Molecular mechanism of origin firing in budding yeast

Abstract

DNA replication plays a critical role in maintaining genome integrity and ensuring normal cell proliferation. In eukaryotes, DNA replication is initiated from multiple sites called origins, distributed along each chromosome. During the G1 phase, the origin recognition complex (ORC) encircles duplex DNA at the origins, serving as a platform to recruit two copies of Cdt1-Mcm2-7 heptamer into a head-to-head MCM double hexamer (DH) with the help of Cdc6. This process is also known as replication licensing or pre-replicative complex (pre-RC) assembly. However, DH remains inactive due to the low kinase activity in the G1 phase. When cells enter the S phase, the kinase activities rise to high levels, which drive origin firing and replication initiation. During this process, Dbf4-dependent kinase (DDK) and S-cyclin-dependent kinase (S-CDK) play essential roles in activating the DH into two active Cdc45-MCM-GINSs (CMGs), upon which two replisomes are established for bi-directional DNA synthesis. While DNA replication initiation has been extensively investigated in the past decades, the molecular mechanism regulating origin firing still remains poorly understood. One reason for this situation can be attributed to the scant high-resolution structures of the replication complexes involved in this process. ii To better understand how DDK recognizes and activates its substrate, DH, we assembled the DH-DDK complex and determined its cryo-electron microscopy (EM) structures. In our structures, DDK docking onto DH is mediated exclusively by Dbf4, which straddles the hexamer interface to engage with the N-terminal domain (NTD) of Mcm2 from one hexamer and the NTDs of Mcm6 and Mcm4 from the opposite hexamer. This unique arrangement places the kinase core of DDK in a strategic position to target its only essential substrate, the N-terminal serine/threonine-rich domain (NSD) of Mcm4, with a higher probability. The kinase core also appears highly dynamic on the DH-DDK, with the potential to target the substrates from Mcm2 and Mcm6. Our mutational analysis further supported this hierarchy in substrate processing by DDK. Our findings provide crucial insights into the roles of DDK on the DH to drive helicase activation. To elucidate the interplay between CMG helicase and the leading strand DNA polymerase epsilon (Polε) during helicase activation and DNA synthesis, we also determined the structure of an in vitro assembled replisome, CMG-Polε-Ctf4-Tof1- Csm3 complex. With this structure, we found that Polε cycles on and off the MCM motor domain of the CMG complex. Disrupting the coupling between Polε and MCM ring significantly inhibits CMG formation and replication initiation. This unexpected result unravels an essential role of Polε in regulating helicase activation. In summary, our studies shed novel light on our understanding of the regulation of DNA replication initiation. The information from our structures also lays a solid foundation for further biochemical and functional characterization of origin firing in yeast and higher eukaryotes.