(A) Example intensity (Cy3, green; Cy5, red) and FRET (blue) time trajectories for a library member TTAA that can form two additional A-T base pairs at 10°C (left), 15°C (center), and 20°C (right). Heatmaps of equilibrium constants (B) and Gibbs free energies (C) for all library members at 10°C (left), 15°C (center), and 20°C (right). Complementary nucleotides are located on the diagonal. (D) Plots of average Gibbs free energies versus temperature for all library members with additional complementary base pairs in none (red), only second (orange), only first (green), or both (blue) variable positions. Error bars represent standard errors of the mean. (E) Comparison of measured Gibbs free energies with mFold predictions at 10°C for all library members (right) or only those that form two additional complementary base pairs (left). In each case, ρ denotes the Pearson correlation coefficient. The red lines in both plots represent the linear fit of the data for library members that form two additional complementary base pairs. Sequences that are substantially more stable than expected on the basis of the nearest-neighbor prediction are highlighted in red. N = 124,387, 139,838, and 99,538 molecules for 10°, 15°, and 20°C, respectively.

(A) Schematic of the FRET assay and design of the construct library with four variable nucleotides in the PAM-distal region. See fig. S2C and table S1 for more details. (B) Heatmap of the equilibrium constant for all library members in the presence of dCas9. The nucleotides in positions 17 and 18 (most PAM-proximal variable nucleotides) are encoded in blocks of four on each axis, and positions 19 and 20 are encoded within each block. The fully matched sequence is located in the upper-left corner. N = 125,697 molecules. (C) Example FRET time trajectories (left) and FRET efficiency histograms after dCas9 addition (right) for four individual members of the library that have between zero and three mismatches. See also fig. S7. (D) Plot of the equilibrium constant as a function of the number of consecutive mismatches starting from the PAM-distal target DNA end (left) or of the position of a single mismatch (right). Error bars represent standard errors of the mean. (E) FRET efficiency histograms after dCas9 addition for library members combined based on the number of consecutive mismatches (red) starting from the PAM-distal (orange) target DNA end. (F) Example intensity (Cy3, green; Cy5, red) and FRET (blue) time trajectories, and Viterbi paths (red) obtained using HMM analysis for two library members. (G) Plots of unwinding (left) and rewinding (right) rates versus mean FRET values for individual library members (black dots) and fit results with a model assuming a constant rewinding or unwinding rate, respectively (red lines).

(A) An agarose gel (left) showing a library of target DNA sequences before and after digestion, and a heatmap (right) of the remaining (uncleaved) fraction of DNA after Cas9 cleavage for 10 s at 4°C for all library members, obtained by NGS. Dashed white rectangle indicates the uncleaved band that was gel-purified and subjected to NGS. The nucleotides in positions 17 and 18 (most PAM-proximal variable nucleotides) are encoded in blocks of four on each axis, and positions 19 and 20 are encoded within each block. The fully matched sequence is located in the upper-left corner. (B) Plot of the uncut fraction obtained by NGS versus mean FRET in the presence of dCas9 for all library members. ρ denotes the Pearson correlation coefficient. (C) Example FRET time trajectories (left) and FRET efficiency histograms after dCas9 addition (right) for sequences with unexpected properties: CATT, GATC, ACAG, and AAGG. Red lines on the right represent fits of FRET efficiency histograms with two (ACAG and AAGG) or three (CATT and GATC) Gaussian peaks. Straight lines in the guide RNA-DNA base-pairing schematics indicate Watson-Crick interactions, while dashed lines indicate potential non–Watson-Crick interactions. Gel-based cleavage normalized to the fully matched sequence is presented next to scissor pictograms. See also fig. S11.

a, Representative donor (green), acceptor (red) and FRET time traces (out of n = 117 traces) showing the unwinding of a single DNA molecule upon continuous ATP uncaging with 4 W cm−2 UV laser power density (purple). b, Representative FRET time traces showing the unwinding of individual DNA molecules at different ATP concentrations in a standard experiment (left, n = 775, 1,388, 145 and 441) or at different uncaging laser intensities in a LAGOON experiment with continuous uncaging (right, n = 85, 178, 153 and 303). c, Histograms of the ratio between Cy5 fluorescence intensities upon 532-nm and 638-nm excitation before and after unwinding in a standard experiment (left, n = 1,513) or before and after continuous uncaging for 1 minute with 0.75 W cm−2 UV laser power density in three FOVs on the same slide in a LAGOON experiment with 10 mM DMNPE-ATP (right, n = 1,454, 1,327 and 1,106). The Cy5532nm/Cy5638nm ratio was chosen instead of FRET because the donor-labeled DNA strand dissociated upon unwinding, leading to the absence of any detectable FRET signal. d, DNA unwinding rates (mean ± s.e.m.) as a function of ATP concentration (n = 637, 775, 1,800, 1,388, 145, 441, 182, 222, 209 and 131) or uncaging laser power density in the presence of 2 mM DMNPE-ATP (n = 85, 141, 134, 169, 178, 252, 104, 94, 186, 153, 210, 305, 303, 229 and 85) upon continuous uncaging. The plot on the right represents an enlarged version of the region marked in yellow in the plot on the left, scaled to overlay the linear sections of two titration curves. e,f, High-throughput smFRET observations of DNA unwinding using LAGOON on a motorized objective-type TIRF setup. e, Representative FRET time traces showing the unwinding of individual DNA molecules during continuous uncaging with approximately 2.5 W cm−2 405-nm laser in the presence of 200 nM T4 helicase, 10 mM DMNPE-ATP and 10 U ml−1 of hexokinase (out of n = 64,243 traces from 112 FOVs). f, Histogram of DNA unwinding rates (n = 158,523 unwinding events). The experiments (e,f) were carried out on a Nikon Eclipse Ti2-E motorized microscope equipped with a CFI Apochromat TIRF 60XC objective. a.u., arbitrary unit.

a, Representation of the labeling scheme for observing the DNA movement at the entry side during nucleosome sliding. The Cy5 fluorophore (red) attached to the exit-side H2B-120C is excited with the 638-nm laser and acts as a FRET donor, whereas the Alexa750 fluorophore (purple) attached to the entry-side DNA acts as a FRET acceptor. b, FRET histograms before (n = 855) and after (n = 726) the addition of Chd1 and 1 mM ATP. c, Representative donor (green), acceptor (red) and FRET (blue) time traces showing the remodeling of individual nucleosomes by Chd1 in the presence of 1 mM (left, n = 145) or 10 µM (right, n = 89) ATP. d, Representative donor (green), acceptor (red) and FRET (blue) time traces showing the remodeling of individual nucleosomes by Chd1 in a LAGOON experiment in the presence of 10 mM DMNPE-ATP with 0.30 W cm−2 (left, n = 93), 1 W cm−2 (middle, n = 91) or 3 W cm−2 (right, n = 72) continuous uncaging. e, A simplified schematic of the single-turnover experiment. Average times are marked for stochastic processes. f, Representative donor (green), acceptor (red) and FRET (blue) time traces showing entry-side DNA movement during nucleosome sliding in the single-turnover regime in the presence of 10 mM DMNPE-ATP (n = 209). The purple dashed lines mark uncaging laser pulses (90-ms duration every 8 seconds, 100 W cm−2 uncaging power density). Red lines in the FRET time traces represent the steps identified in the data using the AutoStepFinder method48. g, Histograms of FRET change in the first observed step upon pulsed ATP uncaging with a pulse duration of 30 ms (top, n = 453), 90 ms (middle, n = 206) or 180 ms (bottom, n = 254). Black lines represent fits of the histograms with two Gaussian peaks. Pulse period, 8 seconds. The asterisk indicates that, for the second peak, its center and width were fixed for the 30-ms and 90-ms histograms to match the parameters derived from the second peak of the 180-ms histogram. a.u., arbitrary unit.

a, Representation of the labeling scheme for observing nucleosome unwrapping upon its invasion by the T7 RNAP. b, FRET histograms before (n = 1,667) and after (n = 1,759) the addition of the T7 RNAP and NTPs. c, Representative donor (green), acceptor (red) and FRET (blue) time traces (n = 294) showing the unwrapping of individual nucleosomes by the T7 RNAP in the presence of NTPs (0.5 mM each). d, FRET histograms from three FOVs on the same slide (n = 349, 256 and 233, respectively) before and after uncaging for 2 minutes with 10 W cm−2 UV laser power in the presence of 2 mM DMNPE-ATP and CTP, GTP and UTP (0.5 mM each). The presence of the acceptor fluorophore (Cy5) was, in this case, probed by direct excitation with the 638-nm laser, which allowed the constructs lacking the acceptor, forming a low-FRET population in the absence of RNAP activity in b, to be excluded from the analysis. e, Representative donor (green), acceptor (red) and FRET (blue) time traces (n = 156) showing the unwrapping of individual nucleosomes by the T7 RNAP in a LAGOON experiment upon continuous uncaging with 10 W cm−2 UV laser power in the presence of 2 mM DMNPE-ATP and CTP, GTP and UTP (0.5 mM each). f, Representative donor (green), acceptor (red) and FRET (blue) time traces showing the unwrapping of individual nucleosomes by the T7 RNAP in a LAGOON experiment upon continuous uncaging with 3 W cm−2 UV laser power in the presence of 1 mM NPE-GTP, CTP, ATP and UTP each (n = 212). g, Schematic of the AFS LAGOON experiment monitoring E. coli RNAP elongation. DM, dichroic mirror; L, lens; LP, long-pass filter. See Methods for a detailed description of the setup. h, Representative raw (gray) and smoothed (with a fast Fourier transform filter, black) RNAP elongation time traces upon intermittent uncaging (n = 11 trajectories). The purple background represents ATP uncaging with a UV laser (~25 mW cm−2). i, Histograms of RNAP elongation rates with or without uncaging. The reaction buffer was supplemented with 1 mM NPE-ATP, CTP, GTP and UTP each. a.u., arbitrary unit.

