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.
Highlights
The influence of HU on dominant lethality of Ins MCM2-7 mutants Supplementary Figure S2 (related to main Figure 3)
Several Ins MCM2-7 mutants release Cdt1 efficiently in the presence of ATP Supplementary Figure S4. wt MCM2-7 and Ins MCM2-7 mutants are getting phosphorylated by DDK with equal efficiency Supplementary Figure S5
Primers used to insert GGSGSG sequence in MCM2-7 subunits Supplementary Table S2: AS499 based yeast strains used in this study Supplementary Table S3: Plasmids used in this study SUPPLEMENTARY REFERENCES
Summary
The influence of HU on dominant lethality of Ins MCM2-7 mutants Supplementary Figure S2 (related to main Figure 3). Several Ins MCM2-7 mutants fractionate by gel-filtration as a hexamer Supplementary Figure S3 (related to main Figure 3). Several Ins MCM2-7 mutants release Cdt1 efficiently in the presence of ATP Supplementary Figure S4 (related to main Figure 5).
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