RESUMO
There has been significant progress in elucidating the mechanisms by which meiotic and mitotic recombination occur. Double-strand breaks in particular have been the object of attention in studies on meiotic gene conversion, site-specific mitotic recombination, the repair of transposon excision and the transformation of cells with linearized DNA. A combination of genetic analysis and physical studies of molecular recombination intermediates have established that double-strand breaks can occur by two different mechanisms.
Assuntos
Recombinação Genética , Animais , Reparo do DNA/genética , Elementos de DNA Transponíveis , DNA de Cadeia Simples/genética , Regulação da Expressão Gênica , Humanos , Meiose/genética , Mitose/genética , Modelos Genéticos , Homologia de Sequência do Ácido NucleicoRESUMO
The study of double-strand chromosome break repair by homologous and nonhomologous recombination is a growth industry. In the past year, there have been important advances both in understanding the connection between recombination and DNA replication and in linking recombination with origins of human cancer. At the same time, a combination of biochemical, genetic, molecular biological, and cytological approaches have provided a clearer vision of the specific functions of a variety of recombination proteins.
Assuntos
Recombinação Genética , Reparo do DNA , Replicação do DNA , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Humanos , Meiose , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Under restricted culture conditions, the growth and division of Saccharomyces cerevisiae was inhibited by the antimitotic drug Colcemid; in contrast, the related drug colchicine had no effect. The difference in the sensitivity of yeast to these two agents was not dependent on their ability to permeate the cell but rather reflected an inherent difference in the affinity of the two drugs for a cellular-binding site. The binding moiety was characterized by gel filtration as a macromolecule of approximately 110,000 mol wt with an affinity constant for Colcemid of 0.5 x 10(4) liters per mole; in addition, this macromolecule was retained by diethylaminoethyl (DEAE) ion exchangers. On the basis of these properties, the Colcemid-binding substance in S. cerevisiae cells was provisionally identified as microtubule subunits.
Assuntos
Sítios de Ligação/efeitos dos fármacos , Colchicina/farmacologia , Mitose/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Fracionamento Celular , Permeabilidade da Membrana Celular , Cromatografia DEAE-Celulose , Cromatografia em Gel , Cromatografia por Troca Iônica , Cromatografia em Papel , Cromatografia em Camada Fina , Colchicina/metabolismo , Microscopia Eletrônica , Microtúbulos/metabolismo , Peso Molecular , Extratos Vegetais/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Fatores de Tempo , TrítioRESUMO
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.
Assuntos
Meiose , Recombinação Genética , Saccharomyces cerevisiae/genética , Reparo do DNA , Replicação do DNA , Enzimas de Restrição do DNA/metabolismo , DNA Fúngico/análise , HeterozigotoRESUMO
Double-strand breaks (DSBs) in Saccharomyces cerevisiae can be repaired by gene conversions or by deletions resulting from single-strand annealing between direct repeats of homologous sequences. Although rad1 mutants are resistant to x-rays and can complete DSB-mediated mating-type switching, they could not complete recombination when the ends of the break contained approximately 60 base pairs of nonhomology. Recombination was restored when the ends of the break were made homologous to donor sequences. Additionally, the absence of RAD1 led to the frequent appearance of a previously unobserved type of recombination product. These data suggest RAD1 is required to remove nonhomologous DNA from the 3' ends of recombining DNA, a process analogous to the excision of photodimers during repair of ultraviolet-damaged DNA.
Assuntos
Reparo do DNA , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/genética , DNA Fúngico/genética , Conversão Gênica , Cinética , Proteínas de Saccharomyces cerevisiae , Deleção de Sequência , Raios UltravioletaRESUMO
A mutant allele of the chromosomal locus corresponding to the cloned actin gene of the yeast Saccharomyces cerevisiae has been constructed by DNA transformation with a hybrid plasmid which integrates into, and thereby disrupts, the protein-encoding sequences of the gene. In a diploid strain of yeast, disruption of the actin gene on one chromosome results in a mutation that segregates as a recessive lethal tightly linked to a selectable genetic marker on the integrated plasmid. The actin gene, therefore, must encode an essential function for yeast cell growth.
Assuntos
Actinas/genética , Saccharomyces cerevisiae/genética , Actinas/fisiologia , Alelos , Clonagem Molecular , DNA Fúngico/genética , DNA Recombinante , Genes Letais , Genes Recessivos , Plasmídeos , Recombinação Genética , Transformação GenéticaRESUMO
Chromosomal double-strand breaks (DSBs) arise after exposure to ionizing radiation or enzymatic cleavage, but especially during the process of DNA replication itself. Homologous recombination plays a critical role in repair of such DSBs. There has been significant progress in our understanding of two processes that occur in DSB repair: gene conversion and recombination-dependent DNA replication. Recent evidence suggests that gene conversion and break-induced replication are related processes that both begin with the establishment of a replication fork in which both leading- and lagging-strand synthesis occur. There has also been much progress in characterization of the biochemical roles of recombination proteins that are highly conserved from yeast to humans.
