Cryo-Electron Microscopy Analysis of the Replicative Helicase
Biochemistry
Cell Biology
Final Report Abstract
The assembly of replication forks is critical for genome stability. DNA replication initiation consists of two parts. First, the core of the replicative helicase is loaded as a double hexamer (DH) on double-stranded DNA but remains initially inactive. Second, two kinases and several activation factors, among them Sld3, Sld7 and Cdc45 get recruited to origins, transfer the helicase onto single-stranded DNA and activate the complex. Crucially, the formation of the MCM2-7, Sld3, Sld7, Cdc45 (CMS) complex has not been structurally analysed and the mechanistic basis of how these proteins facilitate the fundamental process of helicase activation. Here we describe the structural basis of DDK dependent Sld3/7 and Cdc45 recruitment during helicase activation and novel mechanistic insights. Surprisingly, we observed Sld3 densities near the Mcm6 C-terminal domain, distal from the known Sld3 interaction site near the Mcm4 N-terminus. In addition to positioning Sld3 ideally for recruitment of Cdc45, this finding suggests a novel way of initial Sld3 recruitment. Cdc45 interacts with the N-terminal parts of Mcm2 and Mcm5, which resembles very closely its interaction with the active helicase. Contrary to previous assumptions, binding of the two early activation factors resulted in no major conformational change in the core helicase or DNA.
Publications
- Structural mechanism of helicase loading onto replication origin DNA by ORC-Cdc6. (July 2020). Proc Natl Acad Sci USA
Yuan Z, Schneider S, Dodd T, Riera A, Bai L, Yan C, Magdalou I, Ivanov I, Stillman B, Li H, Speck C
(See online at https://doi.org/10.1073/pnas.2006231117)
