Dataset Information

Post-licensing Specification of Eukaryotic Replication Origins by Facilitated Mcm2-7 Sliding along DNA.

ABSTRACT: Eukaryotic genomes are replicated from many origin sites that are licensed by the loading of the replicative DNA helicase, Mcm2-7. How eukaryotic origin positions are specified remains elusive. Here we show that, contrary to the bacterial paradigm, eukaryotic replication origins are not irrevocably defined by selection of the helicase loading site, but can shift in position after helicase loading. Using purified proteins we show that DNA translocases, including RNA polymerase, can push budding yeast Mcm2-7 double hexamers along DNA. Displaced Mcm2-7 double hexamers support DNA replication initiation distal to the loading site in vitro. Similarly, in yeast cells that are defective for transcription termination, collisions with RNA polymerase induce a redistribution of Mcm2-7 complexes along the chromosomes, resulting in a corresponding shift in DNA replication initiation sites. These results reveal a eukaryotic origin specification mechanism that departs from the classical replicon model, helping eukaryotic cells to negotiate transcription-replication conflict.

SUBMITTER: Gros J

PROVIDER: S-EPMC4680849 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Post-licensing Specification of Eukaryotic Replication Origins by Facilitated Mcm2-7 Sliding along DNA.

Gros Julien J Kumar Charanya C Lynch Gerard G Yadav Tejas T Whitehouse Iestyn I Remus Dirk D

Molecular cell 20151119 5

Eukaryotic genomes are replicated from many origin sites that are licensed by the loading of the replicative DNA helicase, Mcm2-7. How eukaryotic origin positions are specified remains elusive. Here we show that, contrary to the bacterial paradigm, eukaryotic replication origins are not irrevocably defined by selection of the helicase loading site, but can shift in position after helicase loading. Using purified proteins we show that DNA translocases, including RNA polymerase, can push budding y ...[more]

PMID: 26656162

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Project description:Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 are well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. Phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO-complex formation and provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO-complex formation.Significance statementEach time a eukaryotic cell divides (by mitosis) it must duplicate its chromosomal DNA exactly once to ensure that one full copy is passed to each resulting cell. Both under-replication or over-replication result in genome instability and disease or cell death. A key mechanism to prevent over-replication is the temporal separation of loading of the replicative DNA helicase at origins of replication and activation of these same helicases during the cell division cycle. Here we define the mechanism by which phosphorylation of the primary DNA binding protein involved in these events inhibits helicase loading. Our studies identify multiple steps of inhibition and provide new insights into the mechanism of helicase loading in the uninhibited condition.

Dataset Information

Post-licensing Specification of Eukaryotic Replication Origins by Facilitated Mcm2-7 Sliding along DNA.

Publications

Post-licensing Specification of Eukaryotic Replication Origins by Facilitated Mcm2-7 Sliding along DNA.

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