BPS2026 - Dynamic pathways supporting bypass of the origin recognition complex by RNA polymerase

In budding yeast, replication begins when the origin recognition complex (ORC) recognizes and binds autonomously replicating sequences (ARS) and then recruits CDC6 and Cdt1 to load the MCM helicase, establishing licensed origins for subsequent activation. This two-step process, temporally separated between G1 (licensing) and S phase (firing), ensures that replication occurs only once per cell cycle. Recent single-molecule studies (Scherr et al., Cell Rep. 2022) uncovered unexpected dynamics of ORC during transcription by RNA polymerase. ORC is not only pushed but also bypassed, suggesting unrecognized mobility. However, the molecular mechanism of ORC bypass remains unclear. One possibility is that ORC exploits secondary DNA-binding sites to transiently “hop” along DNA; alternatively, conformational states may facilitate release and rebinding during transcriptional collisions. To address these questions, I designed three ORC1 truncations: one lacking the basic patch, one preserving a single basic patch, and one containing two basic patches- allowing us to test how these regions modulate ORC’s ability to be pushed or bypassed during transcription. Using single-molecule total internal reflection fluorescence (TIRF) microscopy, which provides spatial and temporal resolution, this work aims to reveal how ORC engages with DNA during collisions with RNA polymerase and define the mechanisms that govern ORC mobility. Together, these studies will shed light on a fundamental yet unresolved question, what are the mechanics of ORC bypass during transcription. Understanding ORC bypass not only provides mechanistic insights into replication origin usage but also reveals broader principles of genome stability in eukaryotic cells.