Li S et al. (2022), Nucleosome-directed replication origin licensin...

XB-ART-59272

Nat Commun 2022 Aug 23;131:4947. doi: 10.1038/s41467-022-32657-7.

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Nucleosome-directed replication origin licensing independent of a consensus DNA sequence.

Li S, Wasserman MR, Yurieva O, Bai L, O'Donnell ME, Liu S.

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The numerous enzymes and cofactors involved in eukaryotic DNA replication are conserved from yeast to human, and the budding yeast Saccharomyces cerevisiae (S.c.) has been a useful model organism for these studies. However, there is a gap in our knowledge of why replication origins in higher eukaryotes do not use a consensus DNA sequence as found in S.c. Using in vitro reconstitution and single-molecule visualization, we show here that S.c. origin recognition complex (ORC) stably binds nucleosomes and that ORC-nucleosome complexes have the intrinsic ability to load the replicative helicase MCM double hexamers onto adjacent nucleosome-free DNA regardless of sequence. Furthermore, we find that Xenopus laevis nucleosomes can substitute for yeast ones in engaging with ORC. Combined with re-analyses of genome-wide ORC binding data, our results lead us to propose that the yeast origin recognition machinery contains the cryptic capacity to bind nucleosomes near a nucleosome-free region and license origins, and that this nucleosome-directed origin licensing paradigm generalizes to all eukaryotes.

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Species referenced: Xenopus laevis
Genes referenced: mmut

