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| Status |
Public on Jun 21, 2024 |
| Title |
Mechanisms of MCM2-7 loading and initial DNA melting at near base-pair resolution [MCM4 & ORC2 ChIP-exo] |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
|
| Summary |
The loading and activation of the replicative helicase MCM2-7 are key events during the G1-S phase transition. In budding yeast, the origin recognition complex (ORC) binds to the conserved DNA elements A and B1 of the autonomously replicating sequence (ARS). This is followed by the consecutive loading of two MCM2-7 hetero-hexamers into a MCM2-7 double-hexamer (DH). In S-phase the MCM2-7 DH is activated, resulting in two Cdc45-MCM-GINS (CMG) helicases that bidirectionally unwind DNA ahead of the replication fork. Here, we show that MCM2-7 helicase loading across the B1 element displaces ORC from origins. This allows ORC binding and helicase loading at lower affinity binding sites and origins throughout the genome. Furthermore, we mapped the sites of initial DNA unwinding genome-wide and show that these sites appear near the N-terminal domains of the MCM2-7 double-hexamer in proximity of the B1 element. Finally, employing a chemical-biology approach, we establish that during helicase activation the Mcm2/5 interface acts as the DNA exit gate for single-stranded-DNA extrusion. Our work identifies that helicase loading follows a distributive mechanism, allowing for equal MCM2-7 loading across the genome and surprisingly finds that DNA unwinding initiates from the helicase N-terminal interface in proximity to the ARS B1 element.
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| |
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| Overall design |
ChIP-Exo 5.0 analysis of Mcm4 and Orc2 from cells arrested in G1 or G2
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| |
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| Contributor(s) |
Reuter LM, Khadayate SP, Speck C |
| Citation(s) |
39181881 |
| |
| Submission date |
Apr 28, 2021 |
| Last update date |
Sep 24, 2024 |
| Contact name |
LMS Bioinformatics Core |
| E-mail(s) |
bioinformatics@lms.mrc.ac.uk
|
| Organization name |
MRC London Institute of Medical Sciences
|
| Department |
Bioinformatics Core
|
| Street address |
Hammersmith Hospital Campus, Du Cane Road
|
| City |
London |
| ZIP/Postal code |
W12 0NN |
| Country |
United Kingdom |
| |
|
| Platforms (1) |
| GPL17342 |
Illumina HiSeq 2500 (Saccharomyces cerevisiae) |
|
| Samples (8)
|
| GSM5269283 |
Mcm4-Flag ChIP-Exo from cells arrested in G1, rep1 |
| GSM5269284 |
Mcm4-Flag ChIP-Exo from cells arrested in G1, rep2 |
| GSM5269285 |
Orc2-Flag ChIP-Exo from cells arrested in G1, rep1 |
| GSM5269286 |
Orc2-Flag ChIP-Exo from cells arrested in G1, rep2 |
| GSM5269287 |
Orc2-Flag ChIP-Exo from cells arrested in G2, rep1 |
| GSM5269288 |
Orc2-Flag ChIP-Exo from cells arrested in G2, rep2 |
| GSM7698558 |
Orc2-Flag ChIP-Exo from cells released from G1-phase (24min), biol rep1 |
| GSM7698559 |
Orc2-Flag ChIP-Exo from cells released from G1-phase (24min), biol rep2 |
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| This SubSeries is part of SuperSeries: |
| GSE240779 |
MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing |
|
| Relations |
| BioProject |
PRJNA725895 |
| SRA |
SRP316778 |
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