Summary DNA replication stress is a major source for genome instability associated with numerous diseases, including cancer. Therefore, understanding the molecular basis of replication stress and, conversely, how accurate, complete, and rapid chromosomal DNA replication is achieved during normal cell proliferation, is crucial for understanding the mechanisms that maintain or threaten genome stability during normal development and disease, respectively. Here we propose experiments that will illuminate both the intrinsic mechanism of the eukaryotic DNA replication machinery and its response to diverse replication stress conditions. Previously, we have generated a fully reconstituted origin-dependent DNA replication system based on purified budding yeast proteins. More recently, we have begun to reconstitute replisomes with purified human proteins. These systems form the central platform for research in our lab. The normal and uninterrupted progression of replication forks is frequently challenged by physical obstacles on the parental DNA. These include non-B-form DNA secondary structures and tightly bound non- histone protein-DNA complexes. While non-B-form DNA secondary structures induce fork stalling in a stochastic and unscheduled manner, the programmed stalling of the replisome at tightly bound protein-DNA complexes can serve important biological functions. How individual physical obstacles on chromosomal DNA induce fork stalling, are overcome by fork restoration mechanisms, or elicit distinct biological outcomes is incompletely understood. To illuminate fork stalling mechanisms, we will investigate the molecular mechanisms by which such physical obstacles impede the eukaryotic replicative DNA helicase, CMG (Cdc45- MCM-GINS). Specifically, we will determine the mechanisms by which G-quadruplexes (G4s) impede CMG progression on the leading strand and the mechanisms by which replication forks stalled at G4s may be restored by accessory DNA helicases and fork remodelers. As an example for programmed fork stalling at protein-DNA complexes, we will investigate the mechanism of unidirectional fork stalling at the replication fork barrier (RFB) derived from both yeast and human rDNA repeats and examine the mechanism of replication termination upon fork convergence at these sites. Stalled replication forks are both inducers and targets of the replication checkpoint, which is essential for the maintenance of genome integrity by stabilizing stalled forks and coordinating fork restoration with cell cycle progression. Continuing our previous work, which has led to the identification of yeast replisomes as direct targets for the checkpoint effector kinase, Rad53, we will determine the mechanism(s) by which the checkpoint controls fork progression.