(A) Example images taken during association (left) and dissociation (right) of LacI-Cy3 to spots on the DNA microarray. Orange circle, Osym operator; red circle, O1 operator; green circle, O2 operator. O3 is not present on the array, because initial experiments with this operator showed no binding over background. (B) Association and dissociation curves for the Osym, O1, and O2 operators (thick lines), and 100 examples of their mutants (faint colors). The dissociation curves for each operator were normalized to the fluorescence count for the first frame of the corresponding dissociation movie (C) Reproducibility of ka (top) and kd (bottom) for individual operators (cyan points) between replicates. The ka values are all normalized to the ka value of Osym in that replicate. Sequences associated with weak binding (fluorescence signal at equilibrium <3% of signal for Osym; gray points). (D) Measured association and dissociation rates for wild-type operators (circles) and their single and double mutants [points are colored by operator density in that (ka,kd) neighborhood; gray points are the same as in (C)]. The value for each operator is a mean from two PBM replicate experiments; see fig. S1B for data from individual replicates.

(A) Predicted effect on the association and dissociation rates if the kon,μ values were different but koff,μ was identical for the different operators. (B) Experimental single-molecule target-site association rate constants ka (7) plotted against the dissociation rate constants kd for the different lac operators. For the crosses, kd was directly measured by single-molecule imaging (14). For the dots, kd was calculated as KD × ka, where the equilibrium constant KD was measured through the repression ratio of gene expression (12, 13). Cyan circle, measured kon,max (see methods). Colored lines, koff,μ-lines, found by evaluating Eq. 1, for individual operators. *Due to the large error in the kd estimate for O3 [68% CI: (−0.08 to 0.24) s−1], the koff,μ-line for O3 is not shown. Error bars are standard errors, obtained by propagating experimental errors. (C) Microscopic dissociation rates koff,μ for the different operators, estimated from the in vivo data with Eq. 1. Error bars are 68% CI, obtained by propagating experimental errors. (D) Energy landscapes (a putative rather than a true reaction coordinate is shown) for the transition from free to bound states (state 1 and state 3, respectively) for the different operators, as determined by the measured KD and koff,μ values. The activation energy on the transition path between the testing state and bound state is not uniquely determined, but the differences in activation energies between the different operators are. The activation energy is equal to −log(koff,μ ) + c, where c is the same constant for all operators (see supplementary text).

(A) Single-molecule colocalization measurements detect association and dissociation for LacI binding to its operators (top) and the predicted effect on association and dissociation rates of changing the salt concentration (bottom). (B) Example single-molecule traces showing binding to and unbinding from the O1 operator at 100 mM NaCl (colored lines), and the normalized association and dissociation curves (black lines) obtained after summing 649 and 777 traces for the association and dissociation experiment, respectively. AU, arbitrary units. (C) Normalized association and dissociation curves for O1 binding at 1 mM (dashed dotted lines), 100 mM (solid lines), and 200 mM NaCl (dashed lines). (D) Measured ka and kd values for O1 binding at different salt concentrations, and a fit to Eq. 1 (red line). The salt concentrations used for the different experiments are in the range of 1 to 250 mM supplemented NaCl (fig. S2, A to D).

(A) Cryo-EM map of the complex between a PARylated nucleosome and ALC1 in the state tightly bound to SHL2 and the H4 tail. The map is shown at a contour level of 0.15 and colored by chain assignment (H3 in blue, H4 in green, H2A in yellow, H2B in red, DNA in gray, and ALC1 in pink). The black ellipse in the dyad view indicates the nucleosome dyad axis. Superhelical locations 0 (dyad), 1, and 3 and the location of the acidic patch are indicated. (B) Atomic model of the complex between a PARylated nucleosome and ALC1 in the state tightly bound to SHL2 and the H4 tail. Same views and same color code as in (A). The black ellipse in the dyad view indicates the nucleosome dyad axis. (C) Same cryo-EM map as in (A), displayed at the same contour level, but in two different orientations, showing the H4 tail interacting with the C-terminal ATPase domain of ALC1. Same color code as in (A). (D) Close-up view of the boxed region in (C). (E) Atomic model shown in the same two orientations as in (C). In addition to the same color code as in (A), the cryo-EM density of the H4 tail is shown as a turquoise transparent surface, at the same contour level as in (A), (C), and (D). (F) Close-up view of the boxed region in (E).

(A) Left: consensus map from 3D refinement in RELION of 43,698 particles, displayed at a contour level of 0.015 and in disc view. Regions of the map assigned to an interaction with the nucleosome acidic patch and to the macro domain are labeled. Right: UMAP visualization of the latent variable distribution of these same 43,698 particle images after training of an 8-dimensional latent variable model with cryoDRGN. Dots indicate all latent space coordinates that are part of the calculated graph traversal, labeled red dots indicate coordinates of maps shown in panels (B) – (G). Black lines are a visual guide and do not represent actual connectivity of the graph traversal. (B – G) Maps sampled at representative steps along the graph traversal shown in (A). All maps are displayed at a contour level of 0.025 and in gyres view.

(A) Left: consensus map from 3D refinement in RELION of 43,698 particles, displayed at a contour level of 0.015 and in disc view. Regions of the map assigned to an interaction with the nucleosome acidic patch and to the macro domain are labeled (same panel as in Figure 3A). Right: same plot as in Figure 3A but sampling different steps along the same graph traversal. Dots indicate all latent space coordinates that are part of the calculated graph traversal, labeled red dots indicate coordinates of maps shown in panels (B) – (G). Black lines are a visual guide and do not represent actual connectivity of the graph traversal. (B) – (G) Maps sampled along the graph traversal shown in (A). All maps are displayed at a contour level of 0.013 and in disc view.

(A) Left: schematic of asymmetric nucleosomes PARylated by PARP1 with and without HPF1. Right: adding ALC1 and ATP to the PARylated FRET-labeled nucleosome results in sliding the histone octamer away from the short linker DNA end, resulting in a decrease of FRET. (B) Saturation curves of nucleosome sliding rates for 10 nM WT nucleosomes versus varying concentrations of ALC1fl. Nucleosome sliding assays were performed after PARylation by 80 nM PARP1 with 25 µM NAD+ in the absence (teal) or presence (orange) of 20 nM of HPF1. Points and error bars represent the mean and standard deviation, respectively, from three independent experiments. (C) Saturation curve of nucleosome sliding rates for 10 nM WT nucleosomes versus varying concentrations of ALC1fl R611A/S612A. Nucleosome sliding assays were performed after PARylation by 80 nM PARP1 and 20 nM HPF1 with 25 µM NAD+. Points and error bars represent the mean and standard deviation, respectively, from three independent experiments. (D, E) Saturation curves of nucleosome sliding rates for 10 nM APM (D) and gH4 (E) nucleosomes versus varying concentrations of ALC1fl. Nucleosome sliding assays were performed after PARylation by 80 nM PARP1 and 20 nM HPF1 with 25 µM NAD+. Points and error bars represent the mean and standard deviation, respectively, from three independent experiments. (F) ATPase activity for 2 µM ALC1fl (teal, orange, and green) or ALC1fl R611A/S612A (blue) in the presence of 250 nM 10-N-10 WT nucleosomes (teal, orange, and blue) or 10-N-10 gH4 nucleosomes (green), activated by PARylation by PARP1 with (orange, blue, and green) or without HPF1 (teal). Bars and error bars represent the mean and standard deviation, respectively, from three independent experiments.

Upon DNA damage, PARP1 or PARP2 and HPF1 deposit PAR chains on histones, causing the rapid recruitment of proteins containing PAR reader domains. Among these proteins, ALC1 selectively binds to PAR chains with its macro domain and recognizes the closest nucleosome by probing its acidic patch. Once the macro domain and linker region of ALC1 jointly recognize a PARylated nucleosome, the ATPase motor is released from their auto-inhibitory effect and can tightly bind to the nucleosomal DNA at SHL2 for productive remodeling. Preferential PARylation of target sites spatially closest to the DNA break causes preferential recruitment of ALC1 on the side of the nucleosome such that remodeling slides this nucleosome away from the break. By setting directionality in remodeling, this mechanism could promote exposure of the lesion to downstream repair factors.

(A) Representation of the histone complex (HisC) cluster in chromosome 2 of Drosophila melanogaster. The HisC consists of tandem arrays of ~100 replication-dependent histone gene units (His-GUs), each encoding the histone proteins H2A, H2B, H3, and H4 and the linker histone H1. (B) The histone deficiency that deletes the entire HisC from chromosome 2 (ΔHisC). Offspring that are homozygous for ΔHisC die at an early embryonic stage (Günesdogan et al., 2010). (C) Scheme of a cross between flies containing ΔHisC and a chromosome 2 balancer expressing GFP (GFP balancer, +). Transgene cassettes (orange boxes) were inserted in both arms of chromosome 3 and consist of three copies of WT His-GU (3×His-GUH3WT) or His-GU in which every histone H3K14 was substituted with arginine (3×His-GUH3K14R), providing a total of 12 copies of transgenic His-GUs. The genotypes of the offspring resulting from the cross are depicted below the arrow. (D) Quantification of the number of surviving embryos from three different genotypes and distinguished from heterozygous individuals by the absence of GFP. WT, N = 1,519; ΔHisC;12xHis-GUH3WT (here called H3WT), N = 2,183; ΔHisC;12xHis-GUH3K14R (here called H3K14R), N = 2,620. Error bars show standard deviation. (E) Stage 14 embryos immunostained for H3K14ac, GFP, and DE-cadherin (DCAD2, control staining). GFP expression distinguishes individuals with transgenic histones that are homozygous for endogenous histones (ΔHisC, GFP negative) from heterozygotes (GFP positive, ΔHisC/+). Nuclei are stained by DAPI. Scale bar, 100 μm. (F) Western blots in early first-instar larvae in the denoted genotypes. Nuclear protein extracts corresponding to approximately 25 larvae were loaded per lane. The graph shows quantification of H3K14ac levels relative to H3. The blot is representative of six independent biological replicates. Two-tailed, unpaired Student’s t test was applied to compare ΔHisC;H3WT with ΔHisC;H3K14R. p < 0.0001. (G) Volcano plot of differentially expressed genes between H3K14R and H3WT, identified by RNA-seq analysis (gray). Genes highlighted in blue and red are changed significantly (fold change ≥ 2-fold, FDR < 0.1) in comparison with H3WT and ΔHisC/+;H3K14R. Common up, n = 10; common down, n = 50. (H) Pie chart showing adult and larval tissues where each of the 50 downregulated genes has the highest expression. Data were retrieved from FlyAtlas and FlyAtlas 2 (Leader et al., 2018). N.A., data not available.

