DOI: 10.5061/dryad.cvdncjt8c
title: Data from: The Origin Recognition Complex requires chromatin tethering by a hypervariable intrinsically disordered region that is functionally conserved from sponge to man

description: The first step towards eukaryotic genome duplication is loading of the
 replicative helicase, the Mcm2-7 complex, onto chromatin. This so-called
 “licensing” step is executed by the Pre-Replication Complex (Pre-RC) whose
 assembly on chromatin is nucleated by the DNA-binding activity of the
 Origin Recognition Complex (ORC). It is thought that metazoan ORC, like
 the yeast complex, is recruited directly to chromatin by its ATP-dependent
 DNA binding and encirclement activity. However, we have previously shown
 that this DNA binding mode is dispensable for chromatin recruitment of fly
 ORC, raising the question of how metazoan ORC binds chromosomes. We show
 here that the intrinsically disordered region (IDR) of fly Orc1 is both
 necessary and sufficient for the recruitment of ORC to chromosomes in vivo
 and demonstrate that this activity is regulated by IDR phosphorylation. In
 vitro studies show that the IDR alone binds DNA and this bestows the ORC
 holocomplex with a high-affinityATP-independentDNA binding mode.
 Interestingly, we find that Orc1 IDRs have diverged so markedly across
 metazoans that they are unrecognizable as orthologs and yet we find that
 these compositionally homologous sequences retain DNA and chromatin
 binding activity down to basal metazoans. Altogether, these data suggest
 that chromatin is recalcitrant to ORC’s ATP-dependent DNA binding activity
 and we propose that this necessitates IDR-dependent chromatin tethering
 which poises ORC to opportunistically encircle nucleosome-free regions as
 they become available. This work reveals a novel step in metazoan
 replication licensing and expands our understanding of disordered protein
 homology and evolution by stretching the relationship between primary
 structure and function.
description: # The Origin Recognition Complex requires chromatin tethering by a
 hypervariable intrinsically disordered region that is functionally
 conserved from sponge to man \##Description of data Notes: \**For
 "EMSA" data each includes replicate images of separate
 experiments and are named according to the construct (e.g.,
 "WT", "D1-D6", or "DBP") and their replicate
 number (I.e., "1-3") \**For imaging data in FIG1A-FIG1B, each
 has the following six components: 1\) a maximum intensity projection of
 the data (file name contains "MAX" description) 2\) the
 background corrected and median filtered data (file name contains
 "BG_MEDIAN" description) 3\) a sub-stack of three images derived
 from item 2 above (file name contains "Substack") 4\) three
 individual images in jpg format from item 3 above (file name contains
 "0000" or "0001" or "0002") \**Imaging data
 in SFIG1E has the following five components 1\) a maximum intensity
 projection of the data (file name contains "MAX" description)
 2\) a sub-stack of three images derived from item 2 above (file name
 contains "Substack") 3\) three individual images in jpg format
 from item 3 above (file name contains "0000" or "0001"
 or "0002") \**Multiple images are associated with the western
 blot data in SFIG7 and images are named according to the primary antibody
 used (e.g., "antiHis" or "antiGFP") followed by the
 sample (e.g., "control" or "Orc1") \**The imaging data
 associated with FIG6F represent a time series of images which are named
 according to the two channels (i.e., "C1" and "C2")
 and their associated 12 images from early to late time points (I.e.,
 "10000" to "10011"). These twenty four images are
 .jpg. The time points between the two channels are matched. The parent
 image stack (before separation of individual time points) is also included
 as a tif file and is named according to the channel (as above). FIG1A:
 spinning disk imaging data of wild-type Orc1 in fly embryos FIG1B:
