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The organization of nucleosomes into chromatin and their accessibility are shaped by local DNA mechanics and modulated by histone variants. Conversely, nucleosome positions shape genetic variations, which may originate from mismatches during replication and chemical modification of DNA. To investigate how DNA mismatches and histone variants affect the mechanical stability and the exposure of nucleosomal DNA, we used an optical trap combined with single-molecule FRET, and a single-molecule FRET cyclization assay. We found that a single base-pair mismatch enhances DNA bendability and nucleosome mechanical stability. The increase in force required for DNA unwrapping from the histone core is observed for single base-pair mismatches placed at three tested positions: at the inner turn, at the outer turn, or at the junction of the inner and outer turn of the nucleosome. Yeast nucleosomes are mechanically less stable and more symmetrical in the outer turn unwrapping compared to Xenopus nucleosomes. H2A.Z histone variants display no measurable difference in mechanical unwrapping patterns compared to canonical yeast nucleosomes. The results support a model where nucleosomal DNA accessibility is reduced by mismatches, potentially explaining the preferred accumulation of single nucleotide substitutions in the nucleosome core and serving as the source of genetic variation during evolution and cancer progression.

A universe of transcription factors (TFs), cofactors, as well as chromatin remodeling and modifying enzymes combine or compete on chromatin to control transcription. Measuring quantitatively how these proteins dynamically interact is required in order to formulate models with predictive ability to elucidate transcription control mechanisms. Single molecule tracking (SMT) provides a powerful tool towards this goal: it is a fluorescence microscopy approach that measures the location and mobility of individual TF molecules, as well as their rates of association with and dissociation from chromatin in the physiological context of the living cell. Here we review SMT principles, and discuss key TF properties uncovered by live-cell SMT, such as fast turnover (seconds), and formation of clusters that locally increase activity.

Accurate chromosome segregation relies on the specific centromeric nucleosome–kinetochore interface. In budding yeast, the centromere CBF3 complex guides the deposition of CENP-A, an H3 variant, to form the centromeric nucleosome in a DNA sequence-dependent manner. Here, we determine the structures of the centromeric nucleosome containing the native CEN3 DNA and the CBF3core bound to the canonical nucleosome containing an engineered CEN3 DNA. The centromeric nucleosome core structure contains 115 base pair DNA including a CCG motif. The CBF3core specifically recognizes the nucleosomal CCG motif through the Gal4 domain while allosterically altering the DNA conformation. Cryo-EM, modeling, and mutational studies reveal that the CBF3core forms dynamic interactions with core histones H2B and CENP-A in the CEN3 nucleosome. Our results provide insights into the structure of the budding yeast centromeric nucleosome and the mechanism of its assembly, which have implications for analogous processes of human centromeric nucleosome formation., Chromosome segregation requires the association of the kinetochore protein complex with a specialized nucleosome at the centromere. Here, the authors present cryo-EM and mutational studies that provide insights into the structure of the budding yeast centromeric nucleosome and how the centromere CBF3 protein complex guides its formation.

Nucleosomal histone H2A is exchanged for its variant H2A.Z by the SWR1 chromatin remodeler, but the mechanism and timing of histone exchange remain unclear. Here, we quantify DNA and histone dynamics during histone exchange in real-time using a three-color single-molecule FRET assay. We show that SWR1 operates with timed precision to unwrap DNA with large displacement from one face of the nucleosome, remove H2A-H2B from the same face, and rewrap DNA, all within 2.3 seconds. Such productive DNA unwrapping requires full SWR1 activation and differs from unproductive, smaller-scale DNA unwrapping caused by SWR1 binding alone. On an asymmetrically positioned nucleosome, SWR1 intrinsically senses long-linker DNA to preferentially exchange H2A.Z on the distal face as observed in vivo. The displaced H2A-H2B dimer remains briefly associated with the SWR1-nucleosome complex and is dissociated by histone chaperones. These findings reveal how SWR1 coordinates DNA unwrapping with histone dynamics to rapidly and accurately place H2A.Z at physiological sites on chromatin.One-Sentence SummaryMulticolor single-molecule FRET reveals how SWR1 unwraps DNA to exchange nucleosomal H2A-H2B for H2A.Z-H2B.

