Exploring the roles of Rrm3 in replisome progression through G4 sequences and mutational avoidance.

DNA replication, the molecular process by which a cell’s genetic material is accurately duplicated during cell division, is essential for the growth and proliferation of all living organisms. A macromolecular complex termed the replisome replicates genomic DNA in eukaryotes. Some genomic regions tend to form highly stable secondary structures, such as G quadruplex (G4), which can form structural blocks to the replisome. To enable DNA replication through G4 rich genomic regions, another set of enzymes termed G4 helicases are required to unwind these structures. Rrm3 is one of the most important enzymes in this family of helicases and enables efficient replication through a variety of challenging genomic regions. However, despite this knowledge, little is known regarding the importance of Rrm3 for promoting replication through G4 rich sequences. Here, we will build upon a novel single-cell high-resolution imaging assay we developed for assessing the direction and efficiency of DNA replication progression at a specific chromosomal locus in live yeast cells. We will use our system to determine the rules by which replisome progression through G4 sequences is regulated by Rrm3. The novel single-cell imaging experiments will be complemented by biochemical studies using purified proteins and defined G4 sequences to examine Rrm3 helicase activity in vitro. Finally, we will examine the importance of Rrm3 for mutational avoidance using in vivo and in vitro mutational assays. Overall, this study will significantly deepen our understanding of the cellular and biochemical functions of Rrm3 and pave the way for future studies to elucidate the critical balance between helicases and replisome progression during DNA replication that protects genome stability.