A break in both strands of the DNA double helix is one of the most harmful DNA lesions because if not properly repaired it leads to mutations, chromosome rearrangements or cell death. The safest way to repair a DNA double-strand break (DSB) is through a process called homologous recombination (HR). The known mechanisms of HR utilize an intact copy of the DNA sequence as a template to retrieve the information lost at the site of a DSB. However, recent results, which will be expanded in this study, indicate a new role of RNA in this process of DNA repair. Therefore, the study will provide important new biological insights to better understanding the physiological function of RNA in DNA repair, and the impact of RNA on genome maintenance and evolution. A postdoctoral fellow, a graduate student and several undergraduate students will be involved in the research. The work and findings of this proposal will be integrated in class topics and activities for many graduate and undergraduate students. Through the inclusion of a Research Experience for Teachers (RET), the project will also target students from a local, 100%-minority High School to support student interest and participation in Science, Technology, Engineering and Math (STEM) programs.
To characterize the mechanism of RNA-DNA recombination, novel genetic systems will be developed to study repair of chromosomal double-strand breaks (DSBs) by homologous transcript RNA in budding yeast cells. One Objective of this project will be to identify the DNA polymerization function/s that use RNA as template in RNA-DNA recombination. Another focus will be to examine whether the process of DSB repair by transcript RNA can be visualized in cells arrested at two distinct points in the cell cycle: before and after DNA replication. It was shown that DSB repair by transcript RNA in yeast cells is initiated efficiently, but it is quickly suppressed by cellular ribonuclease H enzymes. Here, refined genetic systems to detect DSB repair by RNA will be utilized to determine the efficiency of DSB repair by transcript RNA in wild-type and ribonuclease-defective cells. Overall, the work of this project will help to better understand the conditions and mechanism in which transcript RNA is a preferred direct template for DSB repair in cells.
This project is funded by the Genetic Mechanisms Program in the Division of Molecular and Cellular Biosciences.