(A) Overlap analysis between unique H3K14ac peaks, no ChIP-seq peaks, no H3K14ac, and overlapped peaks with the 50 commonly downregulated genes in H3K14R embryos. (B, C, and E) Genome browser snapshots showing two downregulated genes (CG13492 and CG18585) and one unaffected gene (CG3309) with ChIP-seq tracks for H3K14ac, H3K9ac, H3K27ac, and H3K4me3 (immunoprecipitation [IP]/input) and RNA-seq signal (H3WT and H3K14R). All tracks show reads per million. (B’ and C’) In situ hybridization in stage 16 embryos (anterior to the left) using antisense probes for CG13492 and CG18585. Expression of these genes is restricted to the midgut. An asterisk marks the GFP antisense probe signal, which was used to distinguish between homozygous and heterozygous ΔHisC animals. Scale bars, 50 μm. (D) Mean RNA-seq expression fold change in H3K14R versus H3WT and H3K14R versus ΔHisC/+;H3K14R for genes associated with unique H3K14ac, no H3K14ac, or overlapped ChIP-seq peaks. Error bars show standard error. (F) ATAC-seq fold change in H3K14R versus H3WT stage 15 embryos at gene bodies of the 50 commonly downregulated genes, genes downregulated 1.5-fold, unchanged genes, and genes upregulated 1.5-fold in RNA-seq. Error bars show standard error. (G) ATAC-seq fold change at gene bodies with unique H3K14ac peaks, no H3K14ac peaks, and peaks overlapping other histone marks. H3K14ac unique versus no H3K14ac, p = 3.94E−111; H3K14ac unique versus overlapping peaks, p = 1.93E−129. Error bars show standard error. (H) CUT&Tag promoter (±100 bp around the TSS) fold change for RNA Pol II (Rpb3 antibody) occupancy in mutant versus WT (w1118) stage 15 embryos compared with RNA-seq data. Error bars show standard error. (I) RNA Pol II promoter fold change (CUT&Tag) at genes with unique H3K14ac peaks, no H3K14ac peaks, and peaks overlapping other histone marks. H3K14ac unique versus no H3K14ac, p = 5.1E−4; H3K14ac unique versus overlapping peaks, p = 4.3E−5. Error bars show standard error. Statistical tests were performed by two-sided t tests.

(A) Representation of a cell in mitosis that has replicated its DNA and undergoes FLP-mediated recombination between homologous chromosomes where an FRT sequence is present (pink box, chromosome 2) (i) and illustration of, from top to bottom, heterozygous cells that have not recombined and show intermediate levels of GFP (dark green), cells WT for the HisC that show strong levels of GFP (bright green, twin spots), and homozygous ΔHisC cells that do not express GFP (black, mutant clones) (ii). All cells contain 12 copies of H3WT or H3K13R transgenes inserted on chromosome 3. (B) Third-instar wing imaginal discs containing H3WT or H3K14R mutant clones (white arrows, no GFP expression) and twin spots (white arrowheads, strong GFP expression) stained for H3K14ac. (C) Wings from adult flies containing H3WT or H3K14R mutant clones and percentage of flies showing defects in the wing margin (lack of bristles, notches, and necrosis). N of flies counted: H3WT, 223; H3K14R, 221. Scale bar, 200μm. (D) Expression of Cut and Senseless in the wing disc of third-instar larvae with H3WT or H3K14R mutant clone cells (no GFP expression). (E) Expression of Patched and Spalt in the wing disc of third-instar larvae with H3WT or H3K14R mutant clone cells (no GFP expression). White arrows indicate regions where H3K14R or H3WT clones are observed. Merged panels show the expression of Cut, Senseless, Patched, or Spalt in mutant clones. Dashed squares indicate the region that is magnified in the rightmost panels in (D) and (E). Scale bars in (B), (D), and (E), 50 μm.

(A) Genomic distribution of H3K14ac ChIP-seq peaks in w1118 third-instar larvae wing disc. (B) Venn diagram indicating the number of total and unique H3K14ac ChIP-seq peaks and those overlapping H3K27ac (Vizcaya-Molina et al., 2018) and H3K4me2/3 (Schertel et al., 2015). (C) Pie charts showing the genomic distribution of unique H3K14ac peaks, overlapped peaks, and peaks with no H3K14ac. (D–F) Genome browser snapshots showing the ChIP-seq signal for H3K14ac, H3K27ac, and H3K4me2/3 (IP/input, except H3K27ac) in the H3K14 target genes (D) cut and senseless, (F) pebbled/hindsight, and in other active genes in the wing disc (E) patched, salr, and salm). All tracks show reads per million. (G) Projection image of Pebbled/Hindsight in the wing disc of third-instar larvae with H3WT or H3K14R mutant clone cells (no GFP expression). White arrows indicate regions where H3K14R or H3WT clones are observed. Asterisk points to a cell expressing Pebbled outside of the dorsal-ventral boundary. Dashed squares indicate the region that is magnified in the rightmost panels. Scale bar, 50 μm.

(A) Volcano plot showing Drosophila S2 cell nuclear extract proteins enriched in H3K14ac peptide affinity purification relative to unmodified H3 peptides. The −log10 Benjamini-Hochberg-corrected p value is plotted against the log2 fold change: H3K14ac/H3K14 unmodified peptide pull-down. Proteins with an FDR of less than 0.05 and a fold change of at least 100% are considered hits and labeled red. (B) Table listing proteins preferentially bound by the H3K14ac peptide and schematic drawings of the Brahma-associated protein (BAP) and Polybromo-BAP (PBAP) complexes in Drosophila. Subunits enriched by the H3K14ac peptide are labeled with a red star. (C) Affinity purification from w1118 embryo nuclear extracts using unmodified (K14un) or acetylated (K14ac) H3 peptide followed by western blot. The blot is representative of two independent biological replicates. (D) Fluorescence anisotropy of TAMRA-labeled unmodified or H3K14ac peptide in the presence of varying amounts of Brm. (E) Expression of Brm in the wing disc of w1118 (top) control and in nub-Gal4>UAS-brm-RNAi (bottom) third-instar larvae. The dashed line indicates the expression domain of Nub. (F) Expression of Cut, Senseless, Patched, and Wingless in the wing disc of nub-Gal4>UAS-brm-RNAi third-instar larvae. (G) Expression of Polybromo in the wing disc of w1118 (top) control and in nub-Gal4>UAS-polybromo-RNAi (bottom) third-instar larvae. (H) Expression of Cut, Senseless, Patched, and Wingless in the wing disc of nub-Gal4>UAS-polybromo-RNAi third-instar larvae. (I) H3K14ac and expression of Cut and Wingless in the wing disc of ptc-Gal4>UAS-gcn5-RNAi third-instar larvae. (J) Quantification of Cut and Wingless signal within and outside the ptc domain (dashed lines). (K) H3K14ac and expression of Cut and Wingless in the wing disc of ptc-Gal4>UAS-chameau-RNAi third-instar larvae. (L) Quantification of Cut and Wingless signal within and outside the ptc domain. (M) Cut and Wingless expression in H3K14R mutant clones. (i)–(iv) in (E)–(H) show the same wing disc. Scale bars in (E)–(M), 50 μm.

(A) Venn diagram indicating the number of total and unique H3K14ac ChIP-seq peaks and those overlapping H3K9ac, H3K27ac, and H3K4me3 in human mesenchymal stem cells (MSCs) from ENCODE data (Moore et al., 2020). (B) Genome browser snapshots showing two genes with H3K14ac unique peaks (NPY and GATA3) with ChIP-seq tracks for H3K14ac, H3K9ac, H3K27ac, and H3K4me3 (IP/input) and RNA-seq signal. All tracks show reads per million. (C) MSC genes with the unique H3K14ac chromatin state are more strongly expressed in these cells compared with human embryonic stem cells (ESCs) and neural stem cells (NSCs). Error bars show standard error. (D) Schematic model showing active genes containing canonical histone modifications associated with gene expression and H3K14-target genes that lack these promoter modifications and are decorated with H3K14ac in exons.

(A–D) ALC1−/− cells are sensitive to PARPi and MMS. (A) CRISPR-mediated inactivation of ALC1 in eHAP. Immunoblot of WCEs in ALC1+/+ and ALC1−/− cells, probed for ALC1. hnRNPA1 was used as a loading control. (B) Schematic representation of survival assays using CellTiter-Glo. (C and D) Reduced survival of eHAP ALC1−/− cells after treatment with indicated genotoxin. Data are mean ± SEM normalized to untreated cells (n = 3 biologically independent experiments). Solid lines show a nonlinear least-squares fit to a four-parameter dose-response model. (E and F) PARP trapping contributes to Olaparib sensitivity in ALC+/+ and ALC1−/− cells. (E) Immunoblot of WCEs versus chromatin in ALC1+/+ and ALC1−/− cells following indicated treatments, probed for ALC1, PARP1, and PARP2. α-tubulin was used as a loading control for cytoplasmic fraction. Histone H3 was used as a loading control for chromatin fraction (data are representative of n = 3 biologically independent experiments). (F) Rescue of Olaparib sensitivity in inducible CAS9 (iCAS9) ALC1+/+ and ALC1−/− eHAP-expressing PARP1 sgRNA following 72 h Dox induction. Data are mean ± SEM normalized to untreated cells (n = 3 biologically independent experiments). Solid lines show a nonlinear least-squares fit to a four-parameter dose-response model. (G) Rescue of Olaparib sensitivity in iCAS9 ALC1−/− eHAP-expressing PARP2 sgRNA following 72 h Dox induction. Data are mean ± SEM normalized to untreated cells (n = 3 biologically independent experiments). Solid lines show a nonlinear least-squares fit to a four-parameter dose-response model. (H) Rescue of Olaparib sensitivity in iCAS9 ALC1+/+ and ALC1−/− eHAP-expressing PARG sgRNA following 72 h Dox induction. Data are mean ± SEM normalized to untreated cells (n = 3 biologically independent experiments). Solid lines show a nonlinear least-squares fit to a four-parameter dose-response model. (I) Rescue of Olaparib sensitivity in iCAS9 ALC1+/+ and ALC1−/− eHAP-expressing 53BP1 sgRNA following 72 h Dox. Data are mean ± SEM normalized to untreated cells (n = 3 biologically independent experiments). Solid lines show a nonlinear least-squares fit to a four-parameter dose-response model. (J) Representative images (n = 3 biologically independent experiments) of clonogenic survival assays in ALC1+/+ and ALC1−/− iCAS9 cells expressing indicated sgRNA following 72 h Dox ± 250 nM Olaparib. (K) Quantification of clonogenic survival assays in ALC1+/+ and ALC1−/− iCAS9 cells expressing indicated sgRNAs following 72 h Dox ± 250 nM Olaparib. Data are mean ± SEM normalized to non-treated ALC1+/+ NT sgRNA (n = 3 biologically independent experiments). (L) Olaparib sensitivity is associated with defective nucleosome remodeling. ALC1+/+ and ALC1−/− eHAP transduced with indicated constructs. Data are mean ± SEM normalized to untreated cells (n = 3 biologically independent experiments). Solid lines show a nonlinear least-squares fit to a four-parameter dose-response model.

