Remodels The Structure Of Chromatin Research Articles

Genome-wide regulation of Pol II, FACT, and Spt6 occupancies by RSC in Saccharomyces cerevisiae

RSC (remodels the structure of chromatin) is an essential ATP-dependent chromatin remodeling complex in Saccharomyces cerevisiae. RSC utilizes its ATPase subunit, Sth1, to slide or remove nucleosomes. RSC has been shown to regulate the width of the nucleosome-depleted regions (NDRs) by sliding the flanking nucleosomes away from NDRs. As such, when RSC is depleted, nucleosomes encroach NDRs, leading to transcription initiation defects. In this study, we examined the effects of the catalytic-dead Sth1 on transcription and compared them to those observed during acute and rapid Sth1 depletion by auxin-induced degron strategy. We found that rapid depletion of Sth1 reduces recruitment of TBP and Pol II in highly transcribed genes, as would be expected considering its role in regulating chromatin structure at promoters. In contrast, cells harboring the catalytic-dead Sth1 (sth1-K501R) exhibited a severe reduction in TBP binding, but, surprisingly, also displayed a substantial accumulation in Pol II occupancies within coding regions. The Pol II occupancies further increased upon depleting endogenous Sth1 in the catalytic-dead mutant, suggesting that the inactive Sth1 contributes to Pol II accumulation in coding regions. Notwithstanding the Pol II increase, the ORF occupancies of histone chaperones, FACT and Spt6 were significantly reduced in the mutant. These results suggest a potential role for RSC in recruiting/retaining these chaperones in coding regions. Pol II accumulation despite substantial reductions in TBP, FACT, and Spt6 occupancies in the catalytic-dead mutant could indicate severe transcription elongation and termination defects. Such defects would be consistent with studies showing that RSC is recruited to coding regions in a transcription-dependent manner. Thus, these findings imply a role for RSC in transcription elongation and termination processes, in addition to its established role in transcription initiation.

Functional interplay between Mediator and RSC chromatin remodeling complex controls nucleosome-depleted region maintenance at promoters

Chromatin organization is crucial for transcriptional regulation in eukaryotes. Mediator is an essential and conserved co-activator thought to act in concert with chromatin regulators. However, it remains largely unknown how their functions are coordinated. Here, we provide evidence in the yeast Saccharomyces cerevisiae that Mediator establishes physical contact with RSC (Remodels the Structure of Chromatin), a conserved and essential chromatin remodeling complex that is crucial for nucleosome-depleted region (NDR) formation. We determine the role of Mediator-RSC interaction in their chromatin binding, nucleosome occupancy, and transcription on a genomic scale. Mediator and RSC co-localize on wide NDRs of promoter regions, and specific Mediator mutations affect nucleosome eviction and TSS-associated+1 nucleosome stability. This work shows that Mediator contributes to RSC remodeling function to shape NDRs and maintain chromatin organization on promoter regions. It will help in our understanding of transcriptional regulation in the chromatin context relevant for severe diseases.

Histone protein surface accessibility dictates direction of RSC-dependent nucleosome mobilization.

Chromatin remodeling enzymes use energy derived from ATP hydrolysis to mobilize nucleosomes and alter their structure to facilitate DNA access. The Remodels the Structure of Chromatin (RSC) complex has been extensively studied, yet aspects of how this complex functionally interacts with nucleosomes remain unclear. We introduce a steric mapping approach to determine how RSC activity depends on interaction with specific surfaces within the nucleosome. We find that blocking SHL + 4.5/–4.5 via streptavidin binding to the H2A N-terminal tail domains results in inhibition of RSC nucleosome mobilization. However, restriction enzyme assays indicate that remodeling-dependent exposure of an internal DNA site near the nucleosome dyad is not affected. In contrast, occlusion of both protein faces of the nucleosome by streptavidin attachment near the acidic patch completely blocks both remodeling-dependent nucleosome mobilization and internal DNA site exposure. However, we observed partial inhibition when only one protein surface is occluded, consistent with abrogation of one of two productive RSC binding orientations. Our results indicate that nucleosome mobilization requires RSC access to the trailing but not the leading protein surface, and reveals a mechanism by which RSC and related complexes may drive unidirectional movement of nucleosomes to regulate cis-acting DNA sequences in vivo.

