Speaker
Description
In eukaryotes, linear chromosomes face the "end-replication problem" due to the inherent limitations of the degradation of RNA primers involved in lagging-strand synthesis. To maintain genomic integrity and prevent gradual loss of genetic information in the terminal, organisms utilize telomeres—specialized TG-rich 3’ overhangs DNA structure (G-strand). In budding yeast, telomere maintenance involves G-strand elongation by telomerase, followed by C-strand, the complementary strand, fill-in by the polα-primase complex. However, the regulatory transition between these steps remains poorly understood. This process requires coordination between two key single-strand binding proteins: the canonical Replication Protein A (RPA) and the telomere-specific Cdc13. While RPA is thought to initially coat the lagging strand during replication, it must be displaced by Cdc13 to facilitate telomerase recruitment and end protection. Using single-molecule fluorescence methods, we characterized the real-time displacement of RPA by Cdc13 on various telomere-like 3’-terminating ssDNA overhang substrates, including TG30 and TG15-T15-3’ and T15-TG15-3’. Our results reveal that Cdc13 displaces RPA in a dose-dependent manner through two distinct kinetic pathways: a "fast" displacement characterized by immediate RPA dissociation upon Cdc13 loading, and a "slow" displacement involving a stable Cdc13-RPA-DNA ternary intermediate. These two pathways correspond to end-mediated and ss/dsDNA-junction-mediated displacement mechanisms, with distinct kinetics. These findings suggest that RPA-to-Cdc13 handoff is a regulated process influenced by DNA orientation.
