DNA replication is an essential biochemical process that underlies genetic inheritance. In eukaryotic cells, this process is carried out by a multi-subunit protein complex called replisome. The core of replisome is made up of six-subunit protein ring Mcm2–7, which contains weak DNA helicase activity. In order to fully activate the helicase activity, protein co-activators of Cdc45 and GINS have to be recruited to assemble a replicative DNA helicase called the CMG (Cdc45-Mcm2–7-GINS) complex. The active CMG subsequently unwinds the parental DNA duplex to provide single-stranded DNA templates for replicative DNA polymerases. The precise mechanism for CMG assembly and activation is still poorly understood. Recent studies using model organisms suggested the importance of protein called Sld3/Treslin for CMG assembly.

The aim of the project was to understand in detail how human Treslin participates in the assembly of the CMG helicase. The interactions of Treslin with Cdc45, Mcm2–7, and DNA were characterised by a series of biochemical, biophysical, and structural biology experiments.

Cdc45–Treslin interaction was shown to be conserved from budding yeast to human, in which it is mediated by a domain called Sld3/Treslin Homology Domain (SHD). However, human Treslin contains an additional Cdc45-binding site—next to the C-terminus of SHD— in an intrinsically disordered region referred to as Treslin ’extension’ (Treslinext). Treslinext was also demonstrated to interact with Mcm2–7 ring and DNA. S-phase Cyclin-Dependent Kinase (S-CDK) appeared to regulate Treslin’s interaction with Cdc45 and Mcm2–7, in which it targets a number of potential S-CDK phosphorylation sites present in Treslinext. As a part of this project, low-resolution Cdc45–Treslin and medium-resolution of MCM single hexamer 3D maps were obtained through single-particle cryo-electron microscopy (cryo-EM) analyses.

The results provide molecular basis for Treslin’s role in the assembly of the CMG complex. Treslin acts as a molecular chaperone for Cdc45 recruitment to the DNA-bound Mcm2-7 ring. Treslin’s role during CMG assembly may involve its binding ability to the Mcm2–7 and DNA. Furthermore, the regulatory role of S-CDK in Treslin’s interactions with Cdc45 and Mcm2–7 suggests a link of the CMG assembly control to the cell cycle in human.