(A) Schematic of screening pipeline. (B) Volcano plot of p value versus log-fold change (LFC), iCAS9 eHAP NT gRNA non-treated versus NT gRNA + 250 nM Olaparib. (C) Immunoblot of WCEs in eHAP iCAS9 NT gRNA and ALC1 gRNA from 3 independent biological replicates following 144 h Dox, probed for ALC1; α-tubulin was used as a loading control. (D) Volcano plot of p value versus LFC, iCAS9 eHAP NT gRNA + 250nM Olaparib versus ALC1 gRNA + 250 nM Olaparib.

(A–E) Loss of ALC1 is synthetic lethal with HRD and leads to PARPi hypersensitivity. (A) Immunoblot of WCEs from DLD-1 WT and BRCA2−/− cells following transduction with LentiCRISPR NT sgRNA and ALC1 sgRNA and clonal selection (no BRCA2/ALC1 double knockouts were recovered), probed with BRCA2 and ALC1. α-tubulin was used as a loading control. (B) Olaparib colony survival in DLD-1 BRCA2+/+ ALC1+/+, BRCA2−/− ALC1−/−, BRCA2−/− ALC1+/+, and BRCA2−/− ALC1Low expression. Data are mean ± SEM normalized to untreated cells (n = 3 independent biological experiments). Solid lines show a nonlinear least-squares fit to a four-parameter dose-response model. (C) Survival in ALC1+/+ and ALC1−/− eHAP cells transfected with BRCA1-targeting short interfering RNAs (siRNAs). Cell survival was measured using CellTiter-Glo. Data are mean ± SEM normalized to ALC1+/+ cells (n = 3 independent biological experiments). (D) Survival in ALC1+/+ and ALC1−/− eHAP cells transfected with BRCA2-targeting siRNAs. Cell survival was measured using CellTiter-Glo. Data are mean ± SEM normalized to ALC1+/+ cells (n = 3 independent biological experiments). (E) Olaparib survival in ALC1+/+ and ALC1−/− eHAP transfected with non-targeting or BRCA2-targeting siRNAs. Data are mean ± SEM normalized to untreated cells (n = 3 independent biological experiments). Solid lines show a nonlinear least-squares fit to a four-parameter dose-response model. (F) Quantification of a crystal violet proliferation assay in parental and ALC1-deleted BARD1AID/AID cells ± IAA. Data are mean ± SD (n = 3 independent biological experiments). (G) Quantification of a crystal violet proliferation assay in parental and ALC1-deleted 53BP1−/−BARD1AID/AID cells ± IAA. Data are mean ± SD (n = 3 independent biological experiments). (H) ALC1+/+ and ALC1−/− eHAP cells transduced with indicated ALC1 constructs were transfected with non-targeting, BRCA1-targeting, or BRCA2-targeting siRNAs. Cell survival was measured using CellTiter-Glo. Data are mean ± SEM normalized to ALC1+/+ cells for each siRNA (n = 3 independent biological experiments). (I) Immunoblot of WCEs in ALC1+/+ and ALC1−/− iCAS9 eHAP cells transduced with ATM sgRNA following 72 h Dox, probed with antibodies against ALC1 and ATM. α-tubulin is used as a loading control. (J) Representative images (n = 3 biologically independent experiments) of clonogenic survival assays in ALC1+/+ and ALC1−/− iCAS9 cells expressing NT and ATM sgRNA following 72 h Dox ± 250 nM Olaparib. (K) Quantification of clonogenic survival assays in ALC1+/+ and ALC1−/− iCAS9 cells expressing NT sgRNA and ATM sgRNA following 72 h Dox ± 250 nM Olaparib. Data are mean ± SEM normalized to non-treated ALC1+/+ NT sgRNA (n = 3 biologically independent experiments). (L) Olaparib survival of ALC1+/+ and ALC1−/− iCAS9 cells transduced with NT sgRNA and ATM sgRNA following 72 h Dox. Data are mean ± SEM normalized to untreated cells (n = 3 independent biological experiments). Solid lines show a nonlinear least-squares fit to a four-parameter dose-response model. (M) Hazard ratio analysis of breast cancer patients from TGCA according to ALC1 and BRCA2 expression. (N) KM survival analysis of BRCA2low breast cancer patients from TGCA according to ALC1 expression. ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

(A–D) Knockdown of BRCA1/2 in ALC1−/− cells results in genome instability. (A) Representative micrographs of metaphase spreads in ALC1+/+ and ALC1−/− eHAP transfected with the indicated siRNA. (B) Quantification of the number of aberrant chromatids per metaphase in ALC1+/+ and ALC1−/− eHAP transfected with indicated siRNA. Data are mean ± SEM (n = 3 independent biological experiments). (C) Quantification of number of micronuclei per primary nucleus from ALC1+/+ and ALC1−/− eHAP cells transfected with non-targeting, BRCA1-targeting, and BRCA2-targeting siRNAs. Data are means from individual experiments; bar represents median (n = 3 independent biological experiments). (D) Lower: scheme of the nucleotide labeling and S1 nuclease treatment strategy used for gap detection at the replication fork. Upper: Representative DNA fiber immunofluorescence images from ALC1−/− eHAP cells transfected with the indicated siRNAs and treated or not with S1 nuclease.Scale bars represent 100 μm. (E) Boxplot showing mean IdU/CldU ratio in ALC1+/+ and ALC1−/− eHAP transfected with the indicated siRNAs and treated or not with S1 nuclease. Data from 500–600 fibers/condition are represented as mean ± SD (2 technical replicates from 2 independent biological experiments). (F) Representative micrographs of ALC1+/+ and ALC1−/− eHAP cells transfected with non-targeting siRNAs stained with RAD51 antibody, EdU click-iT, and DAPI. Scale bar, 10 μm. (G) Quantification of nuclear RAD51 foci in CSK pre-extracted EdU+ and EdU− ALC1+/+ and ALC1−/− eHAP cells transfected with indicated siRNAs 72 h following knockdown. Data are means from individual experiments; bar represents median (n = 3 biologically independent experiments). (H) Immunoblot of WCEs in ALC1+/+ and ALC1−/− cells transfected with non-targeting, BRCA1-targeting, or BRCA2-targeting siRNAs, probed with ATM pSer1981, ATM, CHK2, CHK1-pSer345, total CHK1, RPA-pSer33, total-RPA, γH2AX, PARP1, cleaved caspase-3, BRCA2, BRCA1, and ALC1. α-tubulin was used as a loading control. ns, p > 0.05; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

(A) ALC1+/+ and ALC1−/− eHAP are not sensitive to dU. Data are mean ± SEM normalized to untreated cells (n = 3 independent biological experiments). (B) ALC1−/− cells are sensitive to formyl-dU. ALC1+/+ and ALC1−/− eHAP transduced with indicated constructs. Data are mean ± SEM normalized to untreated cells (n = 3 biologically independent experiments). (C) Immunoblot of WCEs in ALC1+/+ and ALC1−/− cells transduced with indicated constructs, probed for ALC1. α-tubulin was used as a loading control. (D and E) SMUG1 knockout rescues ALC1-dependent formyl-dU sensitivity. (D) Immunoblot of WCEs in ALC1+/+ and ALC1−/− iCAS9 cells transduced with NT sgRNA and SMUG1 sgRNA following 72 h Dox, probed with ALC1 and SMUG1. Ponceau was used as a loading control. (E) Formyl-dU survival of ALC1+/+ and ALC1−/− iCAS9 cells transduced with NT sgRNA and SMUG1 sgRNA following 72 h Dox. Data are mean ± SEM normalized to untreated cells (n = 3 independent biological experiments). (F) Formyl-dU survival of ALC1+/+ and ALC1−/− eHAP cells transfected with non-targeting or BRCA2-targeting siRNAs. Data are mean ± SEM normalized to untreated cells (n = 3 biologically independent experiments). (G) Formyl-dU survival of ALC1+/+ and ALC1−/− eHAP cells expressing NT or UBC13 sgRNA following 72 h Dox. Data are mean ± SEM normalized to untreated cells (n = 3 biologically independent experiments). (H) Formyl-dU survival of ALC1+/+ and ALC1−/− eHAP cells expressing NT or ATM sgRNA following 72 h Dox. Data are mean ± SEM normalized to untreated cells (n = 3 biologically independent experiments). (I) Is PARylation upstream of formyl-dU (red star) removal by SMUG1? (J) PARylation by PARPs occurs downstream of SMUG1. Immunoblot of WCEs in ALC1+/+ SMUG1+/+, ALC1+/+ SMUG1−/−, ALC1−/− SMUG1+/+, and ALC1−/− SMUG1−/− cells with indicated treatments, probed for ALC1, SMUG1, and anti-PAR binding reagent. α-tubulin was used as a loading control. (K) Schematic illustrating (BER) repair of formyl-dU (red star). The monofunctional glycosylase SMUG1 catalyzes the removal of formyl-dU, creating an abasic (AP) site. The endonuclease APEX1 catalyzes the incision of the DNA backbone, leaving a 5′-deoxyribose phosphate. (L) Immunoblot of WCEs in cells with indicated genotypes probed for ALC1 and APEX1. α-tubulin was used as a loading control. (M) Formyl-dU survival of ALC1+/+ APEX1+/+, ALC1+/+ APEX1−/−, ALC1−/− APEX1+/+, and ALC1−/− APEX−/− cells. Data are mean ± SEM normalized to untreated cells (n = 3 independent biological experiments). (N) MMS survival of ALC1+/+ APEX1+/+, ALC1+/+ APEX1−/−, ALC1−/− APEX1+/+, and ALC1−/− APEX1−/− cells. Data are mean ± SEM normalized to untreated cells (n = 3 independent biological experiments). (O) Olaparib survival of ALC1+/+ APEX1+/+, ALC1+/+ APEX1−/−, ALC1−/− APEX1+/+, and ALC1−/− APEX1−/− cells. Data are mean ± SEM normalized to untreated cells (n = 3 independent biological experiments). Solid lines show a nonlinear least-squares fit to a four-parameter dose-response model. (P) Is incision by APEX1 required for ALC1 recruitment? (Q) ALC1 recruitment is upstream of incision by APEX1. Immunoblot of CSK chromatin fractionation in cells with indicated genotype ± formyl-dU treatment, probed for ALC1, PARP1, SMUG1, and APEX1. α-tubulin and histone H3 were used as loading controls. (R) Representative images (n = 3 biologically independent experiments) of clonogenic survival assays in ALC1+/+ APEX1+/+, ALC1+/+ APEX1−/−, ALC1−/− APEX1+/+, and ALC1−/− APEX1−/− eHAP cells transfected with non-targeting or BRCA2-targeting siRNAs ± 50 nM Olaparib. (S) Quantification of clonogenic survival assays in ALC1+/+ APEX1+/+, ALC1+/+ APEX1−/−, ALC1−/− APEX1+/+, and ALC1−/− APEX1−/− eHAP cells transfected with non-targeting or BRCA2-targeting siRNAs ± 50 nM Olaparib. Data are mean ± SEM normalized to non-treated ALC1+/+ APEX1+/+ (n = 3 biologically independent experiments).