Sth1, the ATPase subunit of the RSC chromatin remodeler has important roles in stress response and DNA damage repair in the pathogenic fungi Candida albicans

Candida albicans, the most prevalent fungal pathogen, exists as a commensal in the human host. It is subjected to myriad physiological stress conditions in different host niches, which jeopardizes its fitness to survive and propagate as an established commensal. C. albicans has highly labile chromatin which gets remodeled in response to the stress conditions to facilitate the expression of several stress-responsive genes. Several epigenetic factors including histone variants, histone modifiers and chromatin remodelers that define the chromatin architecture play crucial roles in the regulation of the stress-responsive genes in this organism. Here we investigated the roles of the ATP-dependent chromatin remodeler RSC (Remodel the Structure of Chromatin) in several stress responses in C. albicans, by targeting the key ATPase component, Sth1, given its profound and similar roles exist in Saccharomyces cerevisiae. We have unraveled the crucial roles of the RSC complex (Sth1) in maintaining cell wall integrity and fighting against osmotic and oxidative stresses. We found that the mutant conditionally depleted of Sth1 was sensitive to the cell wall disrupting agents, and the mutant without exposure to any stressor accumulated higher chitin content in the cell wall as a defense mechanism to restore the cell wall integrity. Further, this was supported by the phosphorylation of MAPK1 protein Mkc1, which happens due to activation of the cell wall integrity pathway PKC1. We also observed the Sth1 mutant to be sensitive to oxidative and osmotic stresses in vitro, which are very important and imparted by the host defense mechanism. This suggests that the mutant could get attenuated and hence become less virulent than the wild-type when loss of function of Sth1 happens. We also found that Sth1 has a crucial role in maintaining genomic integrity as sth1 mutant cells accumulate extensive DNA damages and show the loss in cell viability. Overall this work suggests that Sth1 has an important role in fighting against some of the clinically relevant and physiologically important stresses. It also has a crucial role in fighting against stress to the genomic integrity and hence functions in DNA damage repair.

The RSC (Remodels the Structure of Chromatin) complex of Candida albicans shows compositional divergence with distinct roles in regulating pathogenic traits.

Regulation of gene expression programs is crucial for the survival of microbial pathogens in host environments and for their ability to cause disease. Here we investigated the epigenetic regulator RSC (Remodels the Structure of Chromatin) in the most prevalent human fungal pathogen Candida albicans. Biochemical analysis showed that CaRSC comprises 13 subunits and contains two novel non-essential members, which we named Nri1 and Nri2 (Novel RSC Interactors) that are exclusive to the CTG clade of Saccharomycotina. Genetic analysis showed distinct essentiality of C. albicans RSC subunits compared to model fungal species suggesting functional and structural divergence of RSC functions in this fungal pathogen. Transcriptomic and proteomic profiling of a conditional mutant of the essential catalytic subunit gene STH1 demonstrated global roles of RSC in C. albicans biology, with the majority of growth-related processes affected, as well as mis-regulation of genes involved in morphotype switching, host-pathogen interaction and adaptive fitness. We further assessed the functions of non-essential CaRSC subunits, showing that the novel subunit Nri1 and the bromodomain subunit Rsc4 play roles in filamentation and stress responses; and also interacted at the genetic level to regulate cell viability. Consistent with these roles, Rsc4 is required for full virulence of C. albicans in the murine model of systemic infection. Taken together, our data builds the first comprehensive study of the composition and roles of RSC in C. albicans, showing both conserved and distinct features compared to model fungal systems. The study illuminates how C. albicans uses RSC-dependent transcriptional regulation to respond to environmental signals and drive survival fitness and virulence in mammals.

Specialized RSC: Substrate Specificities for a Conserved Chromatin Remodeler.

The remodel the structure of chromatin (RSC) nucleosome remodeling complex is a conserved chromatin regulator with roles in chromatin organization, especially over nucleosome depleted regions therefore functioning in gene expression. Recent reports in Saccharomyces cerevisiae have identified specificities in RSC activity toward certain types of nucleosomes. RSC has now been shown to preferentially evict nucleosomes containing the histone variant H2A.Z in vitro. Furthermore, biochemical activities of distinct RSC complexes has been found to differ when their nucleosome substrate is partially unraveled. Mammalian BAF complexes, the homologs of yeast RSC and SWI/SNF complexes, are also linked to nucleosomes with H2A.Z, but this relationship may be complex and extent of conservation remains to be determined. The interplay of remodelers with specific nucleosome substrates and regulation of remodeler outcomes by nucleosome composition are tantalizing questions given the wave of structural data emerging for RSC and other SWI/SNF family remodelers.

