RESUMO
Maintaining telomeric DNA at chromosome ends is essential for genome stability. In virtually all organisms the telomerase enzyme provides this function; however, telomerase-independent mechanisms also exist. These latter mechanisms rely on recombination pathways to replenish telomeric DNA and extrachromosomal DNA may also be implicated. Here, we report that in Saccharomyces cerevisiae cells, extrachromosomal circular DNA occurs for both subtypes of telomerase-independent telomere-maintenance mechanisms. This DNA consists of circular molecules of full-length subtelomeric repeat elements in type I cells, and very heterogeneously sized circles of telomeric repeat DNA in type II cells that are at least partially single stranded. Surprisingly, both type I and type II cells can adapt to a loss of the normally essential telomere-capping protein Cdc13p by inducing an alternate and reversible state of chromosome ends. Chromosome capping, therefore, is not strictly dependent on canonical capping proteins, such as Cdc13p, but can be achieved by alternate mechanisms.
Assuntos
DNA Circular/genética , Saccharomyces cerevisiae/genética , Telomerase/genética , Proteínas de Ligação a Telômeros/genética , Telômero/genética , Sobrevivência Celular/genética , Dano ao DNA/genética , Reparo do DNA/genética , DNA Circular/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Herança Extracromossômica , Regulação Fúngica da Expressão Gênica/genética , Instabilidade Genômica/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Telomerase/metabolismo , Telômero/metabolismo , Proteínas de Ligação a Telômeros/metabolismoRESUMO
The neutral/neutral (N/N) two-dimensional (2-D) agarose gel technique is a useful tool for understanding the mechanisms leading to the complete duplication of linear eukaryotic chromosomes. For the yeast Saccharomyces cerevisiae, it has been used to localize and characterize origins of replication as well as fork progression characteristics in a variety of experimental settings. The method involves running a first-dimension gel in order to separate restriction-digested replication intermediates of different mass. A gel slice containing the continuum of replicating DNA is then cut and subjected to a second-dimension gel, such as to resolve replication intermediates of varying topology. The 2-D gel is then blotted and probed to allow an examination of replication intermediates in specific DNA regions.
Assuntos
Replicação do DNA , DNA Fúngico/isolamento & purificação , Eletroforese em Gel Bidimensional/métodos , Saccharomyces cerevisiae/metabolismo , DNA Fúngico/biossíntese , DNA Ribossômico/biossíntese , DNA Ribossômico/isolamento & purificação , Eletroforese em Gel Bidimensional/instrumentação , Micologia/métodos , Origem de ReplicaçãoRESUMO
The concept of telomeres as being the end-part of eukaryotic chromosomes was first described by H. J. Muller and B. McClintock. Their pioneering work opened the path for multiple new researches and assays on a thrilling subject, with implications for various domains such as aging, replication, immortality, and cancer. Yeast has been a model of choice to study telomere length, senescence, telomerase activity, telomere cloning, and sequencing with important new techniques being discovered in this species and adapted afterward for other organisms. The main functions of telomeres include the protection of the genome from deletions, recombination, and degradation, and they are therefore essential for genome stability. Their maintenance is assured by a specific enzyme (telomerase) and it is of vital interest for the organism to maintain their length and specific structure. Multiple assays have been described to analyze telomere length and for yeast, Southern blot analysis of terminal restriction fragments (TRFs) remains one of the most popular ones to get a global picture of the state of telomeres in a given experimental setting. However, growth phenotypes (senescence) and fine-structure analyses of the chromosome terminal DNA are also becoming increasingly important.
Assuntos
Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Telômero/genética , Telômero/metabolismo , Sequência de Bases , Western Blotting , Imunoprecipitação da Cromatina , DNA Fúngico/genética , Inativação Gênica , Dados de Sequência Molecular , Micologia/métodos , Fenótipo , Plasmídeos/genética , Reação em Cadeia da Polimerase , Técnicas do Sistema de Duplo-HíbridoRESUMO
The precise DNA arrangement at chromosomal ends and the proteins involved in its maintenance are of crucial importance for genome stability. For the yeast Saccharomyces cerevisiae, this constitutive DNA configuration has remained unknown. We demonstrate here that G-tails of 12-14 bases are present outside of S phase on normal yeast telomeres. Furthermore, the Mre11p protein is essential for the proper establishment of this constitutive end-structure. However, the timing of extended G-tails occurring during S phase is not affected in strains lacking Mre11p. Thus, G-tails are present on yeast chromosomes throughout the cell cycle and the MRX complex is required for their normal establishment.