(A and B) Processing of endogenous lesions by SMUG1 does not drive synthetic lethality with BRCA1/2 in ALC1-deficient cells. (A) Immunoblot of WCEs in ALC1+/+ and ALC1−/− iCAS9 cells transduced with NT sgRNA or SMUG1 sgRNA following 72 h Dox and transfected with non-targeting or BRCA1/2-targeting siRNAs, probed with ALC1, BRCA1, BRCA2, and SMUG1. Ponceau was used as a loading control. (B) Survival in ALC1+/+ and ALC1−/− iCAS9 cells transduced with NT sgRNA or SMUG1 sgRNA following 72 h Dox and transfected with non-targeting or BRCA1/2-targeting siRNAs. Cell survival was measured using CellTiter-Glo. Data are mean ± SEM normalized to ALC1+/+ cells (n = 3 independent biological experiments). (C–E) The monofunctional glycosylase MPG drives MMS sensitivity in ALC1-deficient cells. (C) The monofunctional glycosylase MPG catalyzes the removal of alkylated bases, creating an abasic (AP) site. (D) Immunoblot of WCEs in ALC1+/+ and ALC1−/− eHAP transfected with non-targeting or MPG-targeting siRNAs, probed for ALC1 and MPG. α-tubulin was used as a loading control. (E) MMS survival of ALC1+/+ ALC1−/− eHAP cells transfected with non-targeting or MPG-targeting siRNAs. Data are mean ± SEM normalized to untreated cells (n = 3 independent biological experiments). (F–H) Processing of endogenous lesions by MPG contributes to synthetic lethality with BRCA1/2 in ALC1-deficient cells. (F) Immunoblot of WCEs in ALC1+/+ and ALC1−/− eHAP transfected with non-targeting, MPG, BRCA1/2-targeting siRNAs, probed with ALC1, BRCA1, BRCA2, and MPG. α-tubulin was used as a loading control. (G) Representative images (n = 3 biologically independent experiments) of clonogenic survival assays in ALC1+/+ and ALC1−/− eHAP cells transfected with non-targeting, MPG, and BRCA1/2-targeting siRNAs. (H) Quantification of clonogenic survival assays in ALC1+/+and ALC1−/− eHAP cells transfected with non-targeting, MPG, and BRCA1/2-targeting siRNAs. Data are mean ± SEM normalized to non-treated ALC1+/+ (n = 3 biologically independent experiments).

(a) FluoRBT combines rotor bead tracking and smFRET. Top left: schematic illustrating an experiment with a DNA hairpin (left) and a B-form to Z-form transition (right). Top right: correlated torque and FRET changes upon B to Z transition in response to twist. Bottom: example DNA hairpin data showing correlated FRET changes and rotor bead movements upon hairpin opening and closing. Adapted with permission from Ref. [37••]. (b) Three-color smFRET sheds light on the coordination of DNA movements during chromatin remodeling. Left: a schematic of the experiment showing the nucleosome labeling scheme and alternating laser excitation used to simultaneously resolve entry-side and exit-side FRET. Dotted circles represent the initial positions of DNA labels, while the arrows indicate the direction of DNA movements during remodeling. Note that the green dye is not excited by the red laser. Right: an example of a remodeling three-color FRET trace that shows the coordination between entry-side and exit-side DNA movements during remodeling. Entry side movement starts first, while exit side moves after an ATP-dependent delay designated tlag. Adapted with permission from Ref. [45•]. (c) Multi-color single-molecule fluorescence microscopy is used to study the transition from initiation to elongation during translation. Top left: cartoon schematic of the ribosome labeling scheme. Top center: ribosome states observed in the experiment. Top right: overlay of the initiation and elongation complexes resolved by cryo-EM. Bottom: example four-color single-molecule fluorescence time trace capturing the transition from the pre-initiation to the elongation complex. Note that 60S fluorophore undergoes photobleaching during the course of the recording. Adapted with permission from Ref. [49••]

(a) Cryo-EM and smFRET were used to establish a conformational pathway leading to the activation of Cas12a upon target binding. Top: Cas12a conformations observed by cryo-EM grouped according to their corresponding FRET states. Bottom: a schematic of the FRET labeling scheme (left) and an example FRET trace demonstrating transitions between different Cas12a conformations. Adapted with permission from Ref. [51••]. (b) The combination of smFRET and structural information was used to uncover the mechanism of transcription inhibition by the antibiotic Lipiarmycin A3. Top: cryo-EM structure of RNA polymerase in complex with Lipiarmycin A3 (red). Bottom: FRET traces demonstrating how RNA polymerase conformational distribution changes upon drug binding. Adapted with permission from Ref. [52••]. (c) Combination of structural and dynamic information was used to clarify the mechanism of histone exchange by SWR1 chromatin remodeler. Top: cryo-EM structure of the SWR1-nucleosome complex demonstrating DNA unwrapping from the nucleosome and the FRET labeling scheme used to probe unwrapping in solution. Bottom: a FRET trace demonstrating nucleosome unwrapping dynamics in the presence of SWR1 and ATP-γ-S. Adapted with permission from Ref. [57••].

a, Left, schematic of DNA constructs with two outer O1 sites (orange), indicating the possible bound states of LacI–R. Top and bottom O1, Alexa Fluor 750 (purple) and Cy5 (red) acceptor fluorophores, respectively. Right, donor fluorescence (green), bottom- (red) and top (purple)-site acceptor fluorescence, as well as bottom (dark blue) and top site (light blue) FRET traces showing the transition of a single LacI dimer initially bound at the bottom site (shaded green) switching to the top site (shaded purple). In the absence of acceptor signals, FRET is set to zero (dashed). b, Left, schematic of DNA constructs with two outer O1 sites (orange) and intervening random (ran) DNA (left) or an additional O3 (brown) (middle) or O1 (right) site. Top and bottom O1, Alexa Fluor 750 (purple) and Cy5 (red) acceptor, respectively. Right, hidden-Markov-model-derived switching rates (Extended Data Fig. 3) are mean ± 95% confidence intervals (n = 1,108, 1,137 and 699 individual molecules from left to right) from at least 10 independent experiments for each construct.

a, Schematic for flow-stretching (top left). Kinetic series of images in horizontal and vertical emission polarization, showing two representative sliding LacI–R molecules when DNA is stretched in the vertical (bottom left) or horizontal (right) direction. Fifty-two and 27 sliding molecules were captured for the vertical and horizontal direction, respectively. b, Schematic of SMCT–FCS setup. Black arrows, direction of communication; green dots, laser pattern during fluorophore (red dot) tracking. c, Simulated traces (inset) of faster (red) and slower (blue) dye rotation and the resulting autocorrelations. PC, personal computer; FPGA, field programmable gate array. d, x (DNA direction) and y coordinate of sliding LacI–MBP–R (blue, 151 molecules) and LacI–R (red, 90 molecules). Ten representative traces are shown (Extended Data Fig. 6a). e, f, Mean normalized autocorrelation of the fluorescence for sliding (e) (n = 86 and 54 informative autocorrelations for LacI–MBP–R and LacI–R, respectively) and stationary (f) (n = 2,273 and 1,064 informative autocorrelations for LacI–MBP–R and LacI–R, respectively) LacI–MBP–R (blue) and LacI–R (red). Diffusion constants are 20% trimmed mean ± s.e.m. g, h, Difference in autocorrelation between LacI–MBP–R and LacI–R for sliding (g) (n = 86 and 54 informative autocorrelations for LacI–MBP–R and LacI–R, respectively) and stationary (h) (n = 2,273 and 1,064 informative autocorrelations for LacI–MBP–R and LacI–R, respectively) molecules. Data in e–h are mean ± s.e.m. Black line in g, best fit to a rotation-coupled sliding model; bootstrapping the trajectories yields an average pitch (p) for the rotation of 39 ± 9 bp (mean ± s.e.m.). i, Theoretical dependence of the pitch on the time lag of maximum difference in the autocorrelations. Yellow area, time regime corresponding to pitch values within the s.e.m of the experimentally measured pitch. j, Best fits of the rotation-coupled sliding model when the pitch is constrained at different levels.

a, Schematic of target search in which LacI combines faithful DNA-groove tracking (1D diffusion) with short and frequent intergroove hops that can bypass a specific binding site (orange). b, The effective rotational (Dr) and translational (D1) diffusion coefficients from SMCT–FCS are used to set parameters (khop(xhop)) in the simulations of the FRET experiments. c, d, Flipping (c) and switching (d) rates calculated from stochastic simulations as a function of the model parameters. Dashed lines, experimentally observed rate of flipping on the same operator (c) and switching between two outer operators with an intervening operator (d). e, Parameter probability densities from experimental uncertainties. Parameters compatible with observed switching (blue) and flipping (orange) rates. Surface transparency scaled according to the probability density for the parameters given the experimental data. The flipping rate surface does not decrease with increasing values of koff,μ, as the simulations yield the maximum possible flipping rate. kon,μ was infinite in these simulations; see Extended Data Fig. 8 for finite values of kon,μ.

