Abstract
In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A–H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3–H4 tetramer, while the vacant H2A–H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3–H4 dimer and extends across the tetramerization interface of the H3–H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3–H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.
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Data availability
Coordinates and cryo-EM map data have been deposited at the Protein Data Bank and Electron Microscopy Data Bank under accession codes EMD-38317 and PDB 8XGC (composite map and the global atomic model), EMD-38316 (global structure, conformation-1), EMD-38314 (global structure, conformation-2), EMD-38313 (optimized local density map of Spt16-MD–histone) and EMD-38315 (optimized local density map of Polε). The raw sequencing data reported in this paper have been deposited in the Genome Sequence Archive at the National Genomics Data Center, Beijing Institute of Genomics (China National Center for Bioinformation), Chinese Academy of Sciences, under accession number GSA CRA012495. The data are publicly accessible online (https://bigd.big.ac.cn/gsa).
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Acknowledgements
We thank K. Labib and T. Formosa for antibodies; and the members of the Biological Cryo-EM Center at the Hong Kong University of Science and Technology (HKUST) for data collection. The Cryo-EM Center at HKUST is supported by a donation from the Lo Kwee Seong Foundation. The computation was supported by the High-Performance Computing Platform of Peking University (PKU). We also thank the cryo-EM platform of PKU and the National Center for Protein Sciences at PKU for technical assistance. The work was supported by the National Natural Science Foundation of China (32321163647 to N.G., 31830048 to Q.L. and 31922036 to N.L.), the National Key R&D Program of China (2019YFA0508900 to Q.L. and 2019YFA0508904 to N.G.), the Beijing Outstanding Young Scientist Program (BJJWZYJH01201910001005) to Q.L., and the Research Grants Council (RGC) of Hong Kong (GRF17119022, GRF17109623, C7009-20GF, and CRS_HKU705/23 to Y. Zhai).
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Y. Zhai, N.G. and Q.L. conceived and supervised the project. Y.G., J. Li, Z.X. and Y. Zhai purified the replisome samples and conducted biochemical assays. J. Lin and X.D.L. performed quantitative MS analysis. D.Y. and Y.G. prepared cryo samples. D.Y., Yingyi Zhang and S.D. collected cryo-EM datasets. N.L. processed images. N.G., N.L. and Y. Zhai built atomic models. Yujie Zhang, J.F. and Q.L. performed eSPAN analyses. Y. Zhai, N.G., N.L., Y.G., Yujie Zhang, Q.L., J. Lin, X.D.L., B.K.T., Y.L. and K.Z. analysed the data, prepared the figures and wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Purification of the endogenous replisome.
a, A flowchart of the procedures for replisome purification from early S chromatin of the yeast strain PSF2-3xFlag. b-c, SDS-PAGE analysis of the glycerol gradient (20-40%) fractions of the replisome sample eluted from anti-Flag affinity purification. The fractions were resolved and visualized by silver staining (b) and immunoblotting of the proteins as indicated (c). Fractions 5-8 containing replisome-FACT-histones were pooled and processed for mass spectrometry analysis. Based on this result, similar fractions containing crosslinked samples after grafix were pooled and processed for further EM analyses. Similar results were obtained at least in two independent experiments. d, The major proteins associated with the endogenous replisome identified by quantitative liquid chromatography mass spectrometry (LC-MS). For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 2 Cryo-EM image processing of the replisome sample.
a, A representative raw cryo-EM image of the replisome sample. b-c, 2D class averages of negative staining (b) and cryo-EM (c) replisome particles. d-i, Workflow of image processing of the replisome cryo-EM images. See Methods for details. j, Corrected (the right line group) and phase randomized (the left line group) FSC curves of the final global and local density maps for the replisome complex. k, The local resolutions of the composite replisome map were colour coded. l-q, Cryo-EM densities for indicated regions of the replisome.
Extended Data Fig. 3 Conformational change in FACT during substrate transaction.
a, c, Different views of the local density map of the FACT-histones complex bound with parental duplex DNA. b, d, same as (a, c) respectively but superimposed with the atomic model. e-f, FACT-DNA interaction. Comparison of the Spt16-DD/Pob3-DD-DNA structure from the replisome (this study) with those from FACT-hexasome (PDB: 6UPK) (e) and RNA Polymerase II-Spt4/5-nucleosome-FACT (PDB: 7NKY) (f). Spt16-DD/Pob3-DD was used as a reference for alignment. g-j, Comparison of the FACT structures from the replisome-histone hexamer-FACT (this study) (g), human FACT-hexasome (PDB: 6UPK) (i), yeast RNA Polymerase II-Spt4/5-nucleosome-FACT (PDB: 7NKY) (j), and RNA polymerase II elongation complex-nucleosome-FACT (Komagataella phaffii) (EC58hex, PDB: 7XTI) (j). Spt16-DD/Pob3-DD was used as a reference for alignment. k-m, Superimposition of the FACT structure from the replisome (this study, g) with those from h-j to highlight the conformational changes in Spt16 bound with different substrates as indicated, respectively. Spt16-DD/Pob3-DD was used as a reference for alignment. n-w, The relevant structures from g-m were aligned using histones as a reference to illustrate the conformational changes in both Spt16 and Pob3 while relocating histones onto Tof1 at replication fork during parental histone recycling. The histones and DNA from the replisome were omitted to highlight the movement of Spt16-MD in r-w.
Extended Data Fig. 4 The histone hexamer is shielded by its chaperones at the replication fork.
a-d, Schematic illustration of the histone-chaperone interaction in the replisome. e-f, Side views of the electrostatic surfaces of the histone hexamer highlighting its associations with Spt16-MD, Mcm2-NTE, and Tof1-HTH for shielding its DNA binding surfaces. i-l, same as (e-f) but superimposed with the nucleosomal DNA from an intact nucleosome (PDB: 1ID3).
