Our group studies mechanisms in genome stability and epigenetics using biochemical, genetic, single molecule and structural approaches. We are particular interested in disease associated with these processes and the development of specific inhibitors for therapeutic purposes.
Our current DNA Replication research focusses on characterising the loading and activation of the replicative helicase. We employ biochemical, genetic, structural, synthetic- and chemical biology approaches to uncover novel mechanism and regulatory principles.
Background on helicase loading
The precise duplication of chromosomal DNA is essential for preserving the genetic complement of the cell. To ensure that chromosomes only replicate once per cell cycle the process of chromosome duplication is divided into discrete steps. The first step is licensing of DNA replication, also termed pre-replicative (pre-RC) complex formation. Pre-RC formation occurs during late mitosis and during G1 phase and results in the loading of the MCM2-7 helicase onto origin DNA. Pre-RC formation involves binding of Cdc6 to the DNA-bound Origin Recognition Complex (ORC) (Speck et al.; NSMB 2005, Speck and Stillman; JBC 2007). The ORC-Cdc6-DNA complex cooperates with Cdt1 to load the MCM2-7 helicase on double-stranded DNA. In 2009 my group published the complete reconstitution of Saccharomyces cerevisiae pre-RC formation with purified proteins (Figure 1). We showed that MCM2-7 is a hexamer before loading and that the complex is transformed into a double-hexamer during pre-RC formation.
Reconstitution of pre-RC formation
A) The assay uses biotinylated DNA bound to streptavidin coated magnetic beads. B) The indicated proteins were incubated with origin DNA. The high salt wash removed ORC, Cdc6 and Cdt1 but kept loaded MCM2-7 associated with DNA. C) Electron micrograph of metal-shadowed DNA with two loaded MCM2-7 double-hexamer complexes.
Evrin, C., Clarke, P., Zech, J., Lurz, R., Sun, J., Uhle, S., Li, H., Stillman, B., Speck, C. (2009). A double-hexameric mcm2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication. Proceedings of the National Academy of Sciences. 106(48): 20240-20245 | abstract
Although the reconstitution was a breakthrough it also raised a number of questions: How is the MCM2-7 ring opened and how does ORC-Cdc6 promote double-hexamer formation? Clearly, the mechanism of helicase loading needs to be addressed, but a deep understanding of the regulation of this process appears equally important. Reduced MCM2-7 loading is observed in genetic diseases or during tumorigenesis and renders cells sensitive to DNA damage and promotes double-strand breaks. On the other hand, the DNA licensing activity must be inactivated prior to S-phase. Failure to inactivate only a single helicase loading complex results in re-replication and severe genomic rearrangements.
Principles of pre-RC formation
- The replicative helicase MCM2-7 is loaded by ORC, Cdc6 and Cdt1 at replication origins into pre-replicative complexes (pre-RC)
- During helicase loading two MCM2-7 hexamers assemble into a double-hexamer bound around double-stranded DNA
- MCM2-7 double-hexamer formation is a multi-step reaction that requires ATP hydrolysis
- CDK affects the stability of the ORC/Cdc6/MCM2-7 pre-RC intermediate for regulated helicase loading and genome stability
- Inhibition of helicase loading affects cancer cell viability and could be of therapeutic value
Loading mechanism of the eukaryotic replicative helicase
Based on the coordinated and distinct structural changes in MCM2-7, which are promoted by ORC, its cofactors Cdc6 and Cdt1, we propose a model that explains how the MCM2-7 double-hexamer assembles onto DNA. ORC binds in an ATP-dependent way to the replication origin (A). In G1 phase, Cdc6 is recruited to ORC and the ORC/Cdc6 complex contributes to origin specificity (B). Cdt1 forms a complex with MCM2-7 and this interaction is required for the binding of Cdt1/MCM2-7 to ORC/Cdc6. Initially only one Cdt1/MCM2-7 heptamer is recruited to ORC/Cdc6. In the absence of Cdc6 ATP-hydrolysis, Cdt1 is greatly stabilized in this initial ORC/Cdc6/Cdt1/MCM2-7 (OCCM) complex (C). ATP-hydrolysis by Orc1 and Cdc6 results in rapid Cdt1 release and formation of an ORC/Cdc6/MCM2-7 (OCM) complex (D). The OCM complex, but not the initial complex, is competent for MCM2-7 dimerization (E). Currently, it is not clear if one or two ORC/Cdc6 complexes participate in MCM2-7 dimerization or when DNA loading occurs. After initial dimerization several important changes occur: the MCM2-7 N-termini are exposed, rendering the complex competent for DDK mediated activation in S-phase; the stable MCM2-7 double-hexamer forms around ds-DNA; ORC and Cdc6 are released, and finally Orc1 ATP-hydrolysis occurs to allow another round of MCM2-7 loading (F).
