GENETIC INSTABILITY IN YEAST

描述：(申请人描述)在各种生物体中，导致DNA复制酶耗尽的突变会增加遗传不稳定的发生率--以高重组缺失率和/或染色体丢失为特征的事件。Werner综合征(WS)的许多细胞表型表明它属于这种遗传缺陷。Werner综合征基因WRN最近被克隆和测序，并被发现编码RecQ家族的DNA解旋酶。申请人提出，在没有WRN解旋酶的情况下进行复制的细胞在DNA中留下损伤，这些损伤由产生基因组重排的重组系统修复。他们计划通过寻找在缺乏酵母WRN解旋酶同源物Sgs1p的情况下可能产生的特定类型的复制缺陷，来探索在酵母中这种可能性。此外，通过研究在缺乏SGS1蛋白的酵母细胞中诱导的重组事件的类型，以及寻找同时与正常的WRN和SGS1解旋酶相互作用的蛋白质，他们希望深入了解在缺乏这种解旋酶的情况下复制机制产生的初级损伤的类型。鉴定出与人类蛋白质具有同源性的酵母蛋白质，以及在酵母中对它们进行遗传学研究的能力，使人们对许多导致人类疾病的过程有了了解。由于酿酒酵母的基因组很小--最近已经全部测序--所以在复制过程中对染色体进行物理分析是非常可行的。在任何其他真核生物中，用于复制启动的顺式作用元件和反式作用蛋白的特征都不像在酵母中那样好。此外，基因组易于操纵使酵母成为研究基本染色体生物学的理想宿主生物。最后，酵母通过双杂交转录报告系统提供了一个用户友好的细胞环境，在其中研究蛋白质与蛋白质的相互作用。

DESCRIPTION: (Applicant's Description) In a variety of organisms, mutations that cause depletion of DNA replication enzymes increase the incidence of genetic instability-- events that are characterized by elevated rates of recombination deletion, and/or chromosome loss. Many of the cellular phenotypes of Werner Syndrome (WS) suggest that it falls into this class of genetic defect. The Werner syndrome gene WRN was recently cloned and sequenced and was found to encode a DNA helicase of the RecQ family. The applicants propose that cells that carry out replication in the absence of the WRN helicase leave behind lesions in the DNA that are repaired by a recombination system that produces the genomic rearrangements. They plan to explore this possibility in yeast, by looking for specific types of replication defects that might be generated in the absence of the yeast WRN helicase homologue, Sgs1p. In addition, by studying the types of recombination events induced in yeast cells deficient for the Sgs1 protein, and by looking for proteins that interact with both the normal WRN and Sgs1 helicases, they hope to gain insight into the types of primary damage generated by the replication machinery in the absence of this helicase. The identification of yeast proteins with homology to human proteins and the ability to study them genetically in yeast has led to an understanding of many processes that contribute to human disease. Because of the small genome size of S. cerevisiae-- recently sequenced in its entirety-- the physical analysis of chromosomes during their replication is quite feasible. In no other eukaryotic organism have the cis-acting elements and trans-acting proteins for replication initiation been as well characterized as they have been in yeast. Moreover, the ease with which the genome can be manipulated makes yeast an ideal host organism in which to study basic chromosome biology. Finally, yeast provides a user-friendly cellular environment in which to study protein-protein interactions by way of the two-hybrid transcriptional reporter system.