The DNA Fiber Assay: An insight into accurate DNA replication
Category: Research Poster
Author(s): Jordi Gomez, Mollie Uhrig, Neelam Sharma, Claudia Wiese
Presenter(s): Jordi Gomez
Mentors(s): Claudia Wiese
Precise DNA replication is critical for all living organisms. Every day, our cells divide and undergo semi-conservative DNA replication to generate identical daughter cells. However, many external and internal factors induce replication stress and pose a threat to the accuracy of the genetic material passed down to daughter cells. To overcome DNA replication stress, our cells have adapted mechanisms to maintain precise DNA replication and avoid disease-promoting mutations. To understand how cells respond to DNA replication stress, we must utilize specialized methods. We use the DNA fiber assay to analyze the dynamics of DNA replication at a single-molecule level. In the DNA fiber assay, exponentially growing human cells are labeled with two thymidine analogs, harvested, dropped on a microscope slide, and lysed. The slide is tilted at a slight angle, and, with the help of gravity, the DNA is then stretched along the slide. Following immunostaining, we can visualize the labeled DNA with fluorescence microscopy. Analysis of the imaged DNA results in a quantification of DNA replication speed and stability. Through this experimental approach, we can compare replication dynamics in various cell types, as well as how different proteins affect replication. Additionally, the DNA fiber assay allows us to analyze the condition of human cells that have been treated with chemotherapeutic drugs, which typically induce replication stress. Our overarching goal is to employ the DNA fiber assay to understand the mechanisms and control of DNA replication, how cells overcome DNA replication stress, and how this translates to human health.