The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during 
prereplicative complex formation

Cecile , Evrin; Fernandez-Cid, Alejandra; Riera, Alberto; Zech, Jürgen; Clarke, Pippa; Herrera, M. Carmen; Tognetti, Silvia; Lurz, Rudi; Speck, Christian

doi:10.1093/nar/gkt1148

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The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation

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Lurz, Rudi
Imaging/Electron Microscopy (Head: Thorsten Mielke), Scientific Service (Head: Christoph Krukenkamp), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Citation

Cecile, E., Fernandez-Cid, A., Riera, A., Zech, J., Clarke, P., Herrera, M. C., et al. (2014). The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation. Nucleic Acids Research (London), 42(4), 2257-2269. doi:10.1093/nar/gkt1148.

Cite as: https://hdl.handle.net/21.11116/0000-0000-70E3-1

Abstract

The replicative mini-chromosome-maintenance 2-7 (MCM2-7) helicase is loaded in Saccharomyces cerevisiae and other eukaryotes as a head-to-head double-hexamer around origin DNA. At first, ORC/Cdc6 recruits with the help of Cdt1 a single MCM2-7 hexamer to form an 'initial' ORC/Cdc6/Cdt1/MCM2-7 complex. Then, on ATP hydrolysis and Cdt1 release, the 'initial' complex is transformed into an ORC/Cdc6/MCM2-7 (OCM) complex. However, it remains unclear how the OCM is subsequently converted into a MCM2-7 double-hexamer. Through analysis of MCM2-7 hexamer-interface mutants we discovered a complex competent for MCM2-7 dimerization. We demonstrate that these MCM2-7 mutants arrest during prereplicative complex (pre-RC) assembly after OCM formation, but before MCM2-7 double-hexamer assembly. Remarkably, only the OCM complex, but not the 'initial' ORC/Cdc6/Cdt1/MCM2-7 complex, is competent for MCM2-7 dimerization. The MCM2-7 dimer, in contrast to the MCM2-7 double-hexamer, interacts with ORC/Cdc6 and is salt-sensitive, classifying the arrested complex as a helicase-loading intermediate. Accordingly, we found that overexpression of the mutants cause cell-cycle arrest and dominant lethality. Our work identifies the OCM complex as competent for MCM2-7 dimerization, reveals MCM2-7 dimerization as a limiting step during pre-RC formation and defines critical mechanisms that explain how origins are licensed.