One-dimensional (1D) target search is a well characterized phenomenon for many DNA binding proteins but is poorly understood for chromatin remodelers. Herein, we characterize the 1D scanning properties of SWR1, a yeast chromatin remodeler that performs histone exchange on +1 nucleosomes adjacent to a nucleosome depleted region (NDR) at promoters. We demonstrate that SWR1 has a kinetic binding preference for DNA of NDR length as opposed to gene-body linker length DNA. Using single and dual color single particle tracking on DNA stretched with optical tweezers, we directly observe SWR1 diffusion on DNA. We found that various factors impact SWR1 scanning, including ATP which promotes diffusion through nucleotide binding rather than ATP hydrolysis. A DNA binding subunit, Swc2, plays an important role in the overall diffusive behavior of the complex, as the subunit in isolation retains similar, although faster, scanning properties as the whole remodeler. ATP-bound SWR1 slides until it encounters a protein roadblock, of which we tested dCas9 and nucleosomes. The median diffusion coefficient, 0.024 μm2/sec, in the regime of helical sliding, would mediate rapid encounter of NDR-flanking nucleosomes at length scales found in cells.

This paper characterizes the actual science performance of the James Webb Space Telescope (JWST), as determined from the six month commissioning period. We summarize the performance of the spacecraft, telescope, science instruments, and ground system, with an emphasis on differences from pre-launch expectations. Commissioning has made clear that JWST is fully capable of achieving the discoveries for which it was built. Moreover, almost across the board, the science performance of JWST is better than expected; in most cases, JWST will go deeper faster than expected. The telescope and instrument suite have demonstrated the sensitivity, stability, image quality, and spectral range that are necessary to transform our understanding of the cosmos through observations spanning from near-earth asteroids to the most distant galaxies., Comment: 5th version as accepted to PASP; 31 pages, 18 figures; https://iopscience.iop.org/article/10.1088/1538-3873/acb293

How pioneer factors interface with chromatin to promote accessibility for transcription control is poorly understood in vivo. Here, we directly visualize chromatin association by the prototypical GAGA pioneer factor (GAF) in live Drosophila hemocytes. Single-particle tracking reveals that the majority of GAF is chromatin-bound, with a stable-binding fraction showing nucleosome-like confinement residing on chromatin for over 2 minutes, far longer than the dynamic range of most transcription factors. These kinetic properties require the full complement of GAF’s DNA-binding, multimerization and intrinsically disordered domains, and are autonomous from recruited chromatin remodelers NURF and PBAP, whose activities primarily benefit GAF’s neighbors such as HSF. Evaluation of GAF kinetics together with its endogenous abundance indicates that despite on-off dynamics, GAF constitutively and fully occupies chromatin targets, thereby providing a temporal mechanism that sustains open chromatin for transcriptional responses to homeostatic, environmental, and developmental signals.

Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of “fluorogenic” compounds—ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone–zwitterion equilibrium constant (KL–Z) is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore—Janelia Fluor 526 (JF526)—with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF526 is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF526 constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging., We developed a general rubric for creating fluorogenic stains, resulting in the novel fluorophore JF526. This dye can be used in numerous imaging modalities including super-resolution microscopy.

Conserved ATP-dependent chromatin remodelers establish and maintain genome-wide chromatin architectures of regulatory DNA during cellular lifespan, but the temporal interactions between remodelers and chromatin targets have been obscure. We performed live-cell single-molecule tracking for RSC, SWI/SNF, CHD1, ISW1, ISW2, and INO80 remodeling complexes in budding yeast and detected hyperkinetic behaviors for chromatin-bound molecules that frequently transition to the free state for all complexes. Chromatin-bound remodelers display notably higher diffusion than nucleosomal histones, and strikingly fast dissociation kinetics with 4-7 s mean residence times. These enhanced dynamics require ATP binding or hydrolysis by the catalytic ATPase, uncovering an additional function to its established role in nucleosome remodeling. Kinetic simulations show that multiple remodelers can repeatedly occupy the same promoter region on a timescale of minutes, implicating an unending ‘tug-of-war’ that controls a temporally shifting window of accessibility for the transcription initiation machinery.