Interaction of RSC Chromatin Remodelling Complex with Nucleosomes is Modulated by H3 K14 Acetylation and H2B SUMOylation  In vivo

Chromatin remodelling complexes are multi-subunit nucleosome translocases that reorganize chromatin in the context of DNA replication, repair and transcription. A key question is how these complexes find their target sites on chromatin. Here, we use genetically encoded photo-crosslinker amino acids to map the footprint of Sth1, the catalytic subunit of the RSC (remodels the structure of chromatin) complex, on the nucleosome in living yeast. We find that the interaction of the Sth1 bromodomain with the H3 tail depends on K14 acetylation by Gcn5. This modification does not recruit RSC to chromatin but mediates its interaction with neighbouring nucleosomes. We observe a preference of RSC for H2B SUMOylated nucleosomes in vivo and show that this modification moderately enhances RSC binding to nucleosomes in vitro. Furthermore, RSC is not ejected from chromatin in mitosis, but its mode of nucleosome binding differs between interphase and mitosis. In sum, our in vivo analyses show that RSC recruitment to specific chromatin targets involves multiple histone modifications most likely in combination with other components such as histone variants and transcription factors.

The Structural Basis for Specific Recognition of H3K14 Acetylation by Sth1 in the RSC Chromatin Remodeling Complex

The Saccharomyces cerevisiae RSC (Remodel the Structure of Chromatin) complex is a chromatin-remodeling complex and plays essential roles in transcription regulation and DNA repair. The acetylation of H3 Lysine14 (H3K14Ac) enhances the RSC retention on nucleosomes and increases the remodeling activity of RSC. However, which RSC component recognizes H3K14Ac remains unclear. Here, we discovered that the bromodomain of the catalytic subunit Sth1 (Sth1BD) possessed the strongest affinity to H3K14Ac among all RSC bromodomains. The Sth1BD specifically recognized the K(Ac)ΦΦR motif (Φ stands for any hydrophobic amino acid), including H3K14Ac and H4K20Ac. We determined the crystal structures of Sth1BD at 2.40Å resolution and Sth1BD-H3K14Ac complex at 1.40Å resolution. The extensive interfaces between Sth1BD and H36-21 facilitate the specific and robust binding of Sth1BD to H3K14Ac. Our studies provide insights into how the RSC complex recognizes H3K14Ac to orchestrate the crosstalk between histone acetylation and chromatin remodeling.

Sth1, the Key Subunit of the RSC Chromatin Remodeling Complex, Is Essential in Maintaining Chromosomal Integrity and Mediating High Fidelity Chromosome Segregation in the Human Fungal Pathogen Candida albicans

Chromatin architecture influences gene expression and makes specialized chromatin domains. Factors including histone variants, histone modifiers and chromatin remodelers that define chromatin architecture impact chromosome related processes in Candida albicans. In this context, we sought to investigate the roles of the ATP-dependent chromatin remodeler, Remodel the Structure of Chromatin (RSC) in chromosome segregation of C. albicans. Sth1 is the key ATPase component of RSC and has profound roles in different cellular processes in Saccharomyces cerevisiae. We demonstrate that STH1 is an essential gene in C. albicans. The depletion of Sth1 induces pseudohyphal cells, abnormal spindle morphology, sensitivity toward anti-mitotic drugs and global cohesion defect suggesting an important role of Sth1 in kinetochore-microtubule related processes in C. albicans. Strikingly, Sth1 is required to maintain clustered kinetochores revealing the fact that RSC is required in kinetochore integrity. Taken together, we show that RSC plays an important role in various chromatin-templated processes including chromosome segregation in C. albicans.

Dynamics of Chromatin and Transcription during Transient Depletion of the RSC Chromatin Remodeling Complex

SummaryNucleosome organization has a key role in transcriptional regulation, yet the precise mechanisms establishing nucleosome locations and their effect on transcription are unclear. Here, we use an induced degradation system to screen all yeast ATP-dependent chromatin remodelers. We characterize how rapid clearance of the remodeler affects nucleosome locations. Specifically, depletion of Sth1, the catalytic subunit of the RSC (remodel the structure of chromatin) complex, leads to rapid fill-in of nucleosome-free regions at gene promoters. These changes are reversible upon reintroduction of Sth1 and do not depend on DNA replication. RSC-dependent nucleosome positioning is pivotal in maintaining promoters of lowly expressed genes free from nucleosomes. In contrast, we observe that upon acute stress, the RSC is not necessary for the transcriptional response. Moreover, RSC-dependent nucleosome positions are tightly related to usage of specific transcription start sites. Our results suggest organizational principles that determine nucleosome positions with and without RSC and how these interact with the transcriptional process.