Riera, A., Tognetti, S., Speck, C., 2014. Helicase loading: How to build a MCM2-7 double-hexamer. Seminars in cell & developmental biology. | abstract
Evrin, C., Fernández-Cid, A., Riera, A., Zech, J., Clarke, P., Herrera, M. C., Tognetti, S., Lurz, R., Speck, C., Feb. 2014. The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation. Nucleic Acids Research 42 (4), 2257-2269. | abstract
The role of Cdt1 in pre-RC formation
In budding yeast, the MCM2-7 helicase needs to be imported into the nucleus prior to pre-RC formation and this reaction is dependent on the Cdt1 protein (A). The question of how Cdt1 recruits MCM2-7 towards ORC/Cdc6 has recently been under intense scrutiny. Using a reconstituted system employing purified proteins we dissected the process in more detail. We and others found that for MCM2-7 recruitment to origin DNA, ORC, Cdc6 and Cdt1 are required (A). Then we described in 2012 that in the absence of ATP-hydrolysis an ORC/Cdc6/Cdt1/MCM2-7 (OCCM) complex is formed. Although all Mcm subunits are highly homologous, containing a C-terminal AAA+ and an N-terminal domain, individual Mcm subunits have specialised functions. In a series of experiments we showed that a C-terminal extension in Mcm6 contains an inhibitory domain, which blocks OCCM formation in the absence of Cdt1. Indeed, MCM2–7-ΔC6, which is missing a conserved Mcm6 C-terminus, can bind to ORC/Cdc6 in a Cdt1-independent manner, while point mutants that interfere with the specific Cdt1-Mcm6 interactions block OCCM formation.
As ATP-hydrolysis during pre-RC formation promotes Cdt1 release, we hypothesized that a productive Cdt1-Mcm6 interaction could be required to induce Orc1 and Cdc6 ATPase activity (B). In fact, we observed that that an ORC/Cdc6/Cdt1/MCM2-7-ΔC6 complex blocked ATP-hydrolysis, and that Cdt1 addition to ORC-Cdc6 was not sufficient to induce ATP-hydrolysis. Furthermore, we found that MCM2-7-ΔC6 also blocked Cdt1 release during pre-RC formation. Thus, the Mcm6-C terminus and Cdt1 have besides their role in nuclear import and auto-inhibition another important function in promoting ATP-hydrolysis and in facilitating Cdt1 release (B).
Fernández-Cid, A., Riera, A., Tognetti, S., Herrera, M. C., Samel, S., Evrin, C., Winkler, C., Gardenal, E., Uhle, S., Speck, C., (2013). An ORC/Cdc6/MCM2-7 complex is formed in a multistep reaction to serve as a platform for MCM Double-Hexamer assembly. Molecular Cell. 50(4):577-88 | abstract
Evrin, C., Fernández-Cid, A., Zech, J., Herrera, M. C., Riera, A., Clarke, P., Brill, S., Lurz, R., Speck, C., (2013). In the absence of ATPase activity, pre-RC formation is blocked prior to MCM2-7 hexamer dimerization. Nucleic Acids Research. 41(5):3162-72 | abstract
The first 3D structure of the eukaryotic replicative helicase in complex with its loader
The eukaryotic replicative helicase, MCM2-7, is a hetero-hexamer, the six distinct subunits of which assemble into a ring shaped complex. The central channel of the ring is wide enough to accommodate double-stranded DNA.
Figuring out how the helicase loader interacts with the large helicase is instrumental to understanding the mechanism of helicase loading and could also inform us about the MCM2-7 double-hexamer formation process. Structural information is key to analysing complex reactions. Unfortunately, crystallographic approaches for the analysis of dynamic and flexible complexes are usually very challenging. However, cryo-electron microscopy (cryo-EM) has the potential to visualize the stable envelope of a protein complex and - in combination with specific labelling approaches - this technique can even pin-point the location of individual subunits within the EM structure.
We have recently employed cryo-EM to study helicase loading in collaboration with Huilin Li (BNL) and Bruce Stillman (CSHL). By using ATPγS, an ATP analogue, which can be only very slowly hydrolysed, we successfully captured a helicase loading intermediate. This complex, which contains all 14 pre-RC polypeptides, reveals for the first time how the eukaryotic helicase interacts with its loader. The structure identifies how ORC/Cdc6 and Cdt1/MCM2-7 interact with each other. The C-terminal section of MCM2-7 latches on the AAA+ ATPase domains of ORC/Cdc6, which leaves the MCM2-7 N-termini available to interact with a second MCM2-7 hexamer, suggesting a route for MCM2-7 double-hexamer assembly. Especially ORC/Cdc6 undergo significant structural changes upon OCCM formation. While ORC/Cdc6 is nearly flat, ORC/Cdc6 within the OCCM adopt a dome shape with the AAA+ domains now pointing towards the MCM2-7 C-termini. Interestingly, the structure shows that Mcm3 and Cdc6, which we and others found to be essential for OCCM formation, are positioned next to each other, although they do not interact directly. Thus, the Mcm3 C-terminus becomes potentially rearranged during initial ORC/Cdc6-Cdt1/MCM2-7 interaction. Most contacts between ORC/Cdc6 and Cdt1/MCM2-7 are on one side of the complex. It remains unknown whether these interactions are required for helicase loading or alternatively function to remodel the complex for MCM2-7 double-hexamer formation.
Sun J, Evrin C, Samel S, Fernández-Cid A, Riera A, Kawakami H, Stillman B, Speck C*, Li H* (2013). Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA. Nature Structure & Molecular Biology. 20(8):944-51 | abstract
Bruce Stillman (President of CSHL), Cold Spring Harbour Laboratory, NY, USA, 2007
Dr. Rudi Lurz / Dr. Thorsten Mielke, Max-Planck Institute for Molecular Genetics, Berlin, Germany, 2006
Prof. Huilin Li, Brookhaven National Laboratory, Brookhaven, NY, USA, 2006