RUI: Mechanisms of Aging in Saccharomyces cerevisiae

在一项由NSF power资助的研究中，研究人员发现酵母DNA2基因对正常寿命至关重要。DNA2编码一种与SGS1和Werner综合征解旋酶有远亲关系的复制和复制后修复解旋酶。4个dna2突变体的平均寿命为4.9 ~ 8.9代，而野生型为23.8代。sgs1和dna2突变体似乎不具有上位性。其他复制突变体的寿命也很短。两株rDNA全部缺失和rDNA由质粒提供的菌株寿命缩短。这些结果导致了酵母DNA老化的复制阻断模型，表明复制性衰老可能是由被阻断的复制分叉的积累引起的。此外，最近发现Y55二倍体在减数分裂中重置了它们的衰老时钟。该项目测试了复制分叉受阻导致的复制衰老模型，并将开始寻找参与重置酵母衰老时钟的基因。通过观察除dna2和核糖体缺失菌株外的复制和复制后修复突变体的大小增加、不育、核仁断裂和rDNA扩增的衰老表型，来验证复制叉阻断假说，看看这些突变体中正常衰老的各个方面是否会加速。如果是这样，这个特殊的过程可能与老化的设定有关。如果rDNA缺失似乎衰老得更快，那么将测试沉默信息调节因子和解旋酶突变的上位性。DNA的结构，特别强调由停止复制分叉导致的结构，将使用纤维- fish， 2D凝胶，脉冲场和正常电泳结合核酸酶消化来检查。此外，在影响出芽不对称的突变体中，正在研究衰老时钟的重置。在另一种减数分裂菌株BR3604中，减数分裂是否影响时钟重置将被确定。如果时钟在减数分裂中被重置，孢子突变体将被用来测试减数分裂中特定步骤在重置中的参与。这项新研究有望进一步加深我们在分子水平上对衰老过程的理解。酵母系统的使用增强了该项目的前景，因为许多分子工具可用于酵母和广泛的现有知识体系。这项研究将在波莫纳学院进行，这是一所本科院校。学生积极参与这项研究是该项目的一个特别吸引人的特点。

In research funded by an NSF POWRE grant awarded to the investigator, the yeast DNA2 gene was found to be essential for normal life span. DNA2 encodes a replicative and post replication repair helicase distantly related to SGS1 and Werner syndrome helicases. Four dna2 mutants have average life spans from 4.9 to 8.9 generations, while wild type is 23.8 generations. The sgs1 and dna2 mutants do not appear to be epistatic. Other replication mutants also have short life spans. Two strains with all rDNA deleted and rDNA supplied by plasmids have shortened life spans. These results led to the replication block model of DNA aging in yeast, suggesting that replicative senescence can result from accumulation of blocked replication forks. In addition, Y55 diploids were recently found to reset their aging clocks in meiosis. This project tests the model of replicative aging resulting from blocked replication forks and will begin a search for genes involved in resetting the yeast aging clock. The replication fork block hypothesis is being tested by observing the aging phenotypes of size increases, sterility, nucleolar fragmentation, and rDNA amplification in replication and post replication repair mutants other than dna2 and in ribosomal deletion strains, to see whether or not aspects of normal aging are accelerated in these mutants. If so, the particular process may be related to aging pacesetting. If the rDNA deletions appear to age faster, then epistasis with mutants of the silent information regulators and helicases will be tested. The structures of the DNA, with particular emphasis on structures that would result from stalled replication forks, will be examined using fiber-FISH, 2D gels, and pulsed field and normal electrophoresis combined with nuclease digestion. In addition, resetting of the aging clock is being examined in mutants affecting budding asymmetry. Whether meiosis affects clock resetting in a different meiosis strain, BR3604, will be determined. If the clock is reset in meiosis, sporulation mutants will be used to test the involvement of specific steps in meiosis in the resetting.This novel research promises to further our understanding at the molecular level of the process of aging. The use of the yeast system enhances the promise of the project because of the many molecular tools available for yeast and the extensive existing body of knowledge about the system. This research will be carried out at Pomona College, an undergraduate institution. The active involvement of students there in this research is an especially attractive feature of the project.