Assuntos
Replicação do DNA , Recombinação Genética , Dano ao DNA , Reparo do DNA , Conversão Gênica , HumanosRESUMO
The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination.
Assuntos
Reparo do DNA/fisiologia , DNA Fúngico/genética , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases , Animais , Dano ao DNA/genética , DNA Helicases , Reparo do DNA/genética , Enzimas Reparadoras do DNA , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Desoxirribonucleases de Sítio Específico do Tipo II/fisiologia , Proteínas Fúngicas/fisiologia , Humanos , Meiose , Proteína Rad52 de Recombinação e Reparo de DNA , Recombinases Rec A/metabolismoRESUMO
Meiosis is distinguished from mitosis by the way double-strand breaks are made and by the synapsis and segregation of homologous chromosomes. Recent studies with the yeast Saccharomyces cerevisiae have identified some of the key players that link homologous recombination to synaptonemal complex formation.
Assuntos
Meiose/fisiologia , Proteínas de Saccharomyces cerevisiae , Proteínas Fúngicas/fisiologia , Proteínas Nucleares , Recombinação Genética , Saccharomyces cerevisiae/fisiologia , Complexo Sinaptonêmico/fisiologiaRESUMO
Mammalian homologues of two important yeast genes involved in DNA double-strand break repair and recombination, RAD51 and RAD54, have been isolated. Knock-out mutations of the genes in mice reveal both reassuring similarities to, and surprising differences from, the analogous mutant phenotypes in yeast.
Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Proteínas de Saccharomyces cerevisiae , Animais , DNA Helicases , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Humanos , Camundongos , Camundongos Knockout , Mutação , Fenótipo , Rad51 Recombinase , Recombinação Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Saccharomyces cells with a single unrepaired double-strand break (DSB) will adapt to checkpoint-mediated G2/M arrest and resume cell cycle progression. The decision to adapt is finely regulated by the extent of single-stranded DNA generated from a DSB. We show that cells lacking the recombination protein Tid1p are unable to adapt, but that this defect is distinct from any role in recombination. As with the adaptation-defective mutations yku70Delta and cdc5-ad, permanent arrest in tid1Delta is bypassed by the deletion of the checkpoint gene RAD9. Permanent arrest of tid1Delta cells is suppressed by the rfa1-t11 mutation in the ssDNA binding complex RPA, similar to yku70Delta, whereas the defect in cdc5-ad is not suppressed. Unlike yku70Delta, tid1Delta does not affect 5'-to-3' degradation of DSB ends. The tid1Delta defect cannot be complemented by overexpressing the homolog Rad54p, nor is it affected in rad51Delta tid1Delta, rad54Delta tid1Delta, or rad52Delta tid1Delta double mutants that prevent essentially all homologous recombination. We suggest that Tid1p participates in monitoring the extent of single-stranded DNA produced by resection of DNA ends in a fashion that is distinct from its role in recombination.
Assuntos
Adaptação Fisiológica , Antígenos Nucleares , Dano ao DNA , DNA Helicases , Proteínas Fúngicas/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Enzimas Reparadoras do DNA , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Fase G2 , Deleção de Genes , Cinética , Autoantígeno Ku , Mitose , Proteínas Nucleares/genética , Saccharomyces cerevisiae/citologiaRESUMO
Eukaryotes have acquired many mechanisms to repair DNA double-strand breaks (DSBs) [1]. In the yeast Saccharomyces cerevisiae, this damage can be repaired either by homologous recombination, which depends on the Rad52 protein, or by non-homologous end-joining (NHEJ), which depends on the proteins yKu70 and yKu80 [2] [3]. How do cells choose which repair pathway to use? Deletions of the SIR2, SIR3 and SIR4 genes - which are involved in transcriptional silencing at telomeres and HM mating-type loci (HMLalpha and HMRa) in yeast [4] - have been reported to reduce NHEJ as severely as deletions of genes encoding Ku proteins [5]. Here, we report that the effect of deleting SIR genes is largely attributable to derepression of silent mating-type genes, although Sir proteins do play a minor role in end-joining. When DSBs were made on chromosomes in haploid cells that retain their mating type, sir Delta mutants reduced the frequency of NHEJ by twofold or threefold, although plasmid end-joining was not affected. In diploid cells, sir mutants showed a twofold reduction in the frequency of NHEJ in two assays. Mating type also regulated the efficiency of DSB-induced homologous recombination. In MATa/MATalpha diploid cells, a DSB induced by HO endonuclease was repaired 98% of the time by gene conversion with the homologous chromosome, whereas in diploid cells with an alpha mating type (matDelta/MATalpha) repair succeeded only 82% of the time. Mating-type regulation of genes specific to haploid or diploid cells plays a key role in determining which pathways are used to repair DSBs.
Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/fisiologia , Genes Fúngicos/fisiologia , Genes Fúngicos Tipo Acasalamento , Histona Desacetilases , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae , Transativadores/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Modelos Genéticos , Mutagênese , Sirtuína 2 , Sirtuínas , Fatores de Tempo , Transativadores/genética , Transcrição GênicaRESUMO
Telomeres are functionally distinct from ends generated by chromosome breakage, in that telomeres, unlike double-strand breaks, are insulated from recombination with other chromosomal termini [1]. We report that the Ku heterodimer and the Rad50/Mre11/Xrs2 complex, both of which are required for repair of double-strand breaks [2-5], have separate roles in normal telomere maintenance in yeast. Using epistasis analysis, we show that the Ku end-binding complex defined a third telomere-associated activity, required in parallel with telomerase [6] and Cdc13, a protein binding the single-strand portion of telomere DNA [7,8]. Furthermore, loss of Ku function altered the expression of telomere-located genes, indicative of a disruption of telomeric chromatin. These data suggest that the Ku complex and the Cdc13 protein function as terminus-binding factors, contributing distinct roles in chromosome end protection. In contrast, MRE11 and RAD50 were required for the telomerase-mediated pathway, rather than for telomeric end protection; we propose that this complex functions to prepare DNA ends for telomerase to replicate. These results suggest that as a part of normal telomere maintenance, telomeres are identified as double-strand breaks, with additional mechanisms required to prevent telomere recombination. Ku, Cdc13 and telomerase define three epistasis groups required in parallel for telomere maintenance.
Assuntos
Antígenos Nucleares , DNA Helicases , Reparo do DNA , Endodesoxirribonucleases , Exodesoxirribonucleases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Telômero/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Replicação do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Autoantígeno Ku , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Telomerase/metabolismo , Telômero/genéticaRESUMO
The study of yeast mating-type (MAT) gene switching has provided insights into several aspects of the regulation of gene expression. MAT switching is accomplished by a highly programmed site-specific homologous recombination event in which mating-type-specific sequences at MAT are replaced by alternative DNA sequences copied from one of two unexpressed donors. The mating-type system has also provided an opportunity to study both the genetic regulation of gene silencing by alterations in chromatin structure, and the basis of preferential recombination between a recipient of genetic information and one of several possible donors.
Assuntos
Proteínas Fúngicas/genética , Conversão Gênica , Regulação Fúngica da Expressão Gênica , Rearranjo Gênico , Genes Fúngicos , Peptídeos/genética , Feromônios/genética , Saccharomyces cerevisiae/genética , Alelos , Fator de Acasalamento , Mitose , Modelos Genéticos , Recombinação Genética , Reprodução/genética , Saccharomyces cerevisiae/fisiologiaRESUMO
Double-strand chromosome breaks can arise in a number of ways, by ionizing radiation, by spontaneous chromosome breaks during DNA replication, or by the programmed action of endonucleases, such as in meiosis. Broken chromosomes can be repaired either by one of several homologous recombination mechanisms, or by a number of nonhomologous repair processes. Many of these pathways compete actively for the repair of a double-strand break. Which of these repair pathways is used appears to be regulated developmentally, genetically and during the cell cycle.
Assuntos
Reparo do DNA/fisiologia , DNA , Recombinação Genética , Alelos , Animais , Cromátides , Dano ao DNA/genética , Replicação do DNA , DNA de Cadeia Simples , Regulação da Expressão Gênica , Meiose , MitoseRESUMO
The yeast Saccharomyces can switch its mating type by a highly choreographed recombination event in which 'a' or 'alpha' sequences at the mating-type (MAT) locus are replaced by opposite mating-type sequences copied from one of two donors, HML and HMR, located near the two ends of the same chromosome III. MAT alpha cells 'know' to choose HML, while MAT alpha cells preferentially recombine with HMR. Donor preference is regulated by a 250 bp recombination enhancer, that controls recombination of the entire left arm of chromosome III. Recent studies have shown how this locus-control region is turned on and off.
