Defining New Functions in DNA Replication and DNA Damage Response Genes

科学目标：在出芽酵母酿酒酵母中，突变菌株的全基因组收集已被广泛用于大规模阐明其生物学功能。由此产生的信息已被用于将基因置于通路中，确定不同通路之间的交叉点，并将基因功能分配给新的蛋白质。这些研究的一个缺陷源于这些全基因组集合中的突变试剂的性质，其中基因功能已通过删除或条件耗尽而失活。由于许多（如果不是全部的话）蛋白质执行不止一种功能，这种突变可能是多效性的。一个潜在的解决方案是采用功能分离突变，消除蛋白质的单一生物功能。然而，在过去，即使对单个基因来说，这类特殊等位基因的鉴定也是一个逻辑障碍。本研究项目基于一种新开发的大规模分离功能分离等位基因的策略，该策略将应用于涉及DNA复制和DNA损伤反应的一组相互关联的基因。由于这些基因中很少有突变被分离出来，这项研究应该在DNA复制和DNA损伤反应途径中定义新的功能。更广泛的影响：本研究项目的科学目标与两个更广泛的影响密切相关。首先，由此产生的突变将广泛应用于酵母群落，特别关注将这些新试剂纳入全基因组系统分析。由功能分离错义突变构建的遗传网络可能会发现以前未发现的界面，这些界面在先前使用现有突变菌株的系统分析中被遗漏。其次，该项目将严重依赖初级研究人员（本科生和高中生），他们将负责生成功能分离突变面板。这种经验将使非常初级的研究人员能够在重要的研究项目中发挥核心作用，并向他们介绍指导在生物医学研究界发挥的关键作用。

Scientific goals: In the budding yeast S. cerevisiae, genome-wide collections of mutant strains have been employed extensively to elucidate biological function on a large scale. The resulting information has been used to place genes in pathways, to identify points of intersection between different pathways and to assign gene function to novel proteins. One flaw of these studies stems from the nature of the mutant reagents in these genome-wide collections, where gene function has been inactivated by either deletion or conditional depletion. Since many, if not all, proteins execute more than one function, such mutations are potentially pleiotropic. A potential solution is to employ separation-of-function mutations that eliminate a single biological function of a protein. However, identification of this particular class of alleles in the past has been a logistic hurdle even for single genes. This research project is based on a newly developed strategy for large-scale isolation of separation-of-function alleles which will be applied to a set of inter-related genes involved in DNA replication and response to DNA damage. Since very few mutations have been isolated for many of these genes, this research should define new functions in the DNA replication and DNA damage response pathways. Broader impacts: The scientific goals of this research project are closely intertwined with two broader impacts. First, the resulting mutations will be made widely available to the yeast community, with a particular focus on incorporation of these new reagents into genome-wide systems analysis. Genetic networks constructed from separation-of-function missense mutations are likely to uncover previously unappreciated interfaces that were missed in prior systems analysis which employed currently available mutant strains. Second, this project will rely heavily on entry-level researchers (undergraduate and high school students) who will be responsible for generating the panels of separation-of-function mutations. This experience will allow very junior researchers to play a central role in a significant research project and also introduce them to the critical function that mentoring plays in the biomedical research community.