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Replication licensing regulated by an intrinsically disordered region of origin recognition complex

ABSTRACT: Replication licensing regulated by an intrinsically disordered region of origin recognition complex

PROVIDER: PRJNA1093011 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR28491780_1.fastq.gz	Fastqsanger.gz
	SRR28491780_2.fastq.gz	Fastqsanger.gz
	SRR28491781_1.fastq.gz	Fastqsanger.gz
	SRR28491781_2.fastq.gz	Fastqsanger.gz
	SRR28491782_1.fastq.gz	Fastqsanger.gz

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Replication licensing regulated by an intrinsically disordered region of origin recognition complex

Project description:In eukaryotes, the hetero-hexameric origin recognition complex (ORC) is responsible for assembling Mcm2-7 complex into a head-to-head double hexamer (DH), forming pre-replicative complex (pre-RC) that licenses origin DNA for replication initiation. This process is tightly controlled throughout the cell cycle to ensure accurate duplication of the genome. Here we show that the N-terminal intrinsically disordered region (IDR) of the yeast Orc2 subunit plays a critical role in promoting pre-RC assembly. We found that removing a short segment (residues 175-200) from Orc2-IDR or mutating a key isoleucine (194) in this region significantly inhibits replication initiation across the genome and causes cell death. Although the Orc2-IDR mutants can still assemble the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) intermediate on DNA, the assembled mutant OCCM exhibits impaired ATP hydrolysis, preventing its conversion into Mcm2-7-ORC (MO) complex and subsequent DH formation. Interestingly, our in vitro assays showed that adding the Orc2-IDR peptide in the pre-RC reactions can rescue this defect. Furthermore, phosphorylation of this Orc2-IDR motif by S-cyclin dependent kinase (S-CDK) blocks its binding to Mcm2, leading to defective pre-RC assembly. Our findings provide crucial mechanistic insights into the multifaceted roles of ORC in modulating MCM loading to support origin licensing during the G1 phase and its regulation to restrict origin firing within the S phase.

2024-09-18 | GSE262693 | GEO

Replication licensing regulated by a short linear motif within an intrinsically disordered region of origin recognition complex.

Project description:In eukaryotes, the origin recognition complex (ORC) faciliates the assembly of pre-replicative complex (pre-RC) at origin DNA for replication licensing. Here we show that the N-terminal intrinsically disordered region (IDR) of the yeast Orc2 subunit is crucial for this process. Removing a segment (residues 176-200) from Orc2-IDR or mutating a key isoleucine (194) significantly inhibits replication initiation across the genome. These Orc2-IDR mutants are capable of assembling the ORC-Cdc6-Cdt1-Mcm2-7 intermediate, which exhibits impaired ATP hydrolysis and fails to be convered into the subsequent Mcm2-7-ORC complex and pre-RC. These defects can be partially rescued by the Orc2-IDR peptide. Moreover, the phosphorylation of this Orc2-IDR region by S cyclin-dependent kinase blocks its binding to Mcm2-7 complex, causing a defective pre-RC assembly. Our findings provide important insights into the multifaceted roles of ORC in supporting origin licensing during the G1 phase and its regulation to restrict origin firing within the S phase.

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The intrinsically disordered region of SPIN1 regulates its function by phase separation.

Project description:The intrinsically disordered region of SPIN1 regulates its function by phase separation.

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Regulation of Src tumor activity by its N-terminal intrinsically disordered region

Project description:The membrane anchored Src tyrosine kinase is involved in numerous pathways and its deregulation is involved in human cancer. Our knowledge on Src regulation relies on crystallography, which revealed intramolecular interactions to control active Src conformations. However, Src contains a N-terminal intrinsically disordered unique domain (UD) whose function remains unclear. Using NMR, we reported that UD forms an intramolecular fuzzy complex involving a conserved region with lipid-binding capacity named Unique Lipid Binding Region (ULBR), which could modulate Src membrane anchoring. Here we show that the ULBR is essential for Src’s oncogenic capacity. ULBR inactive mutations inhibited Src transforming activity in NIH3T3 cells and in human colon cancer cells. It also reduced Src-induced tumor development in nude mice. An intact ULBR was required for MAPK signaling without affecting Src kinase activity nor sub-cellular localization. Phospho-proteomic analyses revealed that, while not impacting on the global tyrosine phospho-proteome in colon cancer cells, this region modulates phosphorylation of specific membrane-localized tyrosine kinases needed for Src oncogenic signaling, including EPHA2 and Fyn. Collectively, this study reveals an important role of this intrinsically disordered region in malignant cell transformation and suggests a novel layer of Src regulation by this unique region via membrane substrate phosphorylation.

2022-01-14 | PXD030006 | Pride

Intrinsically disordered regions regulate RhlE RNA helicase functions in bacteria

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Ordered and disordered regions of the Origin Recognition Complex direct differential in vivo binding at distinct motif sequences.

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Project description:The intrinsically disordered region of SPIN1 regulates its function by phase separation [RNA-seq]

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The intrinsically disordered region of SPIN1 regulates its function by phase separation [ChIP-seq]

Project description:The intrinsically disordered region of SPIN1 regulates its function by phase separation [ChIP-seq]

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Recruitment of mRNAs to P granules by gelation with intrinsically-disordered proteins (RNAseq datasets)

Project description:RNA granules are cellular condensates that contain RNAs and proteins. The mechanism that drive the recruitment of many mRNAs to RNA granules are not fully understood. Here we characterize the assembly and transcriptome of the germ (P) granules of C. elegans. We find that mRNAs are recruited into P granules by condensation with the intrinsically-disordered protein MEG-3. MEG-3 binds ~500 mRNAs in C. elegans embryos in a sequence-independent manner that favors mRNAs with low ribosome coverage. Translational stress causes additional mRNAs to localize to P granules and translational activation correlates with P granule exit for two mRNAs coding for germ cell fate regulators. MEG-3/RNA condensates assembled in vitro are gel-like and trap mRNAs in a non-dynamic state. Our observations reveal similarities between P granules and stress granules and identify gelation with intrinsically-disordered proteins as a sequence-independent mechanism to enrich low-translation mRNAs in RNA granules

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