The origin recognition complex (ORC) is essential for initiation of eukaryotic chromosome replication as it loads the replicative helicase—the minichromosome maintenance (MCM) complex—at replication origins1. Replication origins display a stereotypic nucleosome organization with nucleosome depletion at ORC-binding sites and flanking arrays of regularly spaced nucleosomes2,3,4. However, how this nucleosome organization is established and whether this organization is required for replication remain unknown. Here, using genome-scale biochemical reconstitution with approximately 300 replication origins, we screened 17 purified chromatin factors from budding yeast and found that the ORC established nucleosome depletion over replication origins and flanking nucleosome arrays by orchestrating the chromatin remodellers INO80, ISW1a, ISW2 and Chd1. The functional importance of the nucleosome-organizing activity of the ORC was demonstrated by orc1 mutations that maintained classical MCM-loader activity but abrogated the array-generation activity of ORC. These mutations impaired replication through chromatin in vitro and were lethal in vivo. Our results establish that ORC, in addition to its canonical role as the MCM loader, has a second crucial function as a master regulator of nucleosome organization at the replication origin, a crucial prerequisite for efficient chromosome replication.
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A chromatinized origin reduces the mobility of ORC and MCM through interactions and spatial constraint
Nature Communications Open Access 23 October 2023
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All data generated or analysed during this study are included in this published article (and its supplementary information files). The raw and processed files from the high-throughput sequencing data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) with the accession number GSE209681.
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The authors thank H. Blum and S. Krebs (LAFUGA) for high-throughput sequencing; E. Oberbeckmann for her support during the initial phases of the project; E. Kocar and G. Linder for help in purifying some proteins; S. Härtel for growing cells and preparing powder for protein preparations; J. Diffley for strains and plasmids; A. Singh and F. Müller-Planitz for strains and for sharing unpublished results; F. Bleichert for sharing Drosophila ORC; A. Costa and O. Willhoft for discussing results; and J. Kurat for critical input and for carefully reading the manuscript. This work was funded by the Deutsche Forschungsgemeinschaft (DFG)—the German Research Foundation—project ID 213249687—SFB 1064 to C.F.K., P.K. and B.P. and PF794/5-1 to B.P. Work in the B.P. laboratory is supported by the Max-Planck-Gesellschaft and the German Aerospace Center (DLR).
The authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 2 Effects of chromatin factors on nucleosome positioning at origins in SGD chromatin.
Composite plots of MNase-seq data as in Fig. 1f, but for SGD chromatin incubated without or with the indicated histone chaperones a) and chromatin remodelers b). Averages of n = 2 independent replicates are plotted for a) as well as for SWI/SNF or SWR1, whereas averages of n = 3 replicates are plotted for the other remodelers.
Extended Data Fig. 3 Effects of chromatin factors in combination with ORC on nucleosome positioning at origins in SGD chromatin.
As Extended Data Fig. 2, but for SGD chromatin incubated without or with the indicated histone chaperones a) and chromatin remodelers b) and wild type ORC. Averages of n = 2 independent replicates are plotted for a) as well as for the SWI/SNF and SWR1 experiments, whereas for the Fun30 and RSC experiments, n = 3 replicates were plotted. SGD chromatin was the same as in Extended Data Fig. 2.
Extended Data Fig. 4 Effects of histone modifications and chromatin density on the ORC/remodeler mechanism.
a) As Fig. 1h, but for SGD chromatin assembled with recombinant yeast histone octamers (n = 1). b) As Fig. 1h, but with SGD chromatin assembled with our standard (high and a medium) assembly degree. Shown is the average of n = 2 biological replicates. The linker 1 length was determined by measuring the distance between the first and second nucleosomal peak, either upstream or downstream of the alignment point, and subtracting 147 bp as previously described20. The average and variation of both upstream and downstream linker 1 lengths as well as +1 and −1 distances to ACS were calculated. The linker 1 length for the Chd1 experiment with medium assembly degree SGD chromatin was measured only upstream the ACS, since peak calling downstream of the ACS was not reliable due to array irregularities for unknown reasons.
