Bipartite GINS binding mode of TopBP1 to activate the metazoan Mcm2-7 replicative helicase
Genome replication must be complete, accurate and occur exactly once during each cell cycle to generate two faithful copies of the DNA, one for each emerging daughter cell. This is at the heart of genetic homeostasis over the successive cell generations.
Replication initiation is the first step of replication and regulates exactly when and where replication takes place. In yeast, the molecular processes and the main proteins involved in initiation have been described. The loading of the Mcm2-7 helicase takes place in G1-phase and its activation occurs in the S-phase. In the first step, origin licensing, the Mcm2-7 helicase is loaded onto the origin of DNA and the pre-replicative complex (pre-RC) is formed. Mcm2-7 is helicase-inactive at that stage. The second step is origin firing, the pre-RCs are converted to the helicase-active CMG (Cdc45-Mcm2-7-GINS) and replisomes are formed. Activation of the Mcm2-7 helicase in S-phase begins with the stepwise assembly of a transient preinitiation complex (pre-IC), and GINS and Cdc45 are recruited to the Mcm2-7 during the pre-IC formation. In yeast, Sld3-Sld7, the orthologue of Treslin-MTBP in vertebrate, mediates the recruitment of Cdc45 to the Mcm2-7. GINS, Dpb11, Sld2 and Polε form pre-loading complex (pre-LC) and this complex recruits GINS to the Mcm2-7. When helicase-active CMG complex is formed, Sld3-Sld7, Dpb11 and Sld2 leave the complex and mature replisomes are formed. Although the origin firing factors are conserved between yeast and vertebrates, the molecular details of the recruitment of Cdc45 and GINS to the Mcm2-7 are poorly investigated in vertebrates due to the lack of in vitro-reconstitution efforts. In this project, we use recombinant proteins to reveal molecular details of the TopBP1-GINS interaction, providing insight how TopBP1 contributes to GINS recruitment to the inactive Mcm2-7 helicase to form helicase-active CMG.
In our research, we conducted biochemical and structural studies, which revealed that the helicase activator GINS interacts with TopBP1 through two distinct binding surfaces. The first binding surface involves the previously poorly described GINI domain. We here showed that the GINI domain is located between BRCT3 and BRCT4 domains of TopBP1 and it consists of highly conserved amino acids among metazoans. Our cryo-EM model demonstrated that GINI domain forms an short -helix structure and this -helix structure is crucial for the interaction with GINS. The second binding surface is located on TopBP1-BRCT4 which is a novel binding site for GINS. Both of these binding surfaces interact with the opposite ends of the A domain of the GINS subunit Psf1, and their cooperation is necessary for a stable biochemical interaction between TopBP1 and GINS. Moreover, we carried out rescue experiments for DNA replication in TopBP1 depleted Xenopus egg extract by adding-back of either recombinant TopBP1-BRCT0-5-WT or recombinant TopBP1 with different sets of mutations in either interface. These rescue experiments showed that this cooperation between GINI and BRCT4 domains is also required during replication origin firing. Furthermore, we here propose that the interaction between TopBP1 and GINS is not compatible simultaneously with the binding of Polε to GINS when GINS is already bound to Mcm2-7-Cdc45. Based on our TopBP1-GINS model, we propose that three molecular processes are coordinated: The arrival of DNA polymerase epsilon, the ejection of TopBP1, and the integration of GINS into Mcm2-7-Cdc45 during replication origin firing.