Function and Regulation of DNA Replication Origin Firing Factors

ABSTRACT DNA replication is a highly dynamic and deeply regulated process during which thousands of replication origins will fire. The efficiency of these origin firing events is highly variable across genomic regions and changes throughout S-phase. This variability in origin firing efficiency carries profound implications, as its deregulation leads to genome instability and cancer. Despite its critical importance, our current understanding of the fundamental mechanisms governing these variable origin firing efficiencies at different genomic locations and times during S phase remains surprisingly limited. This proposal’s overall objective is to address critical gaps in understanding the regulation of origin firing efficiency, focusing on the recruitment of two essential origin firing factors called TICRR/TRESLIN and MTBP, to replication origins throughout the cell cycle. The research proposal seeks to complete three specific aims: 1) Determine the mechanisms for replication origin selection by TICRR and MTBP; 2) Define roles for transcription factors and chromatin modified in determining where replication factors bind and replication origins fire; 3) Uncover how TICRR/TRESLIN and MUVB-MYBL2 functionally interact to coordinate mitotic entry with the completion of DNA replication. The applicant has created a series of human cell lines that will serve as ideal models for elucidating the mechanism of action for specific replication initiation proteins through approaches including flow cytometry-based assays, Cut&Run, Optical Replication Mapping, ChIP-qPCR and SLAM-Seq. In Aim 1, the applicant will examine the specific contributions of TICRR and MTBP to origin site specification as cells progress from G1 to early and late S-phase. Aim 2 will test whether origin site specification depends on physical interactions, which the applicant discovered, between TICRR and a transcription factor and histone acetylation reader proteins. Aim 3 will examine the coordination of gene activation of G2/M genes with the completion of DNA replication using both cultured human cells and zebrafish embryos. This coordination is crucial for cell cycle progression, yet it remains under-researched, highlighting its potential impact. The overall research in this proposal is innovative because it examines a mechanism by which cells actively control the activation of replication origins and coordinate the completion of DNA replication with mitotic entry. Successful achievement of the aims presented in this grant will be significant because it will help answer long-standing, fundamental questions about how the genome is accurately and completely replicated. Importantly, the studies are focused on delineating mechanisms that may be manipulated for treating diseases such as cancer.