Complete and faithful duplication of the genome is a fundamental prerequisite for cell division in development and tissue renewal. The protein machinery that replicates DNA is part of a highly integrated protein network that maintains genomic stability in all cells. Errors and malfunctions in genome maintenance can result in loss of cell viability and are responsible in large part for diseases such as cancer, including virus-associated cancers. However, major gaps remain in our knowledge of how the mammalian genome is duplicated, how this process is regulated, and how malfunctions are corrected. The long-term goal of the proposed research is to elucidate in molecular detail the mechanisms that control DNA replication in mammalian cells. Chromosomal DNA replication begins with the synthesis of RNA primers that can be extended by a DNA polymerase. In prokaryotes, a dynamic nanomachine known as a 'primosome' couples duplex DNA unwinding with RNA primer synthesis, but eukaryotic primosomes remain elusive. Analysis of simian virus 40 (SV40) DNA replication provides the first insight into a eukaryotic primosome that can be reconstituted with purified recombinant proteins in vitro. The SV40 primosome consists of the viral helicase large T antigen (Tag), the host DNA polymerase alpha-primase (pol-prim), and the host single-strand DNA-binding protein RPA. Our previous work suggests that a network of at least six pairs of physical interactions of the viral helicase Tag with human pol-prim and RPA gives rise to primosome activity. Similarly, a conserved vertebrate helicase B (HELB/HDHB) interacts with pol-prim and RPA, and displays primosome activity in vitro. HDHB has been implicated in chromosomal replication and in replication restart after DNA damage in human cells. The proposed research program is designed (1) to develop a comprehensive mechanistic understanding of the SV40 primosome at the atomic level, and (2) to extend this mechanistic analysis to the HDHB primosome activity and determine whether this activity is linked to HDHB-mediated replication restart after DNA damage. We anticipate that these simple primosome mechanisms will serve as useful models for identifying and understanding the complex primosome(s) that initiates chromosomal replication in eukaryotes and others that may restart replication forks arrested by DNA damage.