RESUMEN
The FLP recombinase promotes site-specific recombination in the 2 micrometer circle of Saccharomyces cerevisiae. FLP recognizes a 48 bp target site (FLP recombination target, or FRT) consisting of three 13 bp protein binding sites, or symmetry elements, flanking an 8 bp spacer region. Efficient recombination also occurs with DNA substrates that have minimal FRT sites, consisting only of the spacer and two surrounding 13 bp symmetry elements arranged in inverse orientation; thus, the wild-type spacer sequence is the main asymmetric feature of the minimal recombination site. FLP carries out recombination with many minimal target sites bearing symmetric or asymmetric mutant spacer sequences; however, the overall directionality of recombination defined in terms of inversion or excision of a DNA domain is determined by spacer-sequence asymmetry. In order to evaluate the potential influence of spacer-sequence asymmetry on structures formed during early steps in recombination, we used electron microscopy to investigate the structure of the FLP synaptic complex, which is the intermediate protein-DNA complex involved in site pairing and strand exchange. Using linear substrate DNAs that have minimal FRTs with wild-type spacer sequences, we find that 85 to 90% of the FLP synaptic complexes examined contain the two FRTs aligned in parallel. This strong preference for parallel site alignment stands in contrast with prevailing models for lambda integrase-class recombination systems, which postulate antiparallel site alignment, and results from biophysical studies on synthetic, immobile four-way DNA junctions. Our results show that the strong preference for parallel alignment can be attributed to conformational preferences of Holliday junctions present in the synaptosome.
Asunto(s)
ADN Nucleotidiltransferasas/química , ADN de Hongos/química , Recombinación Genética , Sitios de Unión , ADN Nucleotidiltransferasas/metabolismo , ADN Nucleotidiltransferasas/ultraestructura , ADN de Hongos/metabolismo , ADN de Hongos/ultraestructura , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Conformación de Ácido Nucleico , Conformación Proteica , Saccharomyces cerevisiaeRESUMEN
A method of evaluating the photoneutron fluence in the maze of accelerator facilities due to room scattered neutrons is presented. Measurements were made to demonstrate that the room scattered fluence is reduced by a factor of 2pi in going from the treatment room to the inner maze entrance.
Asunto(s)
Neutrones , Aceleradores de Partículas , Protección Radiológica , Rayos gamma , Matemática , Fotones , Monitoreo de Radiación , Dispersión de RadiaciónRESUMEN
Most normal diploid human cells do not express telomerase activity and are unable to maintain telomere length with ongoing cell divisions. We show that the length of the single-stranded G-rich telomeric 3'-overhang is proportional to the rate of shortening in four human cell types that exhibit different rates of telomere shortening in culture. These results provide direct evidence that the size of the G-rich overhang is not fixed but subject to regulation. The potential ability to manipulate this rate has profound implications both for slowing the rate of replicative aging in normal cells and for accelerating the rate of telomere loss in cancer cells in combination with anti-telomerase therapies.
Asunto(s)
Senescencia Celular , Telómero/química , Mama/metabolismo , Células Cultivadas , ADN/metabolismo , Endotelio Vascular/ultraestructura , Epitelio/ultraestructura , Fibroblastos/ultraestructura , Guanina/química , Humanos , Pulmón/metabolismo , Modelos GenéticosRESUMEN
Telomeres protect the ends of linear chromosomes from degradation and abnormal recombination events, and in vertebrates may be important in cellular senescence and cancer. However, very little is known about the structure of human telomeres. In this report we purify telomeres and analyze their termini. We show that following replication the daughter telomeres have different terminal overhangs in normal diploid telomerase-negative human fibroblasts. Electron microscopy of those telomeres that have long overhangs yields 200 +/- 75 nucleotides of single-stranded DNA. This overhang is four times greater than the amount of telomere shortening per division found in these cells. These results are consistent with models of telomere replication in which leading-strand synthesis generates a blunt end while lagging-strand synthesis produces a long G-rich 3' overhang, and suggest that variations in lagging-strand synthesis may regulate the rate of telomere shortening in normal diploid human cells. Our results do not exclude the possibility that nuclease processing events following leading strand synthesis result in short overhangs on one end.