The mammalian genome is a highly organized structure where distinct chromosomal domains occupy discrete territories and position in a non-random fashion. Genome organization also determines the proper acquisition of various biological processes, including DNA replication. Errors in DNA replication can catastrophically degrade both the genetic and the epigenetic integrity of chromosomes, contributing to genome instability that drives tumorigenesis. Chromatin modifiers are the major determinants of chromatin architecture as well as accessibility to nucleosomal DNA which allows efficient DNA replication. However, the mechanistic understanding of 3-D nuclear chromatin organization orchestrated via chromatin modifiers in mediating stabilization of replication machinery upon replication stress has been poorly studied. My hypothesis is that stalled replication forks, especially at the regions prone to acquire breaks, undergo compartmentalization/re-organization within 3-D nuclear space in response to replication stress to allow efficient fork protection and fork restart ability. My lab uses a multidisciplinary approach for molecular characterization of DNA replication stress in mammalian system, taking advantage of a unique combination of specialized single molecule analysis of replication forks, super-resolution/high-throughput imaging and unique 3-D chromatin conformation capture techniques. Building on my expertise in the study of chromatin organization and DNA replication stress, for this proposal, I will focus on dissecting the role of chromatin architecture in tolerance to replication stress by: a) developing a high-resolution technology to capture novel chromatin interactions ensuing directly at replicating sites in response to replication stress; b) determine the role of chromatin modifiers in mobilizing the genomic loci under replication stress; and c) unravel the significance of re-organization of chromatin architecture in developing resistance towards replication stress in cancer cells. These studies will contribute to an overall understanding of how chromatin architecture adaptations in diseases such as, cancer can develop resistance towards replication stress inducing chemotherapeutic drugs.