SUMMARYTranscription initiation by RNA polymerase II (Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus where thousands of promoters are broadly distributed in chromatin, it is unclear how ten individual components converge on any target to establish the PIC. Here, we use live-cell, single-molecule tracking in S. cerevisiae to document subdiffusive, constrained exploration of the nucleoplasm by PIC components and Mediator’s key functions in guiding this process. On chromatin, TBP, Mediator, and Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently at an average promoter where initiation-coupled disassembly may occur within a few seconds. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Collectively, our study provides a global spatio-temporal model for transcription initiation in live cells.

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription of noncoding RNAs prior to premature termination, in addition to transcription of mRNAs, are responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

Summary The histone chaperone FACT occupies transcribed regions where it plays prominent roles in maintaining chromatin integrity and preserving epigenetic information. How it is targeted to transcribed regions, however, remains unclear. Proposed models include docking on the RNA polymerase II (RNAPII) C-terminal domain (CTD), recruitment by elongation factors, recognition of modified histone tails, and binding partially disassembled nucleosomes. Here, we systematically test these and other scenarios in Saccharomyces cerevisiae and find that FACT binds transcribed chromatin, not RNAPII. Through a combination of high-resolution genome-wide mapping, single-molecule tracking, and mathematical modeling, we propose that FACT recognizes the +1 nucleosome, as it is partially unwrapped by the engaging RNAPII, and spreads to downstream nucleosomes aided by the chromatin remodeler Chd1. Our work clarifies how FACT interacts with genes, suggests a processive mechanism for FACT function, and provides a framework to further dissect the molecular mechanisms of transcription-coupled histone chaperoning.

Transcriptional Cyclin Dependent Kinases modulate RNA Polymerase II function to impact gene expression. Here, we show that CDK13 is mutated in 4% of patient melanomas and mutation or downregulation is associated with poor overall survival. Mutant CDK13 lacks kinase activity and overexpression in zebrafish leads to accelerated melanoma. CDK13 mutant fish and human melanomas accumulate prematurely terminated RNAs that are translated into truncated proteins. CDK13 binds to and regulates the phosphorylation of ZC3H14, a member of the PolyA eXosome Targeting (PAXT) RNA degradation complex. ZC3H14 phosphorylation recruits the PAXT complex to degrade prematurely terminated polyadenylated transcripts in the nucleus. In the presence of mutant CDK13, ZC3H14 phosphorylation is compromised and consequently fails to recruit the PAXT complex, leading to truncated transcript stabilization. This work establishes a role for CDK13 and the PAXT nuclear RNA degradation complex in cancer and has prognostic significance for melanoma patients with mutated or downregulated CDK13.

Histone variant H2A.Z, a universal mark of dynamic nucleosomes flanking gene promoters and enhancers, is incorporated into chromatin by SRCAP (SWR1), an ATP-dependent, multicomponent chromatin-remodeling complex. The YL1 (Swc2) subunit of SRCAP (SWR1) plays an essential role in H2A.Z recognition, but how it achieves this has been unclear. Here, we report the crystal structure of the H2A.Z-binding domain of Drosophila melanogaster YL1 (dYL1-Z) in complex with an H2A.Z-H2B dimer at 1.9-Å resolution. The dYL1-Z domain adopts a new whip-like structure that wraps over H2A.Z-H2B, and preferential recognition is largely conferred by three residues in loop 2, the hyperacidic patch and the extended αC helix of H2A.Z. Importantly, this domain is essential for deposition of budding yeast H2A.Z in vivo and SRCAP (SWR1)-catalyzed histone H2A.Z replacement in vitro. Our studies distinguish YL1-Z from known H2A.Z chaperones and suggest a hierarchical mechanism based on increasing binding affinity facilitating H2A.Z transfer from SRCAP (SWR1) to the nucleosome.

