Abstract: DNA replication restart pathways reload cellular DNA replication complexes onto replication forks that have been prematurely terminated, forming an essential link between DNA repair and replication. The proteins that drive these reactions, referred to as the primosome or the Replication Restart Proteins, recognize the structures of abandoned replication forks and reload the DNA replication machinery specifically at these sites. The replication restart process is regulated to ensure loading fidelity and to avoid over-replication that could arise from initiating replication at improper DNA structures. In spite of the broad biological importance of this process, the mechanisms underlying DNA replication restart and its regulation remain poorly understood. Our proposal combines structural, biochemical, and genetic approaches to define the structural and cellular mechanisms of DNA replication restart. Our overall objective is to determine the mechanisms that govern each step of the process, from recognition of diverse structures of abandoned DNA replication forks, to primosome assembly, and finally to reloading the DNA replication machinery. Aim 1 will define the structures of primosome complexes with replication forks, which will provide new insights into structure-specific recognition of DNA replication forks and the mechanisms that regulate DNA replication restart. Structural advances are coupled to genetic studies that will define the cellular mechanisms of action of primosome proteins in bacteria. Aim 2 blends structural and genetic studies to focus on maturation of primosome complexes and their activities in reloading the replicative helicase back onto replication forks. Completion of our specific aims will define the molecular and cellular mechanisms that mediate and regulate bacterial DNA replication restart.