(A) Toeprint assay on +42 tisB mRNA. Inclusion of 30S, 30S depleted of S1 (ΔS1), and 30SΔS1 complemented with a 3-fold excess of S1, is indicated, as is the presence of tRNAfMet. Arrow: +15 relative to AUG. (B) In vitro translation of +42 3xFLAG-tisB mRNA, with 70S, 70S depleted of S1 (ΔS1), or 70SΔS1 complemented with 3-fold excess of S1. For controls, the presence of IstR-1 is indicated. TisB protein was detected by Western blot. (C) Fluorescence anisotropy variation of 3′-end FAM-labeled +42 mRNA (5 nM) with 30S, 30S depleted of S1 (30S∆S1), or 30S∆S1 complemented with S1 (all at 150 nM). *T-test P values < 0.05. (Bottom) Schematic representation of competition effects on fluorescence anisotropy (see also Fig. 1D). The purple star indicates the location of the 3′-end FAM dye.

5′-end FAM-labeled +42 tisB mRNA (10 nM) was incubated with increasing concentrations of 30S subunits (A) or of protein S1 (C). Curve fitting was done as in Fig. 1. (B) Competition experiments with 5′-end FAM-labeled +42 tisB mRNA (10 nM). 30S subunits were at 150 nM, and additions of IstR-1 (200 nM) or competitor mRNAs (500 nM) are indicated. (D) As experiment in B, but with protein S1 (80 nM) instead of 30S subunits. Calculations are based on triplicates. *T-test P value < 0.05; **P value < 0.005.

(A) Read coverage obtained by deep sequencing of 30S- or S1-protected tisB mRNA fragments. The footprints near the 5′ end, at the standby site, as well as upstream of and within the ORF are highlighted with double-head arrows (purple, blue, red, and black, respectively). (B) Secondary structure of the +42 tisB mRNA with the standby site highlighted (blue), RBS (green), and the tisB ORF (black), and S1 and 30S footprint coverage (from A) are indicated with double-head arrows (same color code).

(A) Schematics of the truncated tisB mRNAs. 5′UTR (gray), standby site (blue), ORF (black), and RBS (green). (B) Toeprint assay on the mRNAs as indicated, incubated alone, with 30S, or 30S+tRNAfMet. The toeprint signal (+15 nt) is indicated, as are AUG and SD positions. (C, Left) In vitro translation of +42 tisB mRNA variants in the presence of S35-Met. Lane “ct”: no mRNA added; asterisk: unspecific label transfer to protein in the extract. See SI Appendix, SI Materials and Methods. (C, Right) Quantitation of band intensities from the gel. The intensity of TisB (+42 mRNA) was set to unity. (D) Competition experiments using 5′-end FAM-labeled +42 tisB mRNA (10 nM) incubated with 80 nM of S1 in the presence or absence of a 50-fold molar excess of the unlabeled mRNAs indicated. (E) Same set-up as in D, but with 30S (150 nM) replacing S1. Anisotropy change calculated from triplicates. *T-test P values < 0.05; **P value < 0.005.

a Cy3 (green), Cy5 (red), and Alexa750 (purple) fluorescence and FRET time traces (exit-side: dark blue; entry-side: light blue) showing sliding of a single nucleosome, after addition of 300 nM Chd1 and 100 μM ATP. The lag time between the onset of entry- and exit-side DNA movements is denoted as tlag. Time traces were recorded with alternating 532 nm and 638 nm laser excitation, as indicated by the areas shaded in green and red, respectively. b Histograms of tlag values (red bars) observed with 100 or 500 μM ATP, fit with a single-exponential distribution (N = 185–220 events). c Dependence of 1/tlag on the ATP concentration. Data are shown as the mean ± SEM (N = 40–340 events). d Left: Cartoon schematic showing the 601-flip nucleosome, with short exit DNA on the TA-poor side and long entry DNA on the TA-rich side of the 601 sequence. Middle: Two representative FRET time traces (exit-side: dark blue; entry-side: light blue) showing sliding of single 601-flip nucleosomes after addition of 300 nM Chd1 and 500 μM ATP. Right: Histogram of tlag values (red bars) from many 601-flip nucleosomes observed with 500 μM ATP, fit with a single-exponential distribution (N = 190 events). e Nucleosome sliding by SNF2h shows the same coordination of entry/exit DNA movements. Middle: Two representative FRET time traces (exit-side: dark blue; entry-side: light blue) showing sliding of single nucleosomes after addition of 1 μM SNF2h and 10 μM ATP. Right: Histogram of tlag values (red bars) constructed from 226 events, fit to a single-exponential distribution (black line). Source data are provided as a Source Data file

a Model of the nucleosome-Chd1 structure (pdb code: 5O9G [ref. 28.]) showing the position of the ATPase motor (brown) relative to 2 nt ssDNA gaps (black) that limit nucleosome sliding. b Native gel electrophoretic mobility assay comparing sliding of nucleosomes with ssDNA gaps at m = 3, m = 5, or m = 8 nt from the entry-side SHL2. Reactions contained 150 nM nucleosome, 200 nM Chd1 and 1 mM ATP. The supershifted bands at the top of each gel represent Chd1-nucleosome complexes. Gels are representative of two or more independent replicates. c Schematic depicting site-specific cross-linking positions on the nucleosome. Using nucleosomes containing two cysteine substitutions, three regions were simultaneously followed. Nucleosome sliding by Chd1, which shifts the histone core toward the side with longer linker DNA (to the right), can be observed by corresponding shifts of DNA cross-linking sites. d, e Time-course experiments showing Chd1-dependent changes in DNA positioning of nucleosomes containing gaps at m = 5 bp (d) and m = 8 bp (e). At each cross-linking site, stacked intensity plots showing time-dependent changes in the distribution of cross-links are given on the left, and the relative intensities of starting versus shifted cross-linking products are plotted on the right. Cross-linking products that rapidly appeared and differed depending on the location of the gap are indicated in orange. Products that formed more slowly over time, which correspond to the preferred phase of the 601 sequence, are shown in blue. Note that consistent with previous work54, cross-linking on the exit side did not show a nucleosome population shifted by +11 bp (blue), likely due to sequence bias in cross-linking. Reactions contained 150 nM nucleosome, 200 nM Chd1, and 1 mM ATP. The time-course for these experiments was 0”, 7”, 15”, 30”, 45”, 60”, 90”, 2’, 4’, 8’, 16’, and 32’. Results are consistent across three separate experiments, two of which use similar time courses. Gel images are shown in Supplementary Fig. 6. Source data are provided as a Source Data file

The remodeller ATPase (brown), located at SHL2, initiates nucleosome sliding on un-shifted DNA (blue). Cycles of hydrolysis translocate DNA at the SHL2 site, drawing DNA onto the entry side of the nucleosome and pushing DNA towards the exit side in ~1 bp steps (shifted DNA shown in red). While some segments of nucleosomal DNA immediately twist to convey the additional bp, other segments absorb the translocated DNA, resulting in local twist defects (+) on the nucleosome. Creation of these twist defects delays the movement of DNA to the exit side.

(A) Left: SAXS data for ALC1fl. Right: same data plotted as q2 × I versus q. (B) Domain homology models. (C) Representative SAXS-derived rigid-body models of ALC1fl (left: ALC1fl-MC, right: ALC1fl-MN), superimposed with the same representative ab initio molecular envelope (gray). Comparisons of the experimental (black dots) with the theoretical scattering (teal) are shown in the right panel. (D) 2D class averages from cryo- and negative stain (“NS”) EM. (E) Theoretical projections of the SAXS-based models (low-pass filtered to 15 Å) in the approximate orientations as shown in (D). (F) Inter-domain cross-links mapped onto the ALC1fl-MC and ALC1fl-MN structures. Lines are colored green versus red for plausible versus non-plausible cross-links.

The SNF2 ATPase (Liu et al., 2017) aligned onto that of ALC1fl-MC (left panel) or ALC1fl-MN (right panel) using the N-lobe or the C-lobe as a guide, respectively. The ATPase lobe of SNF2 used as a guide for alignment is not shown.

(A) Reconstitution of an ALC1cat:ALC1macro complex (ALC1complex). Top: analytical gel filtrations. Bottom: SDS-PAGE of the peak fractions. (B) SDS-PAGE of ALC1complex after Ni-NTA purification. 6xHis-tag bearing ALC1cat was incubated with a version of ALC1macro that lacks the 6xHis-tag (ALC1macroΔ6xHis) and purified using Ni-NTA resin. (C) Co-immunoprecipitation (coIP) assay with plasmids expressing FLAG-tagged macro domain (FLAG-macro), YFP, or an YFP-tagged version of the ATPase (YFP-ALC1Δmacro). Cells were grown in the presence or absence of olaparib, as indicated. Cell lysates (input) were immunoprecipitated with α-FLAG. Immunoblotting was carried out with antibodies as indicated.

(A) Difference plot of HDX data from ALC1fl in the presence and absence of PARylated PARP1. Experimental error is shown in gray, and areas of increased exposure and protection are shaded in red and blue, respectively. Different line colors show different D2O incubation times. Non-covered sequence (Figure S5C) is omitted. (B) Comparison of intra-ALC1 cross-links in non-activated or activated ALC1fl. Gray, green, and magenta lines indicate cross-links that are detected in both cases, only with activated, or only with non-activated ALC1fl, respectively. (C) SAXS-based ab initio and rigid-body models of ALC1cat. (D) The ATPase of SNF2 (Liu et al., 2017) aligned onto that of ALC1cat (cartoon) and of ALC1fl-MC (lines), using the N-lobe as a guide (not shown for SNF2). (E) SAXS-derived pairwise interatomic distance distribution for ALC1cat in the apo (orange) and DNA-bound (green) state, scaled such that the area under the curve matches the observed particle volume.