Hydroxyl-radical footprinting (HRF) is a powerful method for probing structures of nucleic acid-protein complexes with single-nucleotide resolution in solution. To tap the full quantitative potential of HRF, we describe a protocol, hydroxyl-radical footprinting interpretation for DNA (HYDROID), to quantify HRF data and integrate them with atomistic structural models. The stages of the HYDROID protocol are extraction of the lane profiles from gel images, quantification of the DNA cleavage frequency at each nucleotide and theoretical estimation of the DNA cleavage frequency from atomistic structural models, followed by comparison of experimental and theoretical results. Example scripts for each step of HRF data analysis and interpretation are provided for several nucleosome systems; they can be easily adapted to analyze user data. As input, HYDROID requires polyacrylamide gel electrophoresis (PAGE) images of HRF products and optionally can use a molecular model of the DNA-protein complex. The HYDROID protocol can be used to quantify HRF over DNA regions of up to 100 nucleotides per gel image. In addition, it can be applied to the analysis of RNA-protein complexes and free RNA or DNA molecules in solution. Compared with other methods reported to date, HYDROID is unique in its ability to simultaneously integrate HRF data with the analysis of atomistic structural models. HYDROID is freely available. The complete protocol takes ~3 h. Users should be familiar with the command-line interface, the Python scripting language and Protein Data Bank (PDB) file formats. A graphical user interface (GUI) with basic functionality (HYDROID_GUI) is also available.

The James Webb Space Telescope uses the Fine Guidance Controller to achieve pointing accuracy to a millionth of a degree needed for its scientific observations. This closed loop controller includes the Fine Guidance Sensor instrument, the Attitude Control System, and the Fine Steering Mirror, all working together to generate precise attitude updates every 64 ms to stabilize and point the Observatory. It was exercised for the first time with the flight hardware during the cryogenic test at Johnson Space Center. We provide a top level summary of the test, the results, and its performance in preparation for on-orbit operations.

Histone CENP-A-containing nucleosomes play an important role in nucleating kinetochores at centromeres for chromosome segregation. However, the molecular mechanisms by which CENP-A nucleosomes engage with kinetochore proteins are not well understood. Here, we report the finding of a new function for the budding yeast Cse4/CENP-A histone-fold domain interacting with inner kinetochore protein Mif2/CENP-C. Strikingly, we also discovered that AT-rich centromere DNA has an important role for Mif2 recruitment. Mif2 contacts one side of the nucleosome dyad, engaging with both Cse4 residues and AT-rich nucleosomal DNA. Both interactions are directed by a contiguous DNA- and histone-binding domain (DHBD) harboring the conserved CENP-C motif, an AT hook, and RK clusters (clusters enriched for arginine–lysine residues). Human CENP-C has two related DHBDs that bind preferentially to DNA sequences of higher AT content. Our findings suggest that a DNA composition-based mechanism together with residues characteristic for the CENP-A histone variant contribute to the specification of centromere identity.

Nucleosomes are the most abundant protein–DNA complexes in eukaryotes that provide compaction of genomic DNA and are implicated in regulation of transcription, DNA replication and repair. The details of DNA positioning on the nucleosome and the DNA conformation can provide key regulatory signals. Hydroxyl-radical footprinting (HRF) of protein–DNA complexes is a chemical technique that probes nucleosome organization in solution with a high precision unattainable by other methods. In this work we propose an integrative modeling method for constructing high-resolution atomistic models of nucleosomes based on HRF experiments. Our method precisely identifies DNA positioning on nucleosome by combining HRF data for both DNA strands with the pseudo-symmetry constraints. We performed high-resolution HRF for Saccharomyces cerevisiae centromeric nucleosome of unknown structure and characterized it using our integrative modeling approach. Our model provides the basis for further understanding the cooperative engagement and interplay between Cse4p protein and the A-tracts important for centromere function.