Structure and regulation of the human INO80-nucleosome complex.

Access to DNA within nucleosomes is required for a variety of processes in cells including transcription, replication and repair. Consequently, cells encode multiple systems that remodel nucleosomes. These complexes can be simple, involving one or a few protein subunits, or more complicated multi-subunit machines 1 . Biochemical studies2-4 have placed the motor domains of several chromatin remodellers in the superhelical location 2 region of the nucleosome. Structural studies of yeast Chd1 and Snf2-a subunit in the complex with the capacity to remodel the structure of chromatin (RSC)-in complex with nucleosomes5-7 have provided insights into the basic mechanism of nucleosome sliding performed by these complexes. However, how larger, multi-subunit remodelling complexes such as INO80 interact with nucleosomes and how remodellers carry out functions such as nucleosome sliding 8 , histone exchange 9 and nucleosome spacing10-12 remain poorly understood. Although some remodellers work as monomers 13 , others work as highly cooperative dimers11, 14, 15. Here we present the structure of the human INO80 chromatin remodeller with a bound nucleosome, which reveals that INO80 interacts with nucleosomes in a previously undescribed manner: the motor domains are located on the DNA at the entry point to the nucleosome, rather than at superhelical location 2. The ARP5-IES6 module of INO80 makes additional contacts on the opposite side of the nucleosome. This arrangement enables the histone H3 tails of the nucleosome to have a role in the regulation of the activities of the INO80 motor domain-unlike in other characterized remodellers, for which H4 tails have been shown to regulate the motor domains.

Nucleosome eviction in mitosis assists condensin loading and chromosome condensation.

Condensins associate with DNA and shape mitotic chromosomes. Condensins are enriched nearby highly expressed genes during mitosis, but how this binding is achieved and what features associated with transcription attract condensins remain unclear. Here, we report that condensin accumulates at or in the immediate vicinity of nucleosome-depleted regions during fission yeast mitosis. Two transcriptional coactivators, the Gcn5 histone acetyltransferase and the RSC chromatin-remodelling complex, bind to promoters adjoining condensin-binding sites and locally evict nucleosomes to facilitate condensin binding and allow efficient mitotic chromosome condensation. The function of Gcn5 is closely linked to condensin positioning, since neither the localization of topoisomerase II nor that of the cohesin loader Mis4 is altered in gcn5 mutant cells. We propose that nucleosomes act as a barrier for the initial binding of condensin and that nucleosome-depleted regions formed at highly expressed genes by transcriptional coactivators constitute access points into chromosomes where condensin binds free genomic DNA.

A mutation in the Cc.arp9 gene encoding a putative actin-related protein causes defects in fruiting initiation and asexual development in the agaricomycete Coprinopsis cinerea.

Agaricomycetes exhibit a remarkable morphological differentiation from vegetative mycelia to huge fruiting bodies. To investigate the molecular mechanism underlying the fruiting body development, we have isolated and characterized many Coprinopsis cinerea mutant strains defective in fruiting initiation to date. Dikaryon formation in agaricomycetes, which is followed by fruiting development, is governed by the mating type loci, A and B. Recently, mutations in the Cc.snf5 gene, which encodes a putative component of the chromatin remodeling complex switch/sucrose non-fermentable (SWI/SNF), were shown to cause defects in A-regulated clamp cell morphogenesis, as well as in fruiting initiation. Here, we demonstrate that Cc.arp9, which encodes a putative actin-related protein associated with two chromatin remodeling complexes, SWI/SNF and remodels the structure of chromatin (RSC), is also essential for fruiting initiation. In contrast to Cc.snf5 mutants, Cc.arp9 mutants were not defective in clamp cell formation. The effects of mutations in Cc.arp9 and Cc.snf5 on oidia production and the transcriptional expression levels of clp1 and pcc1, which are under the control of the A gene, were also examined. These indicated that Cc.Snf5 is involved in A-regulated pathways, whereas Cc.Arp9 is not apparently. Cc.arp9/Cc.snf5 double-gene disruptants were generated and their phenotypes were analyzed, which suggested a complicated developmental regulation mechanism mediated by chromatin remodeling.

RSC Chromatin-Remodeling Complex Is Important for Mitochondrial Function in Saccharomyces cerevisiae.