Assuntos
Saccharomyces/genética , Elementos Facilitadores Genéticos , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Genes Fúngicos Tipo Acasalamento , Região de Controle de Locus Gênico , Recombinação GenéticaRESUMO
Four new complementation groups of mutations which confer resistance to several amino acid analogs in Saccharomyces cerevisiae are described. These mutants were isolated on medium containing urea as the nitrogen source, in contrast to previous studies that had used medium containing proline. All four resistance to amino acid analog (raa) complementation groups appear to confer resistance by reducing amino acid analog and amino acid uptake. In some genetic backgrounds, raa leu2 and raa thr4 double mutants are inviable, even on rich medium. The raa4 mutation may affect multiple amino acid transport systems, since raa4 mutants are unable to use proline as a nitrogen source. raa4 is, however, unlinked to a previously described amino acid analog resistance and proline uptake mutant, aap1, or to the general amino acid permease mutant gap1. Both raa4 and gap1 prevent uptake of [3H]leucine in liquid cultures. The raa1, raa2, and raa3 mutants affect only a subset of the amino acid analogs and amino acids affected by raa4. The phenotypes of raa1, -2, and -3 mutants are readily observed on agar plates but are not seen in uptake and incorporation of amino acids measured in liquid media.
Assuntos
Aminoácidos/farmacologia , Mutação , Saccharomyces cerevisiae/genética , Aminoácidos/metabolismo , Transporte Biológico , Meios de Cultura , Resistência Microbiana a Medicamentos/genética , Teste de Complementação Genética , Genótipo , Cinética , Leucina/metabolismo , Fenótipo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Novel recombinational repair of a site-specific double-strand break (DSB) in a yeast chromosome was investigated. When the recognition site for the HO endonuclease enzyme is embedded in nonyeast sequences and placed between two regions of homology, expression of HO endonuclease stimulates recombination between the homologous flanking regions to yield a deletion, the apparent product of an intrachromosomal exchange between direct repeats. This deletion-repair event is very efficient, thus preventing essentially all the potential lethality due to the persistence of a DSB. Interestingly, unlike previous studies involving spontaneous recombination between chromosomal repeats, the recombination events stimulated by HO-induced DSBs are accompanied by loss of the sequences separating the homologous regions greater than 99.5% of the time. Repair is dependent on the RAD52 gene. The deletion-repair event provides an in vivo assay for the sensitivity of any particular recognition site to HO cleavage. By taking advantage of a galactose-inducible HO gene, it has been possible to follow the kinetics of this event at the DNA level and to search for intermediates in this reaction. Deletion-repair requires approximately 45 min and is inhibited when cycloheximide is added after HO endonuclease cleavage.
Assuntos
Cromossomos/fisiologia , Reparo do DNA , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/genética , Deleção Cromossômica , Genótipo , Plasmídeos , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae , Especificidade da EspécieRESUMO
We marked a large number of yeast telomeres within their Y' regions by transforming strains with a fragment of Y' DNA into which the URA3 gene had been inserted. A few of the Ura+ transformants obtained were very unstable and were found to contain autonomously replicating URA3-marked circular Y' elements in high copy number. These marked extrachromosomal circles were capable of reintegrating into the chromosome at other telomeric locations. In contrast, most of the Ura+ transformants obtained were quite stable mitotically and were marked at bona fide chromosomal ends. These stable transformants gave rise to mitotically unstable URA3-marked circular Y' elements at a low frequency (up to 2.5%). The likelihood that such excisions and integrations represent a natural process in Saccharomyces cerevisiae is supported by our identification of putative Y' circles in untransformed strains. The transfer of Y' information among telomeres via a circular intermediate may be important for homogenizing the sequences at the ends of yeast chromosomes and for generating the frequent telomeric rearrangements that have been observed in S. cerevisiae.
Assuntos
Replicon , Saccharomyces cerevisiae/genética , Cromossomos/ultraestrutura , Replicação do DNA , DNA Circular/genética , DNA Fúngico/genética , Herança Extracromossômica , Meiose , Mitose , Transformação GenéticaRESUMO
Homothallic switching of the mating type genes of Saccharomyces cerevisiae occurs by a gene conversion event, replacing sequences at the expressed MAT locus with a DNA segment copied from one of two unexpressed loci, HML or HMR. The transposed Ya or Y alpha sequences are flanked by homologous regions that are believed to be essential for switching. We examined the transposition of a mating type gene (hmr alpha 1-delta 6) which contains a 150-base-pair deletion spanning the site where the HO endonuclease generates a double-stranded break in MAT that initiates the gene conversion event. Despite the fact that the ends of the cut MAT region no longer share homology with the donor hmr alpha 1-delta 6, switching of MATa or MAT alpha to mat alpha 1-delta 6 was efficient. However, there was a marked increase in the number of aberrant events, especially the formation of haploid-inviable fusions between MAT and the hmr alpha 1-delta 6 donor locus.