Extended Data Fig. 5 Small displacement of H2A-H2Bproximal by Spt16-MD.
a-c, Comparison of the histone structures from the replisome (a) and an intact nucleosome (PDB: 1ID3) (b). Superimposition of these two structures using (H3-H4)2 as a reference for alignment (c). H3-αN and H2A-docking segment were coated with transparent surface presentation to highlight conformational changes occurring upon these regions in the replisome. d, Same as (c) but with Spt16-MD from the replisome visible to illustrate the steric conflict between Spt16-MD and H3-αN/H2A-docking segment on H3-H4 tetramer. e, Zoomed-in view of the boxed region (red box) in (d) but with the region from the replisome shown only. f, Same as (e) but shown with the H3 from the intact nucleosome (grey) and with Spt16-MD from the replisome superimposed. g, Zoomed-in view of the boxed region (green box) in (d) but with the region from the replisome shown only. The key residues involved in the interaction between Spt16-MD and H2A-docking segment are shown in stick and labelled. h, Same as (g) but shown with the H2A-docking segment from the intact nucleosome (red) and with Spt16-MD from the replisome superimposed. i, Same as (d) but with a 90°-rotation. The (H3-H4)2 tetramer is not shown to highlight a 13°-rotation in the H2A-H2Bproximal upon Spt16-MD binding.
Extended Data Fig. 6 The arrangement of Mcm2-NTE around the evicted histones is pre-determined by Spt16-MD and H2A-H2Bproximal.
a, Structural comparison of the (H3-H4)2/MCM2-NTE interactions from the structures of human (H3-H4)2-MCM2-HBD (PDB:5BNV) and yeast histone hexamer-Mcm2-NTE-Spt16-MD from the replisome (this study). H3-H4 was used as a reference for alignment. h: human; y: yeast; M2: MCM2. b, Same as (a) but with hM2-NTE-1 and yM2-NTE shown only. c, Same as (a) but with y(H3-H4)2 shown in surface presentation and other subunits and motifs in cartoon presentation. d-e, Zoomed-in views of the boxed regions in (c).
Extended Data Fig. 7 eSPAN analysis of the interaction between Tof1 and Mcm2.
a, Snapshot of MNase-seq, MNase-BrdU-IP-seq, H3K4me3-ChIP-seq, H3K4me3-eSPAN, H3K56ac-ChIP-seq, and H3K56ac-eSPAN datasets around replication origins ARS1309 and ARS1310 in wild-type (WT), tof1-3A, tof1ΔHTH, mcm2-6A, and mcm2ΔL. The scale bar represents a 10-kilo base pair (kbp) DNA region. b, DNA content analysis of the eSPAN samples. Cellular DNA content was measured by flow cytometry with PI staining. Similar results were obtained in two independent experiments. See Methods for more details.
Extended Data Fig. 8 The interaction between Tof1 and Mcm2 affects the relative amounts of the newly synthesized histone deposition between the leading and lagging strands.
a, Line plots of the H3K56ac eSPAN bias show the relative amount of new histone deposition between the two daughter strands in wild-type (WT), tof1-3A, and tof1ΔHTH cells. The H3K56ac-eSPAN bias around 139 early replication origins of the autonomous replication sequences (ACSs) was calculated. b, Box plots of the H3K56ac-eSPAN bias around the 139 ACSs in WT, tof1-3A, and tof1ΔHTH mutant cells are shown (n = 139). c, Box plots showing the H3K56ac-eSPAN density on both leading and lagging strands in WT, tof1-3A, and tof1ΔHTH cells (n = 139). d, Line plots of the H3K56ac eSPAN bias around the 139 ACSs in wild-type (WT), mcm2-6A, and mcm2ΔL cells are shown. e, Box plots of the H3K56ac-eSPAN bias around the 139 ACSs in WT and mcm2-6A or mcm2ΔL mutant cells are shown (n = 139). f, Box plots showing the H3K56ac-eSPAN density on both leading and lagging strands in WT, mcm2-6A, and mcm2ΔL cells (n = 139). g-h, Heatmap analysis of the H3K56ac-eSPAN bias around 139 ACSs in WT, tof1-3A, tof1ΔHTH (g), mcm2-6A, and mcm2ΔL (h) cells. Box plots (b, c, e, f) show the median, minimal, maximal, and 25% and 75% quartiles values. P values calculated using two-sided rank-sum Wilcoxon test. Similar results were obtained in two independent experiments. See Methods for more details.
Extended Data Fig. 9 The asymmetric histone partitioning between daughter strands in the disrupted Tof1-Mcm2 interaction cells does not result from defects in DNA replication.
a-d, The statistical results of correlation between BrdU incorporation level and H3K4me3-eSPAN value at each ACS region in WT (a), tof1-3A (b), mcm2-6A (c) and mcm2ΔL (d) cells. Dot scatterplot showing the distribution of the H3K4me3-eSPAN bias at early replication origins (n = 139). e-h, The statistical results of correlation between BrdU incorporation level and H3K56ac-eSPAN value at each ACS region in WT (e), tof1-3A (f), mcm2-6A (g) and mcm2ΔL (h) cells. Dot scatterplot showing the distribution of the H3K56ac-eSPAN bias at early replication origins (n = 139). Similar results were obtained in two independent experiments.
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Li, N., Gao, Y., Zhang, Y. et al. Parental histone transfer caught at the replication fork. Nature 627, 890–897 (2024). https://doi.org/10.1038/s41586-024-07152-2
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DOI: https://doi.org/10.1038/s41586-024-07152-2
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