 spinning disk imaging data of Orc1 delta IDR in fly embryos FIG1C:
 spinning disk imaging data of Orc1 IDR alone in fly embryos FIG1D: excel
 file for quantitation of FIG1A-C FIG1E: spinning disk imaging data for
 S-phase nuclear enrichment of constructs in FIG1A-C FIG1F: spinning disk
 imaging data of Orc1 IDR alone in S2 cells FIG2A: excel file of raw
 fluorescence polarization readings for ORC DNA binding at 300 mM
 KGlutamate FIG2B: excel file of raw fluorescence polarization readings for
 ORC DNA binding at 150 mM KGlutamate FIG2C: excel file of raw fluorescence
 polarization readings for ORC delta IDR at 150 mM KGlutamate FIG3B: excel
 file quantifying spinning disk imaging of Orc1 IDR chromatin partitioning
 in S2 cells FIG3C: coomassie stained SDS-PAGE gel of IDR phosphorylated by
 CDK1/CycA FIG3D: coomassie stained SDS-PAGE gel of IDR phosphorylated by
 CDK2/CycE FIG3E: excel file for mass of intact mass spectrometry of
 phosphorylated IDR FIG3F: excel file for liquid chromatography mass
 spectrometry to identify phosphorylation sites and PDF of sequence logo
 FIG3H: image of agarose gel for EMSA analysis of Orc1 IDR DNA binding
 FIG3I: excel file quantifying EMSA analysis in FIG3H FIG3J: excel file of
 fluorescence polarization analysis of DNA binding by phosphorylated IDR
 FIG3K: excel file of fluorescence polarization analysis of DNA binding by
 phosphorylated IDR (time resolved assay) FIG4B: excel file quantifying
 EMSA analysis of IDR variants FIG4C: excel file analyzing fraction charged
 residues in Orc1 IDR FIG4D: vector graphic of Chi-Score Analysis of
 Drosophila Orc1 IDR FIG5B: csv file of Orc1 IDR ortholog alignment FIG5C:
 vector graphic of sequence similarity scores of Orc1 IDR and AAA orthologs
 FIG5D: vector graphic of compositional homology scores of Orc1 IDR and AAA
 orthologs FIG5E: excel file of ortholog Orc1 IDR isoelectric point (pI)
 FIG6A: image of agarose gel for EMSA analysis of Orc1 IDR ortholog DNA
 binding FIG6B: excel file of phosphorylation sites in Orc1 IDR orthologs
 FIG6C: image of agarose gel for EMSA analysis of Orc1 IDR ortholog DNA
 binding with phosphorylation FIG6D: excel file quantifying partitioning of
 Orc1 IDR orthologs onto chromatin in S2 cells in metaphase FIG6E: excel
 file quantifying partitioning of Orc1 IDR orthologs onto chromatin in S2
 cells through mitosis FIG6F: excel file and image analyzing human Orc1 IDR
 binding to mitotic chromosomes in HeLa cells SFIG1D: excel file of nuclear
 intensity of delta IDR Orc1 in embryos SFIG1E: spinning disk imaging data
 of genomic Orc1 IDR transgene in embryos SFIG2A: coomasie stained SDS-PAGE
 gel of ORC complexes and Orc1IDR SFIG2B: image of agarose gel for EMSA
 analysis of Drosophila Orc1 IDR DNA binding SFIG3A: coomasie stained
 SDS-PAGE gel of ORC holocomplex phosphorylation SFIG3B: coomasie stained
 SDS-PAGE gel of Orc1 IDR phosphorylation SFIG3C: excel file of intact mass
 spectrometry analysis of Orc1 IDR phosphorylation SFIG4A: coomassie
 stained SDS-PAGE gel of purified Orc1 IDR variants SFIG4B-H: images of
 agarose gel for EMSA analysis of Drosphila Orc1 IDR variant DNA binding
 SFIG5: vector graphic of Chi-Score comparison of compositional homology
 between Orc1 IDR orthologs and other IDRs SFIG6A: coomassie stained
 SDS-PAGE gel of purified Orc1 IDR orthologs SFIG6B-H: text file of raw
 data for fluorescence polarization DNA binding of Orc1 IDR orthologs
 SFIG6I: text file of binding affinity (Kd) data for Orc1 IDR orthologs
 SFIG7: images of western blot analysis HsOrc1-IDR-eGFP chromatin spin down
 assay \##Description of the file structure Data in this upload are
 organized based on the figures in which they appear in the forthecoming
 publication of this work in the journal Nucleic Acids Research, article
 number NAR-01896-J-2023.R1. \##Code/Software All image quantification was
 completed using Fiji (is just ImageJ) version 2.1.0/1.53c