In Saccharomyces cerevisiae and higher eukaryotes, the loading of the replicative helicase MCM2-7 onto DNA requires the combined activities of ORC, Cdc6, and Cdt1. These proteins load MCM2-7 in an unknown way into a double hexamer around DNA. Here we show that MCM2-7 recruitment by ORC/Cdc6 is blocked by an autoinhibitory domain in the C terminus of Mcm6. Interestingly, Cdt1 can overcome this inhibitory activity, and consequently the Cdt1-MCM2-7 complex activates ORC/Cdc6 ATP-hydrolysis to promote helicase loading. While Cdc6 ATPase activity is known to facilitate Cdt1 release and MCM2-7 loading, we discovered that Orc1 ATP-hydrolysis is equally important in this process. Moreover, we found that Orc1/Cdc6 ATP-hydrolysis promotes the formation of the ORC/Cdc6/MCM2-7 (OCM) complex, which functions in MCM2-7 double-hexamer assembly. Importantly, CDK-dependent phosphorylation of ORC inhibits OCM establishment to ensure once per cell cycle replication. In summary, this work reveals multiple critical mechanisms that redefine our understanding of DNA licensing.

The regulated loading of the replicative helicase minichromosome maintenance proteins 2-7 (MCM2-7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2-7 hexamers into one double hexamer around dsDNA. Although the MCM2-7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. Here, we establish that the Saccharomyces cerevisiae MCM2-7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, we show that ORC-Cdc6-Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, we demonstrate that the MCM2-7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2-7 complex formation prior to MCM2-7 double hexamer formation. Our study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation.

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SummaryIn silico models for Pseudomonas putida KT2440 metabolism predict 68 genes to be essential for growth on minimal medium. In this study a genomewide collection of single-gene P. putida KT2440 knockouts was generated by mini-Tn5 transposon mutagenesis and used to identify genes essential for growth in minimal medium with glucose. Our screening of the knockout library allowed us to rescue mutants for 48 different knockouts that were conditionally essential for growth on minimal medium. The in vivo screening showed that 24 of these mutants had a insertion in genes proposed to be conditionally essential based on in silico models, whereas another 24 newly implicated conditionally essential genes have been found. For 10 of the in silico proposed conditionally essential genes not found in the screening, knockout mutants were available at the Pseudomonas Reference Culture Collection. These mutants were tested for conditional growth on minimal medium, but none of them was shown to be essential, suggesting that the in silico proposal was inaccurate. Among the set of identified conditionally essential genes were a number of genes involved in the biosynthesis of certain amino acids and vitamins. Auxotrophs for all amino acids predicted by the in silico models were found and, in addition, we also found auxotrophs for proline, serine, threonine and methionine, as well as auxotrophs for biotin, nicotinate and vitamin B12 that were not predicted in silico. Metabolic tests were performed to validate the mutants' phenotypes. Auxotrophies for L-Arg, L-Leu, L-Pro and L-Cys were bypassed by external addition of the corresponding D-amino acids, suggesting the existence of number of D-to L-amino acid racemases encoded by the KT2440 genome. Therefore, the in vivo high-throughput analysis presented here provides relevant insights into the metabolic cross-road of biosynthetic pathways in this microorganism, as well as valuable information for the fine tuning of current in silico metabolic models.