Funktionelle in vitro- und in vivo-Analyse der ringförmigen replikativen Helikase MCM2-7 zur Identifizierung von Proteinuntereinheiten, die eine Ringöffnung ermöglichen und damit das Laden und Ablösen der Helikase von DNS kontrollieren.
Zusammenfassung der Projektergebnisse
DNA replication is a central part of the cell cycle. As a cell prepares to undergo cell division it needs to duplicate its genome. In order to ensure a faithful duplication this process is tightly controlled and requires many specialised proteins. One of these is a heterohexameric protein complex, the replicative helicase MCM2-7. MCM2-7 unwinds the double helix at the replication fork, i. e. it separates the DNA strands in a zipper-like fashion before they get duplicated. To play this role the ring-shaped helicase complex comprising the six subunits Mcm2 through Mcm7 needs to be opened and loaded onto DNA. This project aimed at the identification of the gate at which the complex opens to allow MCM2-7 to be loaded onto DNA. Towards this end we added the dimerisable protein domains FKBP and FRB to each of the six subunits thereby creating a rapamycin-dependent, conditional linkage between each two subunits. Such modified complexes were studied in an assay that tested their loadability. In the absence of rapamycin all complexes were able to be loaded onto DNA. In contrast in the presence of rapamycin all but one complex were loaded onto DNA. The one complex unable to be loaded contained a linkage between Mcm2 and Mcm5. Therefore, since blocking the interface between Mcm2 and Mcm5 prevents loading, it is the ‘DNA entry gate’ of the complex. This system of the rapamycin-dependent blocking of the Mcm2/Mcm5 interface was then also tested in vivo employing a rapamycin-tolerant S. cerevisiae strain. The wild-type (wt) genes of Mcm2 and Mcm5 were replaced by the genes of the fusion proteins, frb-mcm2 and fkbp-mcm5. In growth experiments addition of rapamycin and thus the closing of the interface abolished growth of those cells. Intriguingly, expression of a FKBP-like protein, Fpr1, competed with the Mcm-attached FKBP domain for the common binding site. Consequently, in these cells Fpr1 bypassed the blocking effect of Rapamycin and viability was restored. Cell cycle analysis identified that the cells containing the mutations behave in the absence of rapamycin like wild-type cells. The very same culture containing rapamycin grew comparably until they reached S phase. At this point the culture proceeded only very slowly through S phase. Employing a technique that identifies chromatin-bound proteins we were able to show that these cells were void of chromatin-associated MCM2-7. By biochemical analysis we then investigated at which point of the multi-step helicase loading process the helicase complex is opened. Using the FKBP/FRB mutants it was found that the loading of the helicase MCM2-7 onto double-stranded DNA occurs earlier than previously thought. The insertion of DNA takes place before ATP hydrolysis and subsequent release of Cdt1 at the stage of the initial complex formation of helicase loader with the MCM2-7 helicase.
Projektbezogene Publikationen (Auswahl)
- An ORC/Cdc6/MCM2-7 complex is formed in a multistep reaction to serve as a platform for MCM double-hexamer assembly. Molecular Cell, Vol. 50.2 013, Issue 4, pp. 457-458.
Fernández-Cid A, Riera A, Tognetti S, Herrera MC, Samel S, Evrin C, Winkler C, Gardenal E, Uhle S, Speck C
(Siehe online unter https://doi.org/10.1016/j.molcel.2013.03.026) - Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1- MCM2-7 bound to DNA. Nature Structural & Molecular Biology, Vol. 20. 2013, pp. 944–951.
Sun J, Evrin C, Samel SA, Fernández-Cid A, Riera A, Kawakami H, Stillman B, Speck C, Li H
(Siehe online unter https://doi.org/10.1038/nsmb.2629) - A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2–7 onto DNA.
Genes & Development, Vol. 28.2014, pp. 1653-1666.
Stefan A. Samel, Alejandra Fernandez-Cid, Jingchuan Sun, Alberto Riera, Silvia Tognetti, M. Carmen Herrera, Huilin Li and Christian Speck1
(Siehe online unter https://doi.org/10.1101/gad.242404.114)