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	Fig. 1. Model for origin licensing in Saccharomyces cerevisiae vs. in most other eukaryotes.a In a chromatinized genome, ORC searches for nucleosomes and stably associates with them regardless of whether a nearby ARS consensus sequence (ACS) exists. ORC then loads MCM double hexamers in conjunction with Cdc6 and Cdt1 at nucleosomal sites in G1 phase. Due to its small genome, S. cerevisiae may have evolved ARS-dependent origins in order to limit ORC binding to specific sequences and thus avoid replication-transcription conflicts24, which does not generalize to higher eukaryotes. b Origins in most eukaryotes lack a consensus ACS motif, but still utilize ORC which is known to bind nucleosomes. We demonstrate in this study that S.c. ORC harbors a cryptic ability to bind nucleosomes and direct MCM DH formation within nucleosome-free regions (NFRs) that lack ARS consensus sequences. We propose that this mechanism is the normal process for most eukaryotes, whose origin sites are chiefly defined by the nucleosomal architecture. In this model, ACS confers origin specificity in S.c. by facilitating ORC binding to nucleosomes proximal to a nucleosome-free ARS sequence. We note that MCM DHs are recruited to origins via a “single ORC flipping” or “two ORC” mechanism10–13, and that our model is compatible with either scenario.
	Fig. 2. A single-molecule platform to study eukaryotic replication initiation.a Cartoon of the λARS1 DNA template. The inserted ARS1 element is illustrated in the inset box. b Schematic of the single-molecule experimental setup. Channels 1–3 are separated by laminar flow. Beads are optically trapped in channel 1, moved to channel 2 to tether DNA, then moved to channel 3 to characterize the force-extension curve of the tether. Once a correct tether is confirmed, the beads-DNA assembly is moved to channel 4 or 5 containing the proteins. The zoom-in box illustrates the final assembly in the imaging channel (not drawn to scale). c A representative kymograph showing the behavior of Cy3-labeled ORC on λARS1 DNA in the presence of Cdc6. The engineered ARS1 position is indicated. d Fraction of λARS1 DNA tethers that were observed to have at least one ORC bound in the presence or absence of Cdc6. The protein concentrations used in this experiment are: 2 nM for ORC, and 5 nM for Cdc6. The number of tethers analyzed for each condition is indicated. Data are presented as mean values ± SD from three independent experiments. Significance was obtained using an unpaired two-tailed t-test (*p < 0.05). e Fraction of ORC molecules that stably reside at the ARS1 site vs. non-ARS1 sites in the presence or absence of Cdc6. n indicates the number of ORC molecules analyzed for each condition. Source data are provided as a Source Data file.
	Fig. 3. ORC-dependent MCM loading occurs frequently at non-ARS DNA sites.a Cartoon of the single-molecule pre-RC assembly experiment using λARS1 DNA, unlabeled ORC, Cdc6, Cdt1, and LD650-labeled MCM (red). b An example kymograph showing that the MCM fluorescence signal appeared at a non-ARS1 position on DNA. c Fraction of stably bound MCM complexes that were observed at ARS1 vs. non-ARS1 positions. n indicates the number of MCM complexes analyzed. d Example kymographs showing the photobleaching steps (white arrows) of MCM fluorescence at ARS1 and non-ARS1 positions on the DNA tether. e Distribution of the number of photobleaching steps observed in each MCM fluorescence trajectory. n indicates the number of trajectories analyzed. f Fraction of DNA tethers that were observed to harbor at least one fluorescent MCM complex in the presence or absence of ORC. The protein concentrations used in this experiment are: 10 nM for MCM, 2 nM for ORC, and 5 nM for Cdc6. The number of tethers analyzed for each condition is indicated. g Cartoon of the high-salt wash experiment to demonstrate MCM loading on DNA using a mixture of LD650-MCM and Cy3-MCM, unlabeled ORC, Cdc6 and Cdt1. h A representative kymograph showing large-scale mobility of an MCM DH (indicated by the dual-color complex which appeared as yellow) loaded at a non-ARS1 position traversing the entire length of the tethered DNA upon high-salt wash (yellow arrowhead). The other MCM complex dissociated at high salt (white arrowhead). i Fraction of MCM complexes on nucleosome-free DNA that underwent sliding on DNA without dissociation (red), remained stably bound to the DNA position (black), or dissociated into solution (white) upon high-salt wash. n indicates the number of MCM complexes analyzed. Source data are provided as a Source Data file.
	Fig. 4. ORC predominantly binds to nucleosomes over bare DNA.a Cartoon of the λARS1 DNA sparsely loaded with Cy3-labeled S.c. nucleosomes (green) and incubated with LD650-labeled ORC (red) and Cdc6. b A representative kymograph showing a λARS1 DNA tether loaded with multiple nucleosomes (positions indicated by green arrowheads), all of which were located at non-ARS1 sites except one. Each nucleosome was observed to be stably bound by ORC (red). The presence of ORC on the nucleosomes is confirmed by turning off the green laser, which showed only the red fluorescence from ORC; alternatively, turning off the red laser showed only the green fluorescence from the nucleosomes. c Fraction of ORC stably bound to nucleosomes (X.l. or S.c.) located at either the ARS1 site or non-ARS1 sites. n indicates the number of ORC molecules analyzed for each condition. d Fraction of nucleosomes (X.l. or S.c.) within a given DNA tether that were observed to be ORC-bound in the presence of 2 nM ORC and 5 nM Cdc6. The number of tethers analyzed for each condition is indicated. Data are presented as mean values ± SD. Significance was obtained using an unpaired two-tailed t-test (ns, p = 0.41). Source data are provided as a Source Data file.
	Fig. 5. MCMs are recruited by ORC to nucleosomes independently of ARS DNA.a Cartoon (top), an example kymograph (middle) and the corresponding fluorescence intensities (bottom) of the pre-RC assembly experiment using Cy3-labeled X.l. nucleosomes (green), LD650-labeled MCM (red), unlabeled ORC, Cdc6 and Cdt1. Yellow arrowhead in the kymograph indicates the time when the MCM fluorescence signal appeared at the nucleosomal site. b Cartoon (top), an example kymograph (middle) and the corresponding fluorescence intensities (bottom) of the pre-RC assembly experiment using A488-labeled S.c. nucleosomes (blue), LD650-labeled MCM (red), unlabeled ORC, Cdc6 and Cdt1. In both examples in a, b the nucleosomes were at non-ARS1 positions on the DNA. c Fraction of nucleosomes (X.l. or S.c.) that were observed to have colocalized MCM signals in the presence or absence of ORC. n indicates the number of nucleosomes analyzed for each condition. d Fraction of MCM complexes on a nucleosome-loaded (X.l. or S.c.) tether that colocalized with a nucleosome vs. with nucleosome-free DNA. n indicates the number of MCM complexes analyzed. e Fraction of MCM-nucleosome (X.l. or S.c.) colocalization events observed at ARS1 vs. non-ARS1 positions. n indicates the number of events analyzed. f Cartoon (top) and an example kymograph (bottom) of the three-color experiment using A488-labeled S.c. nucleosomes (blue), both LD650-labeled MCM (red) and Cy3-labeled MCM (green), unlabeled ORC, Cdc6 and Cdt1. The colocalization of a dual-color MCM with a nucleosome indicates MCM DH recruitment to the nucleosomal site. Individual lasers were occasionally turned off to confirm the fluorescence signals from the other channels. Source data are provided as a Source Data file.
	Fig. 6. ORC-mediated MCM loading occurs at nucleosomal sites.a Cartoon illustrating the experimental assay that evaluates MCM loading at nucleosomal sites via high-salt wash. b A representative kymograph showing that MCM complexes (red) formed on a DNA tether in the presence of unlabeled ORC, Cdc6 and Cdt1. Upon moving to a high-salt buffer (0.5 M NaCl), a fraction of the MCMs displayed diffusive behavior without dissociation, demonstrating their successful loading onto DNA. MCM diffusion could occur from both bare DNA and nucleosome sites (white arrowheads in the zoomed-in view). Blue arrowheads indicate nucleosome positions, all of which were at non-ARS1 sites in this example. MCM and nucleosome fluorescence signals are also separately shown in gray scale at the bottom. c Fraction of nucleosome-colocalized MCM complexes that underwent diffusion without dissociation (red), remained stably bound to the nucleosome (black), or dissociated into solution (white) upon high-salt wash. n indicates the number of MCM complexes analyzed. Source data are provided as a Source Data file.
	Fig. 7. Genome-wide analysis of ORC/MCM localization and ACS motif scores.a Heatmap of Orc1 ChIP peaks (n = 295) and Mcm2-7 ChIP signal at the corresponding sites. b Examples of Orc1 and Mcm2-7 ChIP data and nucleosome occupancy at six ARS. Red vertical lines represent the location of ACS consensus in these regions. The three ARS on the right contain no consensus with score above 9. c ACS motif score vs. Orc1 ChIP peak strength. The motifs were identified in a 1-kb region near Orc1 ChIP peaks (peak center ±500 bp). All elements with a PWM score >9 are shown here. For the x axis, “1” represents the largest Orc1 ChIP peak, and “295” the weakest. d Fraction of sequences underlying all Orc1 peaks that contain motifs above a certain threshold (varying from 9 to 15). The red arrow represents the recommended cutoff of 11.9. e Same as in d except using a subset of Orc1 peaks that overlap with previously annotated ARS. f Abf1 and Reb1 ChIP peaks analyzed in the same way as in d. The red arrows represent recommended cutoffs for these two factors. g Heatmap of nucleosome occupancy near Orc1 ChIP peaks. The left panel includes 62 peaks containing ACS consensus (PWM > 11.9), with each row aligned at the consensus site. The right panel includes 145 peaks with no consensus above 9, and it was aligned at the center of the ChIP peaks.

References [+] :

Aladjem, Order from clutter: selective interactions at mammalian replication origins. 2017, Pubmed