title: In the absence of ATPase activity, pre-RC formation is blocked prior to MCM2-7 hexamer dimerization.
abstract: The origin recognition complex (ORC) of Saccharomyces cerevisiae binds origin DNA and cooperates with Cdc6 and Cdt1 to load the replicative helicase MCM2-7 onto DNA. Helicase loading involves two MCM2-7 hexamers that assemble into a double hexamer around double-stranded DNA. This reaction requires ORC and Cdc6 ATPase activity, but it is unknown how these proteins control MCM2-7 double hexamer formation. We demonstrate that mutations in Cdc6 sensor-2 and Walker A motifs, which are predicted to affect ATP binding, influence the ORC-Cdc6 interaction and MCM2-7 recruitment. In contrast, a Cdc6 sensor-1 mutant affects MCM2-7 loading and Cdt1 release, similar as a Cdc6 Walker B ATPase mutant. Moreover, we show that Orc1 ATP hydrolysis is not involved in helicase loading or in releasing ORC from loaded MCM2-7. To determine whether Cdc6 regulates MCM2-7 double hexamer formation, we analysed complex assembly. We discovered that inhibition of Cdc6 ATPase restricts MCM2-7 association with origin DNA to a single hexamer, while active Cdc6 ATPase promotes recruitment of two MCM2-7 hexamer to origin DNA. Our findings illustrate how conserved Cdc6 AAA+ motifs modulate MCM2-7 recruitment, show that ATPase activity is required for MCM2-7 hexamer dimerization and demonstrate that MCM2-7 hexamers are recruited to origins in a consecutive process.
authors: DNA Replication Group, MRC Clinical Sciences Centre, Imperial College, London W12 0NN, UK.
authors: Cecile
authors: C
authors: Evrin
authors: Alejandra
authors: A
authors: Fernández-Cid
authors: Juergen
authors: J
authors: Zech
authors: M Carmen
authors: MC
authors: Herrera
authors: Alberto
authors: A
authors: Riera
authors: Pippa
authors: P
authors: Clarke
authors: Shlomo
authors: S
authors: Brill
authors: Rudi
authors: R
authors: Lurz
authors: Christian
authors: C
authors: Speck
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D019943:Amino Acid Substitution; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics; D002460:Cell Line; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry; D004261:DNA Replication; D004271:DNA, Fungal / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D006868:Hydrolysis; D064168:Minichromosome Maintenance Complex Component 7; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry; D051738:Origin Recognition Complex / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D055503:Protein Multimerization; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics
chemical_list: C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C497214:ORC1 protein, S cerevisiae; C497219:ORC4 protein, S cerevisiae; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; C499592:TAH11 protein, S cerevisiae; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; C069199:MCM2 protein, S cerevisiae; C491088:MCM7 protein, S cerevisiae; D064168:Minichromosome Maintenance Complex Component 7
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