(A) Macro domain placement against the juxtaposed ATPase lobe. Surfaces are colored on a red-white-blue gradient from −2 kT/e to +2 kT/e, as calculated by Adaptive Poisson-Boltzmann Solver (APBS) (Baker et al., 2001, Dolinsky et al., 2007). Cyan sphere: PAR-binding site, from an alignment with the Af1521 macro domain (Karras et al., 2005). (B–E) CoIP assays to probe macro domain-ATPase motor interactions. As indicated, cells were transfected with plasmids expressing mutated (B) or WT (C) FLAG-tagged macro domain (FLAG-macro), YFP-tagged ATPase motor (YFP-ALC1Δmacro) bearing mutations on the C-terminal (D) or N-terminal (E) ATPase lobe, YFP-tagged ALC1 lacking both macro domain and the N-terminal ATPase lobe (YFP-ALC1ΔNΔmacro) (E), and/or YFP-tagged intact ALC1 bearing macro domain mutations (C). Cells were grown in the presence or absence of olaparib, as indicated. Cell lysates were immunoprecipitated with α-FLAG. The cell lysates (input) and immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. The asterisk denotes a non-specific band.

(A) ATPase activities of ALC1fl WT (teal), R857Q (green), R857E (magenta), and R860W (brown). (B) Remodeling by ALC1fl WT (same as in Figure 1C) compared to R857Q (left), R857E (middle), and R860W (right) in the presence or absence of PARP1/NAD+. Bar graph: relative rates for ALC1fl WT (teal), R857Q (green), R857E (magenta), and R860W (brown). (C) Representative images of U2OS cells expressing WT, R857Q, R857E, and R860W YFP-ALC1, taken at the indicated early time points following laser damage. (D) Kinetics of YFP-ALC1 (left) and YFP-ALC1 R860W (right) association with DNA breaks as quantified from (C). (E) Half-hour time course with representative images of U2OS cells expressing WT, R857Q, R857E, and R860W YFP-ALC1, taken at indicated time points following laser damage. (F) Kinetics of WT, R857Q, R857E, and R860W YFP-ALC1 association with/dissociation from DNA breaks as quantified from (E). (G) Kinetics of YFP-ALC1 R857Q and R857E association with DNA breaks. (H) Kinetics of WT YFP-ALC1 dissociation from DNA breaks. Error bars ± SEM; τ: time constant; scale bars, 10 μm.

a, Domain architecture of WT and AutoN-2RA (R142A and R144A) Snf2h. b, Ensemble remodelling time courses of [WT H4, 78 bp], [WT H4, 40 bp] and [H4Δ1–19, 78 bp] nucleosomes by 40 nM WT ACF (black/grey lines, duplicated from Fig. 1b) and AutoN-2RA ACF (blue/cyan symbols) at 5 µM ATP. c, Dependence of the pause-phase exit rate on the linker DNA length and H4 tail for WT (black) or AutoN-2RA ACF (blue/cyan). *Too slow to be measured. d, Effect of the exogenously added H4-tail peptide on the pause-phase exit rates during remodelling by WT ACF. e, Pause-phase exit rates of nucleosomes lacking the H4 tail during remodelling by WT ACF. In c–f, [ACF] = 10 nM and [ATP] = 20 μM, except that 2 mM of ATP was used in e to make the pause exit rates measurable for nucleosomes lacking the H4 tail. Data are mean ± s.e.m. from at least 100 (c, d) or at least 50 (e) individual nucleosome remodelling traces from three independent experiments.

a, Domain architecture of WT and ΔN-term (residues 1–371 deleted) Acf1. b, Fluorescence anisotropy of dye-labelled H4-tail peptide in the presence of varying amounts of WT (black symbols) or ΔN-term (green symbols) Acf1. Data are mean ± s.e.m. (n = 3 independent experiments). The dissociation constant (Kd) for WT Acf1 is 3 ± 9 nM (error bars, 95% confidence intervals). c, Ensemble remodelling time courses of [WT H4, 78 bp], [WT H4, 40 bp] and [H4Δ1–19, 78 bp] nucleosomes by 40 nM WT ACF (black/grey lines, duplicated from Fig. 1b) and ΔN-term ACF (green/light green symbols) at 5 µM ATP. d, Dependence of the pause-phase exit rate on the linker DNA length for WT ACF (black) or ΔN-term ACF (green). [ACF] = 10 nM and [ATP] = 20 μM. Data are mean ± s.e.m. derived from at least 100 individual nucleosome remodelling traces from three independent experiments. e, Crosslinking of the H4 tail to Acf1 depends on the linker DNA length. The crosslinking products were analysed by SDS–PAGE (left). The Acf1-H4 crosslinking band was absent for ACF without nucleosomes (lane ‘ACF−nucleosomes’). Right: quantification of the H4-crosslinked fractions of Acf1 and Snf2h as a function of linker DNA length. Data are mean ± s.e.m. (n = 3 independent crosslinking experiments). f, Effects of deletion of Itc1 (Acf1 homologue) and its N-terminal region on the growth of yeast cells. Top row: WT. Second row: the itc1 gene is deleted (Δitc1). Third row: the coding sequence of the N-terminal region of Itc1 is deleted (Δitc1-Nterm). Bottom row: the remaining portion of itc1 is deleted from Δitc1-Nterm (rescue-Δitc1). One representative of three independent growth experiments is shown. g, Model for linker DNA length sensing by the ACF complex. DNA: grey lines; histone octamer: beige cylinders; Snf2h: blue/cyan; Acf1: green. The ATPase domain of Snf2h is depicted as a cyan sphere and labelled ‘On’ when active and ‘Off’ when inactive.

(A) Remodeling of nucleosomes with initial exit-linker DNA length of n = 3 bp or n = −3 bp by ISW2. Left: FRET time trace showing ISW2-induced translocation of a single n = 3 bp nucleosome. 6.2 nM ISW2 and 2 μM ATP were added at time zero. Middle: Histogram of the FRET values at translocation pauses constructed from n = 3 bp nucleosomes. Right: FRET histograms of the translocation pauses constructed from n = −3 bp nucleosomes. (B) Remodeling of the n = 3 bp and n = −3 bp nucleosomes by ISW1b. FRET histograms of the pauses for n = 3 bp (left) and n = −3 bp (right) nucleosomes in the presence of 8.8 nM ISW1b and 10–150 μM ATP. (C) Remodeling of the n = 3 bp and n = −3 bp nucleosomes by Isw2p. FRET distribution of the pauses for the n = 3 bp (left) and n = −3 bp (right) nucleosomes in the presence of 69 nM Isw2p and 1 mM ATP. The nucleosome schemes display the footprint of the histone octamer (yellow oval) on the DNA (blue line). Numbers above double-headed arrows shown in the histograms represent mean step sizes.

(A) FRET time trace of a nucleosome indicating the dwell times of the first two translocation phases (t1 and t2). (B) Histogram of t1 and t2 values constructed from many nucleosomes. Fits to the Γ-distribution AtN−1exp(-kt) (black lines) yield N = 6.5 ± 0.4 and k = 0.52 ± 0.04 s−1 for the t1 phase (left) and N = 3.4 ± 0.4 and k = 0.67 ± 0.11 s−1 for the t2 phase (right). (C) FRET time trace, before (gray) and after (blue) 5-point averaging, of a single n = 3 bp nucleosome in the presence of 6.2 nM ISW2, 2 μM ATP, and 2 mM ATP-γ-S. A HMM fit is shown by the red line. The horizontal orange dotted lines indicate 1 bp intervals (derived from the calibration in Figure S1D). (D) Histograms of FRET plateaus from many nucleosomes determined with the HMM analysis. Numbers above double-headed arrows represent mean step sizes.

(A) The pause duration after the first, 7 bp compound step (tp,7) at various ATP-γ-S concentrations and 2 μM ATP. (B) The pause duration between each 1 bp elementary step (tp) at various ATP-γ-S concentrations and 2 μM ATP. All pauses except for the ones after 6, 7, and 8 bp of translocation were pooled to determine tp. As shown in Figure S4, in addition to the 7th pause, the 6th and 8th pauses also appear longer than the remaining ones, likely due to errors in pause identification. The value of tp at 0 μM ATP-γ-S was derived from 1/k value obtained from the Γ-distribution in Figure 3B. (C) Dependence of tp on the ATP concentration at 2 mM ATP-γ-S. At this saturating concentration of ATP-γ-S, all pauses, including the 7th one, had approximately equal durations and were pooled to determine tp. All data are shown as the mean ± standard error of the mean (SEM) (N = 15–100 events)

(A) Schematic of the nucleosome construct used for measuring DNA movement at the entry side. (B) Donor signal (green), acceptor signal (red), and FRET (blue) time traces showing ISW2-induced remodeling after adding 12 nM ISW2 and 2 μM ATP at time zero. The dashed line indicates the onset of FRET change after an initial wait time, twait. (C) Comparison of twait on the entry (yellow bars) and exit sides (purple bars) of the nucleosome under identical enzyme and ATP concentrations. Data are shown as the mean ± SEM (N = 80–220 events).

(A) Schematic of the nucleosome constructs used to monitor DNA movement at the entry side when exit-side translocation is restricted by a 2 nt ssDNA gap. The gap is located m bp away from the SHL2 site (shown as a purple line) such that m bp of DNA can be translocated to the exit side. (B) FRET time traces of single m = 0, 7, and 8 bp nucleosomes (blue, green, and orange lines, respectively) after addition of 12 nM ISW2 and 2 μM ATP at time zero. (C) FRET values before (red bar) and after (blue bars) remodeling by ISW2 as a function of the distance m to the SHL2 site. Because the DNA path on the entry side may involve bending and/or twisting due to the direct interaction with the remodeling enzyme, we do not expect a similar linear dependence of FRET on the linker DNA length as on the exit side where the linker DNA is largely free of enzyme-induced distortion. Data are shown as the mean ± SEM (N = 80–150 nucleosomes).

DNA, histone octamer, and remodeler are shown in black/gray/red, yellow, and blue/green, respectively. The upper and lower DNA gyres are depicted as solid black and dashed gray lines, respectively. Each base pair of DNA translocated to the exit side is shown by a red dot. A cartoon representation of the remodeler is shown as a semitransparent light blue or light green shape, and the locations of the ATPase and linker-DNA-binding domains as blue and green spheres, respectively. ISWI-induced remodeling starts with the ATPase domain translocating DNA from the SHL2 site toward the exit side, 1 bp at a time. The translocation by the ATPase domain generates strain on the entry-side DNA (depicted by magenta/purple coloring of the DNA), which initially remains immobile. After 7 bp of DNA translocation, the accumulated strain is sufficiently strong to trigger an entry-side action, possibly a conformational change of the enzyme, which pushes a 3 bp equivalent of DNA into the nucleosome. This action partially relaxes the strain and allows three additional base pairs of DNA to be translocated to the exit side. This cycle then repeats to allow processive nucleosome translocation.