RSC (Remodel the Structure of Chromatin) is an ATP-dependent chromatin remodeling complex essential for the growth of Saccharomyces cerevisiae. RSC exists as two distinct isoforms that share core subunits including the ATPase subunit Nps1/Sth1 but contain either Rsc1or Rsc2. Using the synthetic genetic array (SGA) of the non-essential null mutation method, we screened for mutations exhibiting synthetic growth defects in combination with the temperature-sensitive mutant, nps1-105, and found connections between mitochondrial function and RSC. rsc mutants, including rsc1Δ, rsc2Δ, and nps1-13, another temperature-sensitive nps1 mutant, exhibited defective respiratory growth; in addition, rsc2Δ and nps1-13 contained aggregated mitochondria. The rsc2Δ phenotypes were relieved by RSC1 overexpression, indicating that the isoforms play a redundant role in respiratory growth. Genome-wide expression analysis in nps1-13 under respiratory conditions suggested that RSC regulates the transcription of some target genes of the HAP complex, a transcriptional activator of respiratory gene expression. Nps1 physically interacted with Hap4, the transcriptional activator moiety of the HAP complex, and overexpression of HAP4 alleviated respiratory defects in nps1-13, suggesting that RSC plays pivotal roles in mitochondrial gene expression and shares a set of target genes with the HAP complex.

Dual tagging as an approach to isolate endogenous chromatin remodeling complexes from Saccharomyces cerevisiae

Affinity isolation has been an essential technique for molecular studies of cellular assemblies, such as the switch/sucrose non-fermentable (SWI/SNF) family of ATP-dependent chromatin remodeling complexes. However, even biochemically pure isolates can contain heterogeneous mixtures of complexes and their components. In particular, purification strategies that rely on affinity tags fused to only one component of a complex may be susceptible to this phenomenon. This study demonstrates that fusing purification tags to two different proteins enables the isolation of intact complexes of remodels the structure of chromatin (RSC). A Protein A tag was fused to one of the RSC proteins and a Twin-Strep tag to another protein of the complex. By mass spectrometry, we demonstrate the enrichment of the RSC complexes. The complexes had an apparent Svedberg value of about 20S, as shown by glycerol gradient ultracentrifugation. Additionally, purified complexes were demonstrated to be functional. Electron microscopy and single-particle analyses revealed a conformational rearrangement of RSC upon interaction with acetylated histone H3 peptides. This purification method is useful to purify functionally active, structurally well-defined macromolecular assemblies.

Chromatin remodelling complex RSC promotes base excision repair in chromatin of Saccharomyces cerevisiae

The base excision repair (BER) pathway is a conserved DNA repair system required to maintain genomic integrity and prevent mutagenesis in all eukaryotic cells. Nevertheless, how BER operates in vivo (i.e. in the context of chromatin) is poorly understood. We have investigated the role of an essential ATP-dependent chromatin remodelling (ACR) complex RSC (Remodels the Structure of Chromatin) in BER of intact yeast cells. We show that depletion of STH1, the ATPase subunit of RSC, causes enhanced sensitivity to the DNA alkylating agent methyl methanesulfonate (MMS) and results in a substantial inhibition of BER, at the GAL1 locus and in the genome overall. Consistent with this observation, the DNA in chromatin is less accessible to micrococcal nuclease digestion in the absence of RSC. Quantitative PCR results indicate that repair deficiency in STH1 depleted cells is not due to changes in the expression of BER genes. Collectively, our data indicates the RSC complex promotes efficient BER in chromatin. These results provide, for the first time, a link between ATP-dependent chromatin remodelling and BER in living cells.

The RSC chromatin remodeling complex has a crucial role in the complete remodeler set for yeast PHO5 promoter opening

Although yeast PHO5 promoter chromatin opening is a founding model for chromatin remodeling, the complete set of involved remodelers remained unknown for a long time. The SWI/SNF and INO80 remodelers cooperate here, but nonessentially, and none of the many tested single or combined remodeler gene mutations could prevent PHO5 promoter opening. RSC, the most abundant and only remodeler essential for viability, was a controversial candidate for the unrecognized remodeling activity but unassessed in vivo. Now we show that remodels the structure of chromatin (RSC) is crucially involved in PHO5 promoter opening. Further, the isw1 chd1 double deletion also delayed chromatin remodeling. Strikingly, combined absence of RSC and Isw1/Chd1 or Snf2 abolished for the first time promoter opening on otherwise sufficient induction in vivo. Together with previous findings, we recognize now a surprisingly complex network of five remodelers (RSC, SWI/SNF, INO80, Isw1 and Chd1) from four subfamilies (SWI/SNF, INO80, ISWI and CHD) as involved in PHO5 promoter chromatin remodeling. This is likely the first described complete remodeler set for a physiological chromatin transition. RSC was hardly involved at the coregulated PHO8 or PHO84 promoters despite cofactor recruitment by the same transactivator and RSC’s presence at all three promoters. Therefore, promoter-specific chromatin rather than transactivators determine remodeler requirements.

