DNA replication in the context of development: Tissue-specific remodeling of the DNA replication fork and dynamic R-loop signatures across embryogenesis
Munden, Alexander Lucas
DNA replication is a fundamental challenge of life. In order to proliferate, every organism must be able to replicate and divide its DNA into daughter cells. Defects in DNA replication can cause in mutations that can result in loss of function or defects in proteins and functional RNAs. DNA replication is meticulously regulated to ensure accurate transmission of genetic and epigenetic data. This exquisitely regulated system undergoes unique challenges during development, including polyploidy, a naive chromatin environment, and contracted cell cycles. In this work, I demonstrated a tissue-specific role for the Rif1 protein in controlling replication fork progression during the endocycles of the Drosophila salivary gland and ovary follicle cells. Next, I adapted a technique to allow quantitative proteomic analysis of the replication fork during embryogenesis. Finally, I turned to a specific obstacle during replication fork progression: R-loops. This three-stranded nucleic acid hybrid plays important roles in regulating chromatin but can also serve as an impediment to replication fork progression. I mapped R-loops genome-wide during early Drosophila embryogenesis, late embryogenesis, and in an established cell line. This work demonstrated that R-loop formation is dynamic across development, this trait is not driven by transcription, that R-loop resolution is important for embryogenesis to proceed, and that R-loops may act to trigger the DNA damage checkpoint at the maternal-to-zygotic transition. Together, this work demonstrated new insight into DNA replication during development.