS81-dependent pathway.
Asunto(s)
Intercambio Genético/genética , Proteínas Nucleares/metabolismo , Ácidos Nucleicos Heterodúplex/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Alelos , Supervivencia Celular , Segregación Cromosómica/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Conversión Génica/genética , Eliminación de Gen , Genotipo , Modelos Genéticos , Homólogo 1 de la Proteína MutL , Proteínas MutL , Proteína 3 Homóloga de MutS , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Esporas Fúngicas/genética , Esporas Fúngicas/metabolismoRESUMEN
Most explanations for the evolutionary maintenance of sex depend on the assumption that sex produces variation by recombining parental haplotypes in the offspring. Therefore, meiosis is expected to be useful only in heterozygotes. We tested this assumption by competing sexual strains of yeast against constitutive asexuals in a hot (37 degrees C) culture for 500 generations, in either heterozygous or homozygous genetic backgrounds. We found that there was an initial cost of sex for all the sexual strains, which was indicated by a sharp increase in the proportion of asexuals after the induction of sex. The cost was larger in the heterozygotes than in the homozygotes, probably because of recombinational load. However, in two of the three heterozygote backgrounds, after the initial success of the asexuals, the remaining sexuals eventually drove them out of the population. These two heterozygotes also suffered the largest initial cost of sex. In the other heterozygote and in the three homozygote backgrounds it appeared to be a matter of chance whether sexuals or asexuals won. The average relative fitness increased in all the strains, but the increase was largest in the two strains that showed both the clearest advantage and the largest cost of sex. We conclude that these results are consistent with the traditional view that sex has a short-term cost but a long-term benefit.
Asunto(s)
Adaptación Biológica/fisiología , Saccharomyces cerevisiae/fisiología , Heterocigoto , Homocigoto , TemperaturaRESUMEN
Mismatches, and the proteins that repair them, play multiple roles during meiosis from generating the diversity upon which selection acts to preventing the intermingling of diverged populations and species. The mechanisms by which the mismatch repair proteins accomplish these many roles include gene conversion, reciprocal crossing over, mismatch repair-induced recombination and anti-recombination. This review focuses on recent studies, predominantly in Saccharomyces cerevisiae, that have advanced our understanding of the details of mismatch repair complexes and how they apply to the diverse roles these proteins play in meiosis. These studies have also revealed unexpected and novel functions for some of the mismatch repair proteins.
Asunto(s)
Reparación del ADN/fisiología , Meiosis , Recombinación Genética , Disparidad de Par Base , Conversión Génica , Células Germinativas/fisiología , Humanos , Ácidos Nucleicos HeterodúplexRESUMEN
The correction of replication errors is an essential component of genetic stability. This is clearly demonstrated in humans by the observation that mutations in mismatch repair genes lead to HNPCC (hereditary non-polyposis colorectal cancer). This disease accounts for as many as 2-3% of colon cancers. Of these, most of them are in the two central components of mismatch repair, MLH1 (mutL homologue 1) and MSH2 (mutS homologue 2). MLH1 and MSH2 function as a complex with two other genes PMS2 and MSH6. Mismatch repair genes, and the mechanism that ensures that incorrectly paired bases are removed, are conserved from prokaryotes to human. Thus yeast can serve as a model organism for analysing mutations/polymorphisms found in human mismatch repair genes for their effect on post-replicative repair. To date, this has predominantly been accomplished by making the analogous mutations in yeast genes. However, this approach is only useful for the most highly conserved regions. Here, we discuss some of the benefits and technical difficulties involved in expressing human genes in yeast. Modelling human mismatch repair in yeast will allow the assessment of any functional effect of novel polymorphisms found in patients diagnosed with colon cancers.
Asunto(s)
Alelos , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Neoplasias Colorrectales/genética , Reparación de la Incompatibilidad de ADN , Saccharomyces cerevisiae/genética , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales Hereditarias sin Poliposis/diagnóstico , HumanosRESUMEN
Meiotic recombination in the yeast Saccharomyces cerevisiae is initiated by programmed double-strand breaks at selected sites throughout the genome (hotspots). alpha-Hotspots are binding sites for transcription factors. Double-strand breaks at alpha-hotspots require binding of transcription factor but not high levels of transcription per se. We show that modulating the production of the transcription factor Gcn4p by deletion or constitutive transcription alters the rate of gene conversion and crossing-over at HIS4. In addition, we show that alterations in the metabolic state of the cell change the frequency of gene conversion at HIS4 in a Gcn4p-dependent manner. We suggest that recombination data obtained from experiments using amino acid and other biosynthetic genes for gene disruptions and/or as genetic markers should be treated cautiously. The demonstration that Gcn4p affects transcription of more than 500 genes and that the recombinationally "hottest" ORFs tend to be Gcn4p-regulated suggest that the metabolic state of a cell, especially with respect to nitrogen metabolism, is a determinant of recombination rates. This observation suggests that the effects of metabolic state may be global and may account for some as yet unexplained features of recombination in higher organisms, such as the differences in map length between the sexes.