The BAH domain of Rsc2 is a histone H3 binding domain

Bromo-adjacent homology (BAH) domains are commonly found in chromatin-associated proteins and fall into two classes; Remodels the Structure of Chromatin (RSC)-like or Sir3-like. Although Sir3-like BAH domains bind nucleosomes, the binding partners of RSC-like BAH domains are currently unknown. The Rsc2 subunit of the RSC chromatin remodeling complex contains an RSC-like BAH domain and, like the Sir3-like BAH domains, we find Rsc2 BAH also interacts with nucleosomes. However, unlike Sir3-like BAH domains, we find that Rsc2 BAH can bind to recombinant purified H3 in vitro, suggesting that the mechanism of nucleosome binding is not conserved. To gain insight into the Rsc2 BAH domain, we determined its crystal structure at 2.4 Å resolution. We find that it differs substantially from Sir3-like BAH domains and lacks the motifs in these domains known to be critical for making contacts with histones. We then go on to identify a novel motif in Rsc2 BAH that is critical for efficient H3 binding in vitro and show that mutation of this motif results in defective Rsc2 function in vivo. Moreover, we find this interaction is conserved across Rsc2-related proteins. These data uncover a binding target of the Rsc2 family of BAH domains and identify a novel motif that mediates this interaction.

Temporal sequence and cell cycle cues in the assembly of host factors at the yeast 2 micron plasmid partitioning locus

The Saccharomyces cerevisiae 2 micron plasmid exemplifies a benign but selfish genome, whose stability approaches that of the chromosomes of its host. The plasmid partitioning locus STB (stability locus) displays certain functional analogies with centromeres along with critical distinctions, a significant one being the absence of the kinetochore complex at STB. The remodels the structure of chromatin (RSC) chromatin remodeling complex, the nuclear motor Kip1, the histone H3 variant Cse4 and the cohesin complex associate with both loci. These factors appear to contribute to plasmid segregation either directly or indirectly through their roles in chromosome segregation. Assembly and disassembly of the plasmid-coded partitioning proteins Rep1 and Rep2 and host factors at STB follow a temporal hierarchy during the cell cycle. Assembly is initiated by STB association of [Rsc8-Rsc58], followed by [Rep1-Rep2-Kip1] and [Cse4-Rsc2-Sth1] recruitment, and culminates in cohesin assembly. Disassembly starts with dissociation of RSC components, is followed by cohesin disassembly and Cse4 exit during anaphase and late telophase, respectively. [Rep1-Rep2-Kip1] persists through G1 of the ensuing cell cycle. The de novo assembly of the ‘partitioning complex’ is cued by the innate cell cycle clock and is dependent on DNA replication. Shared functional attributes of STB and centromere (CEN) are consistent with a potential evolutionary link between them.

A role for chromatin remodellers in replication of damaged DNA

In eukaryotic cells, replication past damaged sites in DNA is regulated by the ubiquitination of proliferating cell nuclear antigen (PCNA). Little is known about how this process is affected by chromatin structure. There are two isoforms of the Remodels the Structure of Chromatin (RSC) remodelling complex in yeast. We show that deletion of RSC2 results in a dramatic reduction in the level of PCNA ubiquitination after DNA-damaging treatments, whereas no such effect was observed after deletion of RSC1. Similarly, depletion of the BAF180 component of the corresponding PBAF (Polybromo BRG1 (Brahma-Related Gene 1) Associated Factor) complex in human cells led to a similar reduction in PCNA ubiquitination. Remarkably, we found that depletion of BAF180 resulted after UV-irradiation, in a reduction not only of ubiquitinated PCNA but also of chromatin-associated unmodified PCNA and Rad18 (the E3 ligase that ubiquitinates PCNA). This was accompanied by a modest decrease in fork progression. We propose a model to account for these findings that postulates an involvement of PBAF in repriming of replication downstream from replication forks blocked at sites of DNA damage. In support of this model, chromatin immunoprecipitation data show that the RSC complex in yeast is present in the vicinity of the replication forks, and by extrapolation, this is also likely to be the case for the PBAF complex in human cells.