Structural and functional analysis of helicase activation mechanisms in budding yeast

Background on pre-initiation complex (pre-IC) assembly

Pre-RC formation requires the absence of cyclin dependent kinase (CDK) activity in G1. Once the pre-RC is formed, S-CDK and DDK activity trigger origin firing. At the same time CDK activity destroys origin competence and in consequence new pre-RCs cannot form anymore on DNA. This kinase-dependent switch guarantees that each origin fires only once – a mechanism that is required for genomic stability. The MCM2-7 double-hexamer is the final product of pre-RC formation and represents the assembly platform for the replication fork. DDK kinase phosphorylates the MCM2-7 DH and promotes binding of Sld3 and Cdc45, resulting in an MCM2-7-Sld3-Cdc45 (MSC) complex. Then CDK kinase dependent phosphorylation of Sld3 and Sld2 promotes the binding of Sld2, Dpb11, GINS and polymerase epsilon (PolE), which facilitates formation of an inactive CMG. MCM2-7, as part of the CMG, is a single-hexamer, suggesting that Sld2, Dpb11, GINS and PolE facilitate double-hexamer splitting. However, the exact functions of Sld3, Sld2, Dpb11 during this process are currently not known. Upon addition of MCM10, the CMG complex becomes activated and DNA unwinding can occur. Then polymerase alpha starts with the synthesis of primers, which consequently become elongated by polymerase delta and epsilon for regular DNA synthesis.

The double-hexamer to CMG transition
Figure 1. The double-hexamer to CMG transition

Riera, A., Barbon, M., Noguchi, Y., Reuter, L. M., Schneider, S., Speck, C. (2017). From structure to mechanism — understanding initiation of DNA replication. Genes & Development 31, 1073-1078.
Abstract | Full text |

Herrera, M. C.*, Tognetti, S.*, Riera, A., Zech, J., Clarke, P., Fernández-Cid, A., Speck, C. (2015). A reconstituted system reveals how activating and inhibitory interactions control DDK dependent assembly of the eukaryotic replicative helicase. Nucleic Acids Research 43, 10238-10250.
*Shared first authorship
Abstract | Full text |

Tognetti, S., Riera, A., Speck, C. (2015) Switch on the engine: how the eukaryotic replicative MCM2–7 helicase becomes activated. Chromosoma 124, 13-26.
Abstract |

Organisation and regulation of the MCM2-7 double-hexamer

The MCM2-7 double-hexamer serves as the assembly platform for the entire replication fork. In 2014 we investigated, as part of an international collaboration, the structure of the MCM2-7 double-hexamer using electron microscopy, which led to a series of discoveries. First, using maltose-binding-protein fusions we discovered the overall subunit organisation of the complex. This demonstrated that the two Mcm2/5 DNA gates within the MCM2-7 double-hexamer are not aligned along the axis, but offset, suggesting a novel mechanism for helicase activation. Second, using biochemical assays, our work demonstrated the double-hexamer cannot hydrolyse ATP, explaining why it is not active for DNA unwinding. Third, we showed that the MCM2-7 double-hexamer, in contrast to the single-hexamer, represents an efficient target for DDK kinase, elucidating how double-hexamer specific complex formation can be initiated.

The organisation of the MCM2-7 double-hexamer
Figure 2. The organisation of the MCM2-7 double-hexamer

Sun, J.*, Fernandez-Cid, A.*, Riera, A.*, Tognetti, S., Yuan, Z., Stillman, B.†, Speck, C.†, Li, H.† (2014). Structural and mechanistic insights into Mcm2–7 double-hexamer assembly and function. Genes & Development 28, 2291-2303.
*Shared first authorship, †Corresponding authors
Abstract | Biomedical Picture of the Day |

The structural basis of the DNA bound MCM2-7 double-hexamer

Here we determined the structure of the MCM2-7 double-hexamer bound to DNA using cryo-electron microscopy at 3.9Å resolution. The work of this international collaboration involving the Li, Stillman and Speck labs, revealed how the MCM2-7 complex specifically interacts with DNA. We observed that the DNA is zigzagged inside the central channel. Several of the Mcm subunit DNA-binding loops interact in a staggered fashion with the DNA, resulting in an approximate step size of one base per subunit. The DNA strands are positioned right in front of the two Mcm2–Mcm5 gates, with each strand being pressed against one gate. The architecture indicates that lagging-strand extrusion initiates in the middle of the DH. We suggest that rotation of the N-terminal rings is required for initial DNA unwinding. Future work will define structural changes that occur during replication fork assembly the define the protein bindings sites on the MCM2-7 double-hexamer.

3D-printed model of the DNA bound MCM2-7 double-hexamer at atomic resolution
Figure 3. 3D-printed model of the DNA bound MCM2-7 double-hexamer at atomic resolution

Noguchi, Y., Yuan, Z., Bai, L., Schneider, S., Zhao, G., Stillman, B., Speck, C., Li, H. (2017). Cryo-EM structure of Mcm2-7 double hexamer on DNA suggests a lagging-strand DNA extrusion model. Proceedings of the National Academy of Sciences of the United States of America 114, E9529-E9538.
Abstract | Full text |

Structural basis of Cdc7-Dbf4 kinase-dependent targeting and phosphorylation of the MCM2-7 double hexamer

The Cdc7 kinase and Dbf4 form complex, the Dbf4-dependent kinase (DDK). The kinase is critical in DNA replication, as DDK activity is essential in activating the MCM2-7 double-hexamer during the G1-S transition. In 2022 we have determined the structure of the budding yeast MCM2-7 double-hexamer in complex with the Cdc7-Dbf4 kinase. We identified that the Dbf4 BRCT domain makes initial contact with Mcm2, which enables recruitment of the kinase complex. We resolved two additional structural variants of the kinase in complex with the helicase. In the first version, Dbf4 makes multiple contacts with Mcm4, enabling Mcm4 N-terminal tail phosphorylation by Cdc7. We used molecular dynamics simulation to explore how the flexible Mcm4 tail is recognised and engaged by the kinase. In the second version, two Mcm2-anchored DDK complexes interact with each other and hover across Mcm6. Thus, this structural variant revealed how Mcm6 can become phosphorylated. In summary, our structures reveal how specific protein-protein interactions enable Cdc7-dependent multisite phosphorylation of the helicase.

Structure of the MCM2-7-Db4-Cdc7 complex
Figure 4. Structure of the MCM2-7-Db4-Cdc7 complex

Saleh, A., Noguchi, Y., Aramayo, R., Ivanova, M. E., Stevens, K. M., Montoya, A., Sunidhi, S., Carranza, N. L., Skwark, M. J., Speck, C. (2022).  The structural basis of Cdc7-Dbf4 kinase dependent targeting and phosphorylation of the MCM2-7 double hexamer.  Nature Communication 13:2915.
Abstract | Full text |