Molecular dissection of CMG helicase assembly in metazoa

The National Institute of General Medical Sciences awarded The Leland Stanford Junior University $152,600 on December 15, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to support molecular dissection of CMG helicase assembly in metazoa.

The research uses single-molecule imaging to determine how CDC45 is loaded onto DNA replication origins in metazoan cells. The investigator will employ real-time tracking in Xenopus laevis nuclear extracts to determine whether two CDC45 molecules bind to origins simultaneously or sequentially, whether the initiation factor TRESLIN-MTBP physically recruits CDC45 or binds first to provide a new epitope for CDC45 loading, and what amino acids mediate the CDC45:TRESLIN interaction interface. This work addresses fundamental mechanisms of DNA replication initiation; mutations in initiation factors cause genomic instability, developmental defects, and cancer predisposition in humans.

The award runs through June 30, 2029, with place of performance in Palo Alto, California. The assistance type is a Project Grant supporting hypothesis-driven basic biomedical research that increases understanding of biological processes underlying disease diagnosis, treatment, and prevention.

Abstract

Initiating DNA replication requires the assembly and activation of the replicative helicase CMG. This process begins in G1 phase when two inactive MCM2-7 hexamers are loaded at DNA replication origins. In s phase, the inactive MCM2-7 double hexamers are loaded with CDC45 and gins to assemble the replicative helicase. The recruitment of CDC45 and gins is gated by a multilayered set of regulation including the activity of the kinases DDK and CDK2, and the recruitment of a variety of scaffolding proteins we call initiation factors. Mutations in initiation factors lead to erroneous replication initiation, genomic instability, DNA damage, and cell cycle progression defects. Patients who harbor such mutations exhibit developmental defects and are predisposed to cancer. The molecular mechanisms that control replication initiation are of fundamental importance to all eukaryotic organisms and have direct implications for human health and disease. Despite their importance, these molecular mechanisms remain poorly understood. To address these challenges, my mentor's lab recently developed a single-molecule imaging platform capable of tracking these molecular interactions in real-time using xenopus laevis nuclear extracts. Specific aims: in aim 1 i will determine how CDC45 is loaded onto replication origins in metazoa. My approach will be broken into three subaims: 1A. Determine if two CDC45 molecules bind to origins simultaneously, or one after another via real-time single-molecule imaging that i mastered as a graduate student. 1B. Determine if the initiation factor treslin-mtbp physically recruits CDC45 to origins, or if IT binds first and provides a new epitope for CDC45 loading. I will do so by performing dual-color imaging with fluorescently labeled CDC45 and treslin-mtbp. 1C. I will determine what amino acids mediate the CDC45:treslin interaction interface. Key somatic mutations in CDC45 that block origin loading cause the developmental disorder meier-grolin syndrome. My alphafold model shows that the most severe of these mutations are in the predicted CDC45:treslin binding interface. By determining how these two proteins interact, i will shed light on why patients with c-terminus mutations have severe CDC45 origin loading phenotypes. In aim 2, i will determine how the cell cycle kinases DDK and CDK2 regulate the step-wise recruitment of initiation factors during CMG helicase assembly. This aim will be broken down into two subaims: 2A. I will determine which initiation factor is recruited to origins via DDK. During s-phase, DDK phosphorylates MCM2- 7 to promote the recruitment of the first initiation factor, either TOPBP1, treslin-mtbp, or both. I will use dual- color single-molecule imaging to determine if TOPBP1 or treslin-mtbp is recruited to origins in a DDK- dependent process. 2B. I will examine how CDK2 regulates the function of key initiation factors. To do so, i will mutate consensus CDK2 sites in TOPBP1, treslin-mtbp, and donson and assess their origin recruitment via single-molecule imaging and ensemble replication assays.