Histone variant H2A.Z-containing nucleosomes exist at most eukaryotic promoters and play important roles in gene transcription and genome stability. The multisubunit nucleosome-remodeling enzyme complex SWR1, conserved from yeast to mammals, catalyzes the ATP-dependent replacement of histone H2A in canonical nucleosomes with H2A.Z. How SWR1 catalyzes the replacement reaction is largely unknown. Here, we determined the crystal structure of the N-terminal region (599-627) of the catalytic subunit Swr1, termed Swr1-Z domain, in complex with the H2A.Z-H2B dimer at 1.78 Å resolution. The Swr1-Z domain forms a 310 helix and an irregular chain. A conserved LxxLF motif in the Swr1-Z 310 helix specifically recognizes the αC helix of H2A.Z. Our results show that the Swr1-Z domain can deliver the H2A.Z-H2B dimer to the DNA-(H3-H4)2 tetrasome to form the nucleosome by a histone chaperone mechanism.

Chz1 is a specific chaperone for the histone variant H2A.Z in budding yeast. The ternary complex formed by Chz1 and H2A.Z-H2B dimer is the major in vivo substrate of Swi2/snif2-related 1 (SWR1), the ATP-dependent chromatin remodeling enzyme that deposits H2A.Z into chromatin. However, the structural basis for the binding preference of Chz1 for H2A.Z over H2A and the mechanism by which Chz1 modulates the histone replacement remain elusive. Here, we show that Chz1 utilizes 2 distinct structural domains to engage the H2A.Z-H2B dimer for optimal and specific recognition of H2A.Z. The middle region of Chz1 (Chz1-M) directly interacts with 2 highly conserved H2A.Z-specific residues (Gly98 and Ala57) and dictates a modest preference for H2A.Z-H2B. In addition, structural and biochemical analysis show that the C-terminal region of Chz1 (Chz1-C) harbors a conserved DEF/Y motif, which reflects the consecutive D/E residues followed by a single aromatic residue, to engage an arginine finger and a hydrophobic pocket in H2A.Z-H2B, enhancing the binding preference for H2A.Z-H2B. Furthermore, Chz1 facilitates SWR1-mediated H2A.Z deposition by alleviating inhibition caused by aggregation of excess free histones, providing insights into how Chz1 controls the bioavailability of H2A.Z to assist SWR1 in promoter-specific installation of a histone mark. Our study elucidates a novel H2A.Z-recognition mechanism and uncovers a molecular rationale for binding of free histone by specialized histone chaperones in vivo., The variant histone H2A.Z is essential for the viability of metazoans. The structure of H2A.Z-H2B histone dimer in complex with the chaperone Chz1 reveals the basis of Chz1’s specificity for recognising H2A.Z. In vitro studies show that Chz1 facilitates H2A.Z deposition by relieving the inhibitory effects of excess free histone dimers.

Conventional acquisition of three-dimensional (3D) microscopy data requires sequential z scanning and is often too slow to capture biological events. We report an aberration-corrected multifocus microscopy method capable of producing an instant focal stack of nine 2D images. Appended to an epifluorescence microscope, the multifocus system enables high-resolution 3D imaging in multiple colors with single-molecule sensitivity, at speeds limited by the camera readout time of a single image.

Summary The ATP-dependent chromatin-remodeling complex SWR1 exchanges a variant histone H2A.Z/H2B dimer for a canonical H2A/H2B dimer at nucleosomes flanking histone-depleted regions, such as promoters. This localization of H2A.Z is conserved throughout eukaryotes. SWR1 is a 1 megadalton complex containing 14 different polypeptides, including the AAA+ ATPases Rvb1 and Rvb2. Using electron microscopy, we obtained the three-dimensional structure of SWR1 and mapped its major functional components. Our data show that SWR1 contains a single heterohexameric Rvb1/Rvb2 ring that, together with the catalytic subunit Swr1, brackets two independently assembled multisubunit modules. We also show that SWR1 undergoes a large conformational change upon engaging a limited region of the nucleosome core particle. Our work suggests an important structural role for the Rvbs and a distinct substrate-handling mode by SWR1, thereby providing a structural framework for understanding the complex dimer-exchange reaction., Graphical Abstract, Highlights • SWR1 consists of four structurally discrete functional modules • Rvb1 and Rvb2 assemble into a single, heterohexameric ring in SWR1 • SWR1 undergoes a large conformational change upon nucleosome binding • SWR1 forms limited contact with the nucleosome core particle, Structural analysis of the 14 subunit SWR1 chromatin remodeler identifies the orientation of its functional modules and reveals conformational changes induced by nucleosome binding.