Regions within the hydrophobic core of the linker-kinase sandwich are in color, and regions outside are in gray.

Helix αC is highlighted in dark blue, the activation loop is shown in brown, and the short helix in the activation loop following the DFG motif is shown in magenta. The dotted line represents an unordered part of the activation loop. The Src family kinase shown here is Hck (PDB code 1QCF; Schindler et al., 1999). The structure of c-Src is very similar (PDB code 2SRC; Xu et al., 1999). Active ZAP-70: PDB code 1U59 (Jin et al., 2004).

(A) Structures of active (kinase domain only; PDB code 1U59; Jin et al., 2004) and inactive ZAP-70, colored according to the crystallographic temperature factors for each atom. The mean value of the temperature factors for all atoms in helix αF (residues 518 to 534) is approximately the same in both structures, as indicated by the similar colors for atoms in this helix. (B) The hydrogen-bonding network centered on the hinge region of the inactive ZAP-70 kinase domain (left). This network is disrupted in active ZAP-70 (right).

(A) Two schematic views of ZAP-70, rotated by 180° with respect to each other, depict the location of residues in ZAP-70 that are most critical for autoinhibition. These hot spot residues are indicated by yellow stars. (B) LAT phosphorylation by ZAP-70 mutants. The Tyr315Ala/Tyr319Ala double mutant is referred to as “YYAA.” Strongly activating alanine mutations at hot spots of the linker-kinase sandwich are highlighted with yellow stars. Control experiments in the presence of Lck are shown in Figure S4.

Detailed view showing the docking of Pro147 in the inter-SH2 linker into the cleft formed by the side chains of Tyr597 and Tyr598. Helix αI is shown as a light brown surface, and Tyr597 and Tyr598 are shown in magenta surface representation.

(A) Left: ITAM-complexed tandem SH2 domains superimposed onto the tandem SH2 unit of inactive ZAP-70, based on helix αL2. The N-terminal SH2 domain of inactive ZAP-70 is not shown. The inter-SH2 linker is green (inactive ZAP-70) or cyan (ITAM-complexed tandem SH2 unit). The doubly phosphorylated ITAM peptide is in purple. Right: A detailed view showing that ITAM binding requires an ∼10° increase in the elbow angle between helices αL2 and αL3 and leads to the disruption of a hydrogen bond between Gln145 and Ser144. (B) Model for activation of ZAP-70 by ITAM motifs. Top: Two views, 90° rotated from each other, showing a superimposition of the ITAM-complexed SH2 domains onto inactive ZAP-70 based on the N-terminal SH2 domain. The ITAM-bound tandem SH2 unit is orange (N-terminal SH2 domain), yellow (C-terminal SH2 domain), and green (inter-SH2 linker). The tandem SH2 domains of inactive ZAP-70 are shown in gray. Bottom: Schematic diagram of conformational changes upon ITAM binding.

(A) Detailed view of the interactions between the regulatory segment and the main body of the kinase domains. In the top panel, the substrate binding residues Glu 96 and Glu 139 (Yang and Schulman, 1999) are shown interacting with Arg 297 from the regulatory segment. The bottom panel shows the regulatory Thr 286 sitting at the C-terminal end of helix αF. Mutation of residues Phe 98 in helix αD and Phe 292 in the regulatory segment activate the kinase strongly (Yang and Schulman, 1999). (B) Comparison of three Ca2+/CaM-activated kinases, CaMKI (Goldberg et al., 1996), twitchin kinase (Hu et al., 1994), and CaMKII. In CaMKII, neither the N lobe nor the ATP binding pocket make any contact with the regulatory segment.

(A) A structural alignment of phosphorylase kinase (PhosK, bound to ATP; Lowe et al., 1997) and CaMKII showing the rotation and translation of helix αD and the interactions of His 282. Note the position of Glu 96 in CaMKII, in a location too distant to interact with ATP. All residue numbering refers to CaMKII. (B and C) After ∼15 ns of molecular-dynamics simulation without the regulatory segment, helix αD rotates spontaneously into the position seen in PhosK. (B) An overlay of the crystal structure of CaMKII (inactive) with an instantaneous structure, at 15.125 ns, from the simulation without the regulatory segment. (C) The same 15.125 ns instantaneous structure, at 15.125 ns, overlaid with PhosK (active).

This schematic diagram illustrates the consequences of structural fluctuations that release either the kinase domains or the coiled-coil interaction. If kinase domains prematurely release from the regulatory segment in the absence of Ca2+/CaM as shown in (A), the coiled-coil strut will prevent the active site from interacting with the regulatory Thr 286. If, as shown in (B), the coiled-coil interaction breaks, the interactions with the regulatory segment will also prevent accidental phosphorylation of Thr 286.

Six autoinhibited kinase-domain dimers were placed around a central association domain as described in the text. The kinases were separated from one another in steps of 5 Å (this variable was defined as “kinase displacement”) and the theoretical solution X-ray scattering was calculated for each resulting model. (A) A value of kinase displacement equal to 0 Å, corresponding to a model where the autoinhibited kinase domains are in the dimer seen in the crystal structure. The resulting model is a disc with a diameter of ∼200 Å and a height of ∼60 Å. (B) A value of kinase displacement equal to 100 Å resulting in a model with diameter of ∼200 Å and a height of ∼200 Å. (C) As the value of kinase displacement increases, the calculated Rg diverges sharply from the experimental value of ∼72 Å. (D) The rings of autoinhibited kinase domains were moved above and below the association domains while simultaneously shrinking the radius of the kinase rings to generate a series of models with essentially equivalent Rg values (matched to the experimental value). The different models can all be described by a single variable, θ, that represents the angle between the midplane of the association domain and a vector connecting the center of an association-domain dimer and the center of mass of the autoinhibited kinase domain. (E) Theoretical X-ray scattering curves were calculated for models corresponding to different values of θ. At a value of θ equal to 0, corresponding to a model in which the individual autoinhibited kinase domains are held together as dimer pairs in an outer ring that is coplanar with the central plane, the curve approximates the actual data much better. This is quantified in (F) where the χ2 statistic calculated by the program Crysol (Svergun et al., 1995), which compares the calculated scattering curve to the experimental one, is optimal at θ = 0.

(A) The holoenzyme model that resulted from the rigid body modeling described above. In green are the more electron-dense C lobes of the kinases and in beige are the N lobes of the kinases. (B) Shown in blue mesh is the SAXS shape reconstruction. The model shown in (A) was fit into the SAXS shape reconstruction as described in the Experimental Procedures.

(A) A schematic diagram of the holoenzyme in its fully autoinhibited form. (B) Ca2+/CaM binds to a regulatory segment of a kinase dimer, releasing the other regulatory segment to bind a second Ca2+/CaM. Release of the dimer pair reveals a basic patch, shown inset with the electrostatic surface overlaid on the structure. This basic patch may then interact with an acidic region on the linker, directing the kinase domains above and below the midplane of the association domain. (C) When an adjacent autoinhibited kinase dimer binds to two Ca2+/CaM, the kinase domains will again be directed above and below the midplane. They will now encounter the previously activated kinase domains, and transphosphorylation of Thr 286 can occur. (D) The addition of Ca2+/CaM to CaMKII causes a change in the shape of the solution SAXS curve. The intensity of the curve without Ca2+/CaM was multiplied by 1.7 so as to overlay the graphs. The Guinier region, shown as an inset, is linear for both curves in a region of s∗Rg = .9 − 1.3. By fitting the Guinier equation (with the program PRIMUS), the value of Rg was determined to be 71.8 ± 0.2 without Ca2+/CaM and 97.5 ± 0.4 when Ca2+/CaM is added. The presence of the linear Guinier region in the s∗Rg region < 1.3 indicates that the samples are not aggregated. (E) The P(r) function, as calculated by the program GNOM (Svergun, 1992), for CaMKII with and without the addition of Ca2+/CaM.

Rates are relative to the catalytic rate of the wild-type protein. The solid black bars represent catalytic rates in the absence of ITAM peptide, and the light-gray bars depict the rates obtained after incubation with saturating amounts of ITAM peptide.

(A) P116 stable lines were incubated with anti-TCR antibody or left unstimulated for 2′, and lysed in 1% Nonidet P-40 lysis buffer. Phosphorylation levels were assessed by immunoblotting for total tyrosine phosphoproteins (pTyr) (B) or with phosphospecific ZAP-70, PLCγ1, Erk, and SLP-76 antibodies (C). Analyses were performed on whole-cell lysates (WCLs) or after immunoprecipitation (IP). The relative expression of the various ZAP-70 mutants are closely matched to the relative expression of the WT protein: YYAA, 0.91; YYFF, 1.44; W131A, 0.97; A141E, 0.87; and KA, 1.26.

P116 lines stably reconstituted with WT or the W131A mutant of ZAP-70 were incubated with anti-TCR antibody over a 15-min period. (A) Cells were fixed at various time points, permeabilized, stained intracellularly with phosphospecific Erk antibody, and analyzed by FACS. (B) Dose response to anti-TCR antibody at the 5′ time point.

ZAP-70–deficient P116 Jurkat cells reconstituted with wild-type or the W131A mutant of ZAP-70 were loaded with the calcium-sensitive dye Indo-1 and stimulated with varying doses of anti-TCR Vβ antibody (C305). Calcium changes were monitored over time.

P116-stable lines were incubated with anti-TCR antibody over a 5-min period and lysed in 1% Nonidet P-40 lysis buffer. Lysates were separated on SDS-PAGE gels and immunoblotted for total tyrosine phosphoproteins (A) and phosphospecific ZAP-70, PLCγ1, Erk, LAT, and total levels of ZAP-70, PLCγ1, and TCRζ chain (B).

Fluorescence anisotropy increases on binding of fluorescently labeled ITAM-ζ1 peptide to wild-type ZAP-70 (A), ZAP-70 W131A (B), and ZAP-70 YYFF (C). (D) Double-reciprocal plot of ITAM-ζ1 binding to ZAP-70. The data corresponding to the binding of ITAM-ζ1 peptide to ZAP-70 WT, W131A, and YYFF are shown as blue, red, and green lines, respectively. The Kd values depicted here are those obtained from a nonlinear curve fit.