Structural mechanism of DNA replication

Project Summary DNA replication is thought to have evolved twice, independently, in Bacteria and Archaea/Eukarya, as the principal components of the replication machinery such as the replicative helicase and the DNA polymerases are not evolutionarily related in the two branches of life. Initiation of eukaryotic DNA replication occurs in G1 phase of the cell cycle, when the initiator Cdc6 binds and activates the Origin Recognition Complex (ORC) to recruit Cdt1-bound Mcm2-7 hexamer, thereby assembling an inactive Mcm2-7 double-hexamer on double stranded DNA and establishing the pre-Replication Complex (pre-RC). At G1-to-S transition, the Mcm2-7 double-hexamer is converted to two active replicative helicases, the Cdc45-Mcm2-7-GINS (CMG) complexes. The CMG helicase and the Ctf4 trimer serve as the hubs to nucleate a replisome to synthesize DNA. Advances in in vitro reconstitutions of origin activation, leading strand and lagging strand DNA synthesis, along with the cryo-EM resolution revolution, have made it feasible to visualize a replisome and tackle the complex replication mechanism. In the previous funding cycle, we have elucidated how Cdc6 binds ORC to activate it and enhance origin specificity, how ORC-Cdc6 recruits and loads Mcm2-7 onto origin DNA in a multi-step process, and how the CMG helicase unwinds DNA duplex at a fork. We have determined the complete structures of all three major enzymes at the replication fork – the leading strand polymerase Pol ε, the lagging strand polymerase Pol δ, and the polymerase-primase complex Pol α. Furthermore, we have revealed entirely unexpected loading mechanisms of the PCNA and 9-1-1 DNA clamps by the clamp loaders RFC and Rad24-RFC, respectively. We have shown how the yeast Yta7 ATPase hexamer disrupts the nucleosome ahead of the replication fork and how the T4 phage helicase hexamer assembles with the primase to form a functional primosome to synthesize a 5-nucleotide RNA primer. Building on these successes, the PI proposes to continue the collaborative and mechanistic study on the replication origin activation mechanism and the replisome architecture. The proposed research is significant because DNA replication is central to cellular growth, and dysregulation of replication leads to uncontrolled proliferation and tumorigenesis.