Dataset Information

The ORC ubiquitin ligase OBI1 promotes DNA replication origin firing.

ABSTRACT: DNA replication initiation is a two-step process. During the G1-phase of the cell cycle, the ORC complex, CDC6, CDT1, and MCM2-7 assemble at replication origins, forming pre-replicative complexes (pre-RCs). In S-phase, kinase activities allow fork establishment through (CDC45/MCM2-7/GINS) CMG-complex formation. However, only a subset of all potential origins becomes activated, through a poorly understood selection mechanism. Here we analyse the pre-RC proteomic interactome in human cells and find C13ORF7/RNF219 (hereafter called OBI1, for ORC-ubiquitin-ligase-1) associated with the ORC complex. OBI1 silencing result in defective origin firing, as shown by reduced CMG formation, without affecting pre-RC establishment. OBI1 catalyses the multi-mono-ubiquitylation of a subset of chromatin-bound ORC3 and ORC5 during S-phase. Importantly, expression of non-ubiquitylable ORC3/5 mutants impairs origin firing, demonstrating their relevance as OBI1 substrates for origin firing. Our results identify a ubiquitin signalling pathway involved in origin activation and provide a candidate protein for selecting the origins to be fired.

SUBMITTER: Coulombe P

PROVIDER: S-EPMC6547688 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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The ORC ubiquitin ligase OBI1 promotes DNA replication origin firing.

Coulombe Philippe P Nassar Joelle J Peiffer Isabelle I Stanojcic Slavica S Sterkers Yvon Y Delamarre Axel A Bocquet Stéphane S Méchali Marcel M

Nature communications 20190603 1

DNA replication initiation is a two-step process. During the G1-phase of the cell cycle, the ORC complex, CDC6, CDT1, and MCM2-7 assemble at replication origins, forming pre-replicative complexes (pre-RCs). In S-phase, kinase activities allow fork establishment through (CDC45/MCM2-7/GINS) CMG-complex formation. However, only a subset of all potential origins becomes activated, through a poorly understood selection mechanism. Here we analyse the pre-RC proteomic interactome in human cells and fin ...[more]

PMID: 31160578

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Project description:During origin licensing, the eukaryotic replicative helicase Mcm2-7 forms head-to-head double hexamers to prime origins for bidirectional replication. Recent single-molecule and structural studies revealed that one molecule of the helicase loader ORC can sequentially load two Mcm2-7 hexamers to ensure proper head-to-head helicase alignment. To perform this task, ORC must release from its initial high-affinity DNA binding site and "flip" to bind a weaker, inverted DNA site. However, the mechanism of this binding-site switch remains unclear. In this study, we used single-molecule Förster resonance energy transfer (sm-FRET) to study the changing interactions between DNA and ORC or Mcm2-7. We found that the loss of DNA bending that occurs during DNA deposition into the Mcm2-7 central channel increases the rate of ORC dissociation from DNA. Further studies revealed temporally-controlled DNA sliding of helicase-loading intermediates, and that the first sliding complex includes ORC, Mcm2-7, and Cdt1. We demonstrate that sequential events of DNA unbending, Cdc6 release, and sliding lead to a stepwise decrease in ORC stability on DNA, facilitating ORC dissociation from its strong binding site during site switching. In addition, the controlled sliding we observed provides insight into how ORC accesses secondary DNA binding sites at different locations relative to the initial binding site. Our study highlights the importance of dynamic protein-DNA interactions in the loading of two oppositely-oriented Mcm2-7 helicases to ensure bidirectional DNA replication.Significance statementBidirectional DNA replication, in which two replication forks travel in opposite directions from each origin of replication, is required for complete genome duplication. To prepare for this event, two copies of the Mcm2-7 replicative helicase are loaded at each origin in opposite orientations. Using single-molecule assays, we studied the sequence of changing protein-DNA interactions involved in this process. These stepwise changes gradually reduce the DNA-binding strength of ORC, the primary DNA binding protein involved in this event. This reduced affinity promotes ORC dissociation and rebinding in the opposite orientation on the DNA, facilitating the sequential assembly of two Mcm2-7 molecules in opposite orientations. Our findings identify a coordinated series of events that drive proper DNA replication initiation.

Project description:Mammalian genomes are replicated in a precise, cell-type-specific order during the S phase, a process that correlates with local transcriptional activity, chromatin modifications, and chromatin architecture. However, the causal relationships between these features and DNA replication timing (RT), particularly during cell fate transitions, remain largely unknown. Here, we employed machine learning to quantify chromatin features — including epigenetic marks, histone variants, and chromatin architectural factors — that best predict local RT under steady-state conditions and RT changes during early embryonic stem (ES) cell differentiation. We found that approximately one-third of the genome exhibits RT changes during differentiation. Chromatin features collectively predicted steady-state RT and RT changes with high accuracy. Notably, histone H3 lysine 4 monomethylation (H3K4me1), catalyzed by KMT2C/D, emerged as a top predictor. Genetic deletion of Kmt2c/d (but not Kmt2c alone) or their enzymatic activities erased genome-wide RT dynamics during cell differentiation. Sites that typically gain H3K4me1 in a KMT2C/D-dependent manner during differentiation failed to transition towards earlier RT, often without affecting transcriptional activation. Further analysis at KMT2C/D binding sites revealed a local requirement for KMT2C/D in promoting DNA replication origin firing. Our findings identify KMT2C/D-dependent H3K4me1 as a functional regulator of RT and origin firing, highlighting a causal relationship between the epigenome and DNA replication that is largely independent of transcription. These insights should be relevant to various diseases associated with KMT2C/D mutations. Mammalian genomes are replicated in a precise, cell-type-specific order during the S phase, a process that correlates with local transcriptional activity, chromatin modifications, and chromatin architecture. However, the causal relationships between these features and DNA replication timing (RT), particularly during cell fate transitions, remain largely unknown. Here, we employed machine learning to quantify chromatin features — including epigenetic marks, histone variants, and chromatin architectural factors — that best predict local RT under steady-state conditions and RT changes during early embryonic stem (ES) cell differentiation. We found that approximately one-third of the genome exhibits RT changes during differentiation. Chromatin features collectively predicted steady-state RT and RT changes with high accuracy. Notably, histone H3 lysine 4 monomethylation (H3K4me1), catalyzed by KMT2C/D, emerged as a top predictor. Genetic deletion of Kmt2c/d (but not Kmt2c alone) or their enzymatic activities erased genome-wide RT dynamics during cell differentiation. Sites that typically gain H3K4me1 in a KMT2C/D-dependent manner during differentiation failed to transition towards earlier RT, often without affecting transcriptional activation. Further analysis at KMT2C/D binding sites revealed a local requirement for KMT2C/D in promoting DNA replication origin firing. Our findings identify KMT2C/D-dependent H3K4me1 as a functional regulator of RT and origin firing, highlighting a causal relationship between the epigenome and DNA replication that is largely independent of transcription. These insights should be relevant to various diseases associated with KMT2C/D mutations.

Dataset Information

The ORC ubiquitin ligase OBI1 promotes DNA replication origin firing.

Publications

The ORC ubiquitin ligase OBI1 promotes DNA replication origin firing.

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