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

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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.