EAGER: Creation and validation of a tool to examine the molecular mechanism of replication fork repair

该项目的总体目标是开发工具来研究如何修复常见的遗传损伤并从细胞中移除。对遗传物质最常见的攻击之一是DNA-蛋白质交联的形成，即DNA和蛋白质之间形成的共价键。其中一些是细胞代谢（包括酒精的加工）不可避免的后果，而另一些则可能是由环境暴露或药剂引起的。这些病变干扰DNA复制，并可能导致基因突变，对细胞存活产生有害后果。在人类中，DNA-蛋白质交联与癌症和过早衰老有关。了解DNA-蛋白质交联是如何修复的，将使我们能够开发出避免与这些病变相关的问题或增强其去除的方法。该项目的更广泛影响是培训来自不同背景的学生如何进行科学研究。 在特定的遗传位点和特定的时间产生DNA蛋白质交联将使研究人员能够研究该位置的事件以实现交联修复。该项目将使用天然产生与DNA交联的蛋白质，脱氧胞嘧啶甲基转移酶（CMeTs），其可以在添加药物5-氮杂胞苷后被捕获。该研究将采用模型遗传生物，啤酒酵母（酿酒酵母）和肠道细菌（大肠杆菌），它们可以在实验室中轻松生长，并且可以使用各种基因组操作工具。为了实现位点特异性，来自细菌限制性系统的CMeT将被工程化以在两种模型生物中表达。也有计划设计dCAS 9-CMeT，其中可以通过表达指导RNA来赋予位点特异性。选择的一个CMeT将仅在DNA的两条链中的一条上产生交联，这对于解决这些模板链在被阻断的复制叉中如何被不同地处理特别有价值。通过分子和遗传分析，该项目将验证工程结构产生DNA-蛋白质交联，阻断复制，并确定它们是否促进遗传不稳定性。该奖项反映了NSF的法定使命，并被认为是值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估的支持。

The overall goal of this project is to develop tools to study how a common genetic lesion is repaired and removed from the cell. One of the most frequent assaults to genetic material is the formation of DNA-protein crosslinks, covalent bonds that form between DNA and protein. Some of these are unavoidable consequences of cellular metabolism, including the processing of alcohol, and others can be induced by environmental exposure or pharmaceutical agents. These lesions interfere with DNA replication and can cause genetic mutations with deleterious consequences to cell survival. In humans, DNA-protein crosslinks have been implicated in causing cancer and premature aging. Understanding how DNA-protein crosslinks are repaired will allow us to develop ways to avoid the problems associated with these lesions or to enhance their removal. A broader impact of this project is to train students from diverse backgrounds on how to conduct scientific research. Producing a DNA protein crosslink at a particular genetic site and at a particular time would allow investigators to study the events that that place to achieve crosslink repair. This project will use proteins that naturally produce crosslinks to DNA, deoxycytosine methyl-transferases (CMeTs), which can be trapped after addition of a drug, 5-azacytidine. The study will employ model genetic organisms, brewer’s yeast (Saccharomyces cerevisiae) and a gut bacterium (Escherichia coli), which can be grown easily in the laboratory and for which a variety of genome manipulation tools are available. To achieve site-specificity, CMeT from bacterial restriction systems will be engineered for expression in both model organisms. There are plans to engineer a dCAS9-CMeT as well, in which site-specificity can be conferred by expression of a guide RNA. One CMeT chosen will produce a crosslink only on one of the two strands of DNA, which is particularly valuable to resolve how these template strands are processed differently in a blocked replication fork. By molecular and genetic analysis, the project will validate that the engineered constructs produce DNA-protein crosslinks, block replication and determine if they promote genetic instability. Mutants in various repair pathways will be assayed for their roles in the process.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.