DNA REPLICATION AND CHROMOSOME STRUCTURE IN YEAST

该补助金的长期目标是了解确保 真核生物染色体的忠实维护，使用酵母 酿酒酵母作为模式生物。 在大多数真核生物中， 染色体端粒由简单重复的DNA组成 例如在 酵母，每个末端有约300个碱基对的C1-3A/TG 1 -3 DNA 染色体 许多生物，包括酵母，也有内部的管道， 端粒序列 其中一些内部纤维束靠近端粒， 而其他的位于更内部的染色体位点。 端粒是 对酵母染色体的稳定维持绝对必要（*）。 虽然端粒序列内部束的功能尚不清楚， C1-3A/TG 1 -3的内部束抑制附近基因的转录， 减少了重组。 转录抑制和减少 内部1 -3A/TG 1 -3区的重组特征是 当纤维束靠近端粒时会加重，这表明内部 纤维束和端粒相互作用。 下一次融资的具体目标 一段涉及端粒和C1 -3内束维持的时期 A/TG1-3 DNA。 第一个目标是开发一个检测端粒的系统- 端粒重组 端粒将被标记为变异重复序列 由突变的端粒酶RNA编码 将变体转移到其他变体 将在野生型和突变细胞中监测端粒。 的假设 端粒-端粒重组允许端粒维持， 将检测主要复制途径端粒酶是否受损。 的 第二个目标是鉴定调节基因之间的重组 C1-3A/TG 1 -3 DNA的内部束。 这些实验将决定 存在限制重组的特定回归因素，如果减少 重组与晚期复制相关。 第三个目标是 确定Piflp解旋酶如何限制现有端粒的长度 并降低端粒从头添加的速率和特异性。 两 主要模型，Piflp是端粒酶的抑制剂，或者Piflp是端粒酶的抑制剂。 将测试重组抑制剂。 此外，功能 酵母属和S.蓬贝将是 确定，和一个2-混合的方法将用于识别Piflp- 相互作用的蛋白质 第四个目的是确定是否有内部的管道， C1-3A/TG 1 -3 DNA与端粒一样，具有非核小体染色质结构 以及这种染色质结构是否因接近端粒而改变。 非整倍体和染色体重排与几乎所有的 人类癌症，衰老，和先天缺陷。 端粒DNA缺失 和/或端粒序列的内部区段之间的重组可以 引发与这些疾病相关的染色体异常 条件 由于染色体的端粒区域与酵母相似， 对人类来说，了解端粒和端粒内部的 在酵母中保持的序列可能与 了解端粒和端粒序列的内部区域是如何 在酵母中维持的可能与理解 人类遗传不稳定的根源。

The long range goal of this grant is to understand processes that ensure the faithful maintenance of eukaryotic chromosomes, using Saccharomyces cerevisiae as a model organism. In most eukaryotes, the very ends of chromosomes, telomeres, consist of simple repetitive DNA. For example, in yeast, there are about 300 bps of C1-3A/TG1-3 DNA at each end of each chromosome. Many organisms, including yeast, also have internal tracts of telomeric sequence. Some of these internal tracts are near telomeres, whereas others are at more internal chromosomal sites. Telomeres are absolutely essential for the stable maintenance of yeast chromosomes (*). Although the function of internal tracts of telomeric sequence is unknown, internal tracts of C1-3A/TG1-3 repress transcription of nearby genes and have reduced recombination. The transcriptional repression and reduced recombination characteristic of internal1-3A/TG1-3 tracts are both accentuated when the tracts are near telomeres, suggesting that internal tracts and telomeres interact. The specific goals for the next funding period concern maintenance of both telomeres and internal tracts of C 1-3 A/TG1-3 DNA. The first aim is to develop a system to detect telomere- telomere recombination. Telomeres will be marked with a variant repeat encoded by a mutant telomerase RNA. The transfer of the variant to other telomeres will be monitored in wild type and mutant cells. The hypothesis that telomere-telomere recombination allows telomere maintenance when the primary replication pathway, telomerase, is impaired will be tested. The second aim is to identify genes that regulate recombination between internal tracts of C1-3A/TG1-3 DNA. These experiments will determine if there are specific regressors that limit recombination and if reduced recombination correlates with late replication. The third aim is to determine how the Piflp helicase limits the length of existing telomeres and reduces the rate and specificity of de novo telomere addition. Two major models, that Piflp is an inhibitor of telomerase or that Piflp is an inhibitor of recombination will be tested. Also, the functional relatedness of PIF-1-like genes from Saccharomyces and S. pombe will be determined, and a 2-hybrid approach will be used to identify Piflp- interacting proteins. The fourth aim is to determine if internal tracts of C1-3A/TG1-3 DNA, like telomeres, have a non-nucleosomal chromatin structure and if this chromatin structure is altered by proximity to a telomere. Aneuploidy and chromosomal rearrangements are associated with virtually all human cancers, with aging, and with birth defects. Loss of telomeric DNA and/or recombination between internal tracts of telomeric sequence can trigger the kinds of chromosomal abnormalities associated with these conditions. Since telomeric regions of chromosomes are similar from yeast to humans, understanding how telomeres and internal tracts of telomeric sequence are maintained in yeast is likely to be relevant to an understanding how telomeres and internal tracts of telomeric sequence are maintained in yeast is likely to be relevant to an understanding of the sources of genetic instability in humans.