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

Cécile Evrin | Zendy; Alejandra FernandezCid | Zendy; Alberto Riera | Zendy; Juergen Zech | Zendy; Pippa Clarke | Zendy; M. Carmen Herrera | Zendy; Silvia Tognetti | Zendy; Rudi Lurz | Zendy; Christian Speck | Zendy

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

Author(s) -

Cécile Evrin,

Alejandra FernandezCid,

Alberto Riera,

Juergen Zech,

Pippa Clarke,

M. Carmen Herrera,

Silvia Tognetti,

Rudi Lurz,

Christian Speck

Publication year - 2013

Publication title -

nucleic acids research

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 9.008

H-Index - 537

eISSN - 1362-4954

pISSN - 0305-1048

DOI - 10.1093/nar/gkt1148

Subject(s) - random hexamer , biology , origin recognition complex , microbiology and biotechnology , pre replication complex , dna replication factor cdt1 , minichromosome maintenance , origin of replication , dna replication , genetics , control of chromosome duplication , eukaryotic dna replication , dna , biochemistry

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.

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