Our focus is on eukaryotic DNA replication and its control, with emphasis on the identification of the protein components involved in these processes. The human replicative helicase, consisting of Cc45-Mcm2-7-GINS (CMG) and the replicative DNA polymerases, catalyze leading and lagging strand syntheses. Our goal is to recapitulate in vitro the in vivo observations suggesting that Pol ε catalyzes leading strand DNA synthesis while Pol δ plus Pol α-primase complex support lagging strand synthesis. We utilize a primed 200-nt circle containing only three nucleotides so leading strands can be measured conveniently using dATP incorporation while lagging strand can be followed by dTTP incorporation. We suggest that the CMG complex is the protein core at the replication fork. Proteins involved in replication, including DNA polymerases, Ctf4, FACT, Tim-Tipin, etc., interact with the CMG complex and constitute the moving replisome. We are investigating these interactions and their influence on the CMG helicase activity both in vitro and in vivo. Alterations in components of the CMG complex play important roles in DNA replication. Specific human mutations in Psf1 (a GINS component) have been detected and their effects on the formation of GINS and CMG are being investigated. A number of these mutations lead to developmental defects; we plan to determine whether alterations in DNA replication in cells isolated from the Psf1 human mutants contribute to the observed phenotypic defects.