Lysine methylation of histones is recognized as an important component of an epigenetic indexing system demarcating transcriptionally active and inactive chromatin domains. Trimethylation of histone H3 lysine 4 (H3K4me3) marks transcription start sites of virtually all active genes. Recently, we reported that the WD40-repeat protein WDR5 is important for global levels of H3K4me3 and control of HOX gene expression. Here we show that a plant homeodomain (PHD) finger of nucleosome remodelling factor (NURF), an ISWI-containing ATP-dependent chromatin-remodelling complex, mediates a direct preferential association with H3K4me3 tails. Depletion of H3K4me3 causes partial release of the NURF subunit, BPTF (bromodomain and PHD finger transcription factor), from chromatin and defective recruitment of the associated ATPase, SNF2L (also known as ISWI and SMARCA1), to the HOXC8 promoter. Loss of BPTF in Xenopus embryos mimics WDR5 loss-of-function phenotypes, and compromises spatial control of Hox gene expression. These results strongly suggest that WDR5 and NURF function in a common biological pathway in vivo, and that NURF-mediated ATP-dependent chromatin remodelling is directly coupled to H3K4 trimethylation to maintain Hox gene expression patterns during development. We also identify a previously unknown function for the PHD finger as a highly specialized methyl-lysine-binding domain.

The maturation of T cells requires signaling from both cytokine and T-cell receptors to gene targets in chromatin, but how chromatin architecture influences this process is largely unknown. Here we show that thymocyte maturation post-positive selection is dependent on the nucleosome remodeling factor (NURF). Depletion of Bptf (bromodomain PHD finger transcription factor), the largest NURF subunit, in conditional mouse mutants results in developmental arrest beyond the CD4+ CD8int stage without affecting cellular proliferation, cellular apoptosis, or coreceptor gene expression. In the Bptf mutant, specific subsets of genes important for thymocyte development show aberrant expression. We also observed defects in DNase I-hypersensitive chromatin structures at Egr1, a prototypical Bptf-dependent gene that is required for efficient thymocyte development. Moreover, chromatin binding of the sequence-specific factor Srf (serum response factor) to Egr1 regulatory sites is dependent on Bptf function. Physical interactions between NURF and Srf suggest a model in which Srf recruits NURF to facilitate transcription factor binding at Bptf-dependent genes. These findings provide evidence for causal connections between NURF, transcription factor occupancy, and gene regulation during thymocyte development.

SummaryHistone variant H2A.Z-containing nucleosomes are incorporated at most eukaryotic promoters. This incorporation is mediated by the conserved SWR1 complex, which replaces histone H2A in canonical nucleosomes with H2A.Z in an ATP-dependent manner. Here, we show that promoter-proximal nucleosomes are highly heterogeneous for H2A.Z in Saccharomyces cerevisiae, with substantial representation of nucleosomes containing one, two, or zero H2A.Z molecules. SWR1-catalyzed H2A.Z replacement in vitro occurs in a stepwise and unidirectional fashion, one H2A.Z-H2B dimer at a time, producing heterotypic nucleosomes as intermediates and homotypic H2A.Z nucleosomes as end products. The ATPase activity of SWR1 is specifically stimulated by H2A-containing nucleosomes without ensuing histone H2A eviction. Remarkably, further addition of free H2A.Z-H2B dimer leads to hyperstimulation of ATPase activity, eviction of nucleosomal H2A-H2B, and deposition of H2A.Z-H2B. These results suggest that the combination of H2A-containing nucleosome and free H2A.Z-H2B dimer acting as both effector and substrate for SWR1 governs the specificity and outcome of the replacement reaction.