Summary. Timely and faithful replication is essential to cellular proliferation in all living systems. Replication malfunction leads to mutations, breaks in the DNA, and cell death, all of which play central roles in human diseases including cancer and the development of antibiotic-resistant bacterial pathogens. Very recently, our laboratories have discovered that conflicts between the replication and transcription machineries increase mutation rates as well as cause significant instability of the replisome complex during the replication process. Our long-term goal is to dissect the mechanisms, cellular responses and biological and medical consequences of transcription-replication conflicts. The objective of this grant is to characterize the structure and stability of the replisome in general, and in particular, in response to transcription-induced conflicts with single-molecule sensitivity. Motivated by our recent observations, the central hypothesis of this proposal is that replisome structure is a critical regulator of replication and a mechanism of conflict avoidance. Our rationale is that we will gain fundamental insight into both the replication process as well as replication conflicts by studying the replisome structure in living cells one conflict at a time with single-molecule sensitivity. Our specific aims combine structural and functional analyses: Aim 1 describes a program to characterize the dynamic organization of the replisome at high-resolution. Aim 2 describes a functional analysis of the mechanisms for exchange, restart and recovery after replisome collapse. Aim 3 describes the test of the hypothesis that cellular organization is key mechanism for reducing replication conflicts. Our preliminary work has already changed the fundamental understanding of the replication process by demonstrating that it is discontinuous. The proposed work is significant since it will continue this program by probing fundamental, but untested assumptions about the structure of the replisome and the cellular mechanisms of conflict resolution and avoidance. The proposed research is innovative because we apply an interdisciplinary approach to study the replisome with single-molecule sensitivity in living cells. No other experiments have yet probed the replisome structure with this resolution in vivo and therefore the work has great potential to reveal both novel and fundamental insights into replisome structure and function.