YEAST 2017

28th International Conference on Yeast Genetics and Molecular Biology (ICYGMB)

August 27 – September 1, 2017
Prague, Czech Republic


Paper ID: 353

Reconstituting chromosome replication

Diffley John

The Francis Crick Institute (United Kingdom)

ABSTRACT

The process by which active CMG (Cdc45-MCM-GINS) DNA helicase is assembled at eukaryotic replication origins ensures precisely one round of genome duplication in each cell cycle. This process comprises two temporally discrete stages: the minichromosome maintenance (MCM) complex is first loaded as a head-to-head double hexamer (DH) encircling duplex origin DNA during G1 phase; ‘firing factors’ then convert each DH into two active CMG helicases during S phase. Bidirectional replication requires the loading of two ring-shaped Minichromosome Maintenance (MCM) helicases around DNA in opposite orientations. MCM loading is orchestrated by binding of the Origin Recognition Complex (ORC) to DNA, but how ORC coordinates symmetrical MCM loading is unclear. Using natural budding yeast origins and synthetic sequences, we show that efficient MCM loading requires binding of two ORC molecules to two ORC binding sites. The relative orientation of these sites, but not the distance between them, is critical for MCM loading in vitro and origin function in vivo. We propose quasi-symmetrical loading of individual MCM hexamers by ORC and directed MCM translocation into double hexamers as a unifying mechanism for the establishment of bidirectional replication in Archaea and Eukarya.

In the second stage, the two strands of origin DNA must be unwound and DH remodelled so that individual MCM hexamers each encircle a single DNA strand to seed formation of bidirectional replisomes. We set out to understand the mechanism of this complex second stage using purified budding yeast proteins. We found that that MCM, which hydrolyses ATP during DH formation, remains stably bound to ADP in the DH. Firing factor recruitment triggers MCM to release ADP. Subsequent ATP binding then promotes stable CMG assembly, which is accompanied by untwisting of one helical turn of DNA per CMG and separation of DH into two discrete but inactive CMG helicases. Mcm10, together with ATP hydrolysis, then triggers further DNA untwisting and activation of the helicase. Our experiments elucidate the mechanism of eukaryotic replicative helicase activation, and show that ATP binding plays an analogous role in origin melting in eukaryotes, viruses and bacteria. 

Keywords:
DNA replication, biochemistry, cell cycle
Presented as:
  Oral presentation [S8-4] in S8 DNA replication, mutation and repair

Institute of Microbiology

YEAST 2017
28th International Conference on Yeast Genetics and Molecular Biology (ICYGMB)

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