Mechanistic insights into the initiation of DNA replication in human cells

Summary This project aims to investigate various aspects of the initiation of DNA replication in human cells. DNA replication is a key process absolutely necessary before each cell division. This process is extremely tightly controlled to ensure its accuracy and timing. The number of licensed origins as well as their firing efficiency defines the ability of cells to timely complete replication, respond to replication stress, and avoid genome instability. Despite recent progress, the initiation of mammalian replication has not been reconstructed in vitro to date, suggesting the existence of origin firing factors and mechanisms that are still not fully described. While in yeast the origins of DNA replication are well defined, their location and regulation in mammalian cells is much more nuanced. Multiple efforts to map origins of replication in cultured cells identified various subsets of replication initiation sites, co-localizing with transcription start sites, as well with G4-structures, lamins, and various chromatin marks. We propose that different subsets of replication origins may require different sets of firing factors for replication initiation, depending on the chromatin context. This project uses a combination of cell biology, genetic, and proteomic approaches to uncover novel factors involved in human origin firing, the order and mechanism of recruitment of specific proteins to the origins, as well as investigates how co-localization of origins with various genomic features affects the mechanisms of their activation. We will use a split-TurboID approach combining transient firing factors with core fork components, as well as location-specific proteins, and identify replication complex (RC)- associated proteins at various stages of origin firing, as well as various chromatin marks and gene elements. We will determine which origin firing factors are common for all replication initiation events, and highlight specific requirements for particular subsets of origins. Given the essential status of the majority of replication proteins, we will leverage mini-auxin-inducible degron system, routinely used in the lab, in combination with cell synchronization approaches, and ATRi-induced massive dormant origin firing, to further investigate the newly discovered firing factors, determine their precise roles in replication initiation, and at which step of the RC assembly/activation they are required. Ultimately, this project will shed light on the complex and diverse process of replication initiation in human cells, and bring us closer to the full understanding of its regulation. Project Number: 1R35GM161292-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Tatiana Moiseeva | Institution: UNIVERSITY OF PITTSBURGH AT PITTSBURGH, PITTSBURGH, PA | Award Amount: $434,212 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award - F Study Section[MRAF] View on NIH RePORTER: https://reporter.nih.gov/project-details/11258706