Extended Data Fig. 5 Effects of remodeler elimination on origin chromatin organization and replication in vivo.
a) Spot dilution assays (10-fold serial dilutions) with the indicated wild type or quadruple knock out (QKO, Δarp8 Δisw1 Δisw2 Δchd1) yeast strains. YPD: yeast extract, peptone, dextrose full medium. b) Composite plots of in vivo MNase-seq data as in Fig. 1a but for wild type (grey background) versus indicated single remodeler deletion mutants (n = 1). Experiments were performed once, but the results confirmed by a different method of rapid remodeler depletion in c). c) As b) but with published data19 for strains before (“+ Remodeler”) versus after (“-Remodeler”) rapid depletion of the indicated remodeler by the degron or the anchor away system. The plotted samples correspond to: GSM3177776 (+INO80), GSM3177777 (-INO80), GSM3177780 (+ISW1a), GSM3177781 (-ISW1a), GSM3177772 (+ISW2), GSM3177773 (-ISW2), GSM3177784 (+Chd1) and GSM3177785 (-Chd1). d) As in c), but for the indicated double combinations of remodeler depletion. The plotted samples correspond to: GSM3452526 (+INO80, ISW2), GSM3452527 (-INO80, ISW2), GSM3452530 (+ISW1a, Chd1) and GSM3452531 (-ISW1a, Chd1). e) As in c), but for the indicated quadruple remodeler depletion. The plotted samples correspond to: GSM3452546 (+INO80, ISW1a, ISW2, Chd1) and GSM3452547 (-INO80, ISW1a, ISW2, Chd1). f) Flow cytometry analyses as in Fig. 2f but for the indicated remodeler deletion mutants.
Extended Data Fig. 6 Orc1 is involved in nucleosome organization at origins and in replication in vivo.
a) Averaged composite plots of biological replicates including the standard error (s.e.m.) between samples of in vivo MNase-seq data as in Fig. 2e but for ORC1 wild type (n = 4) versus orc1 mutant cells, as indicated (orc1-BAH, n = 3; orc1-IDR, n = 2). b) Heat maps as in Fig. 2a but for in vivo chromatin of the same strains as in a). c) Flow cytometry analyses as in Fig. 2f but with the same strains as in a).
Extended Data Fig. 7 Effects of Orc1 mutations on nucleosome positioning at origins with different remodelers.
a) Composite plots of in vitro MNase-seq data as in Fig. 1h but for SGD chromatin incubated with the indicated chromatin remodelers and wild-type ORC (grey background) or the indicated Orc1-mutant ORCs. Averages of n = 2 independent replicates are plotted. b) As in a) but for the indicated Orc1-mutatant ORCs. Averages of n = 2 independent replicates are plotted.
Extended Data Fig. 8 Effects of Orc1 mutations on the interaction with chromatin remodelers and nucleosomes.
a) Outline of the in vitro co-immunoprecipitation assay. b) In vitro co-immunoprecipitations assay as in a) on SGD chromatin with indicated Orc1 wild-type and mutant ORCs and remodelers. Experiments were performed once, with the exception of the INO80 experiment, which was repeated twice, but the result confirmed with ISW1a, ISW2 and, to much lesser extent with Chd1. For gel source data, see Supplementary Fig. 3.
a) Outline of the genome-scale in vitro replication assay as in Fig. 4c, but for naked DNA plasmid origin library templates. b) In vitro replication assay as in Fig. 4d but according to a) with naked DNA instead of SGD chromatin. c) Outline of the in vitro chromatin replication assay as in a) but for chromatinised single locus ARS1 origin templates14. d) In vitro replication assay as in b) but according to c) for SGD chromatin with single locus ARS1 origin templates instead of the origin plasmid library. e) Outline of the genome-scale in vitro chromatin replication assay as in Fig. 4c, but with different remodelers. f) In vitro replication assay as in Fig. 4d but according to d) with the indicated remodelers. Replication reactions on naked DNA were repeated twice and a representative example is shown. Reactions on the ARS1 origin template and reactions with the different remodelers were repeated once but confirmed the results of other assays or replication assays on SGD chromatin templates (Fig. 4d). For gel source data, see Supplementary Fig. 2.
This file contains Supplementary Figs. 1–3 and Supplementary Tables 1 and 2. Supplementary Figures 1–3: Source gel data from main figures and from Extended Data Figs. 1, 8 and 9. Supplementary Table 1: List of yeast strains that were used in this study. Supplementary Table 2: DNA sequences that were used to generate the Orc1 mutants for in vivo and in vitro studies.
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Chacin, E., Reusswig, KU., Furtmeier, J. et al. Establishment and function of chromatin organization at replication origins. Nature 616, 836–842 (2023). https://doi.org/10.1038/s41586-023-05926-8
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