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The Mo-dependent enzyme YiiM enzyme from Escherichia coli is a member of the sulfite oxidase family and shares many similarities with the well-studied human mitochondrial amidoxime reducing component (mARC). We have investigated YiiM catalysis using electrochemical and spectroscopic methods. EPR monitored redox potentiometry found the active site redox potentials to be Mo

The endogenous yeast 2-μm plasmid while innocuous to the host, needs to be properly regulated to avoid a toxic increase in copy number. The plasmid copy number control system is under the control of the plasmid encoded recombinase, Flp1. In case of a drop in 2-μm plasmid levels due to rare plasmid mis-segregation events, the Flp1 recombinase together with the cell’s homologous recombination machinery, produce multiple copies of the 2-μm plasmid that are spooled during DNA replication. The 2-μm plasmid copy number is tightly regulated by controlled expression of Flp1 as well as its ubiquitin and SUMO modification. Here, we identify a novel regulator of the 2-μm plasmid, the ATPase, ubiquitin ligase, Irc20. Irc20 was initially identified as a homologous recombination regulator, and here we uncover a new role for Irc20 in maintaining the 2-μm plasmid copy number and segregation through regulating Flp1 protein levels in the cell.

Fun30 is an ATP dependent chromatin remodeler in budding yeast that is involved in cellular processes important for maintaining genomic stability such as gene silencing and DNA damage repair. Cells lacking Fun30 are moderately sensitive to the Topoisomerase inhibitor camptothecin and exhibit a delay in cell cycle progression in the presence of camptothecin. Here, we show that Fun30 is required to cope with torsional stress in the absence of Top1. Moreover, we show through genetic studies that Fun30 acts in a parallel pathway to Mus81 endonuclease but is epistatic to Tdp1 phosphodiesterase and Rad1 endonuclease in the repair of camptothecin induced DNA damage. More importantly, we show that DNA damage sensitivity of Fun30 deficient cells are enhanced in the absence of RNase H enzymes that remove RNA:DNA hybrids. We believe that, chromatin remodeling by Fun30 may be important in dealing with torsional stress and camptothecin induced DNA damage.

DNA double-strand breaks (DSBs) constitute one of the most cytotoxic forms of DNA damage and pose a significant threat to cell viability, survival, and homeostasis. DSBs have the potential to promote aneuploidy, cell death and potentially deleterious mutations that promote tumorigenesis. Homologous recombination (HR) is one of the main DSB repair pathways and while being essential for cell survival under genotoxic stress, it requires proper regulation to avoid chromosome rearrangements. Here, we characterize the Saccharomyces cerevisiae E3 ubiquitin ligase/putative helicase Irc20 as a regulator of HR. Using purified Irc20, we show that it can hydrolyze ATP in the presence and absence of DNA, but does not increase access to DNA within a nucleosome. In addition, we show that both the ATPase and ubiquitin ligase activities of Irc20 are required for suppressing the spontaneous formation of recombination foci. Finally, we demonstrate a role for Irc20 in promoting Rad51 chromatin association and the removal of Rad52 recombinase from chromatin, thus facilitating subsequent HR steps and directing recombination to more error-free modes.

Sulfite is a neurotoxin, which is detoxified by the molybdenum cofactor (Moco)-dependent enzyme sulfite oxidase (SOX). In humans, SOX deficiency causes the formation of the glutamate analog S-Sulfocysteine (SSC) resulting in a constant overstimulation of ionotropic glutamatergic receptors. Overstimulation leads to seizures, severe brain damage, and early childhood death. SOX deficiency may be caused either by a mutated sox gene or by mutations in one of the genes of the multi-step Moco biosynthesis pathway. While patients affected in the first step of Moco biosynthesis can be treated by a substitution therapy, no therapy is available for patients affected either in the second or third step of Moco biosynthesis or with isolated SOX deficiency. In the present study, we used a combination of behavior analysis and vital dye staining to show that SSC induces increased swimming, seizure-like movements, and increased cell death in the central nervous system of zebrafish larvae. Seizure-like movements were fully revertible upon removal of SSC or could be alleviated by a glutamatergic receptor antagonist. We conclude that in zebrafish SSC can chemically induce phenotypic characteristics comparable to the disease condition of human patients lacking SOX activity.

The SWI/SNF chromatin-remodeling complex contains a bromodomain in its Swi2/Snf2 subunit that helps tether it to acetylated promoter nucleosomes. To study the importance of this bromodomain in the SWI/SNF complex, we have compared the nucleosome-binding and the chromatin-remodeling activities of the SWI/SNF to a mutant complex that lacks the Swi2/Snf2 bromodomain. Here we show that the SWI/SNF complex deleted of the Swi2/Snf2 bromodomain cannot bind to SAGA- or NuA4-acetylated nucleosomes as well as the wild-type complex. Moreover, we show that this reduced binding leads to partial remodeling of these acetylated nucleosome templates by the Deltabromodomain SWI/SNF complex. These results demonstrate that the Swi2/Snf2 bromodomain is required for the full binding and functional activity of the SWI/SNF complex on H3- and H4-acetylated nucleosomes.

The linker histones are involved in the salt-dependent folding of the nucleosomes into higher-order chromatin structures. To better understand the mechanism of action of these histones in chromatin, we studied the interactions of the linker histone H1 with DNA at various histone/DNA ratios and at different ionic strengths. In direct competition experiments, we have confirmed the binding of H1 to superhelical DNA in preference to linear or nicked circular DNA forms. We show that the electrophoretic mobility of the H1/supercoiled DNA complex decreases with increasing H1 concentrations and increases with ionic strengths. These results indicate that the interaction of the linker histone H1 with supercoiled DNA results in a soluble binding of H1 with DNA at low H1 or salt concentrations and aggregation at higher H1 concentrations. Moreover, we show that H1 dissociates from the DNA or nucleosomes at high salt concentrations. By the immobilized template pull-down assay, we confirm these data using the physiologically relevant nucleosome array template.

The SWI/SNF and SAGA chromatin-modifying complexes contain bromodomains that help anchor these complexes to acetylated promoter nucleosomes. To study the importance of bromodomains in these complexes, we have compared the chromatin-remodeling and octamer-transfer activity of the SWI/SNF complex to a mutant complex that lacks the Swi2/Snf2 bromodomain. Here we show that the SWI/SNF complex can remodel or transfer SAGA-acetylated nucleosomes more efficiently than the Swi2/Snf2 bromodomain-deleted complex. These results demonstrate that the Swi2/Snf2 bromodomain is important for the remodeling as well as for the octamer-transfer activity of the complex on H3-acetylated nucleosomes. Moreover, we show that, although the wild-type SWI/SNF complex displaces SAGA that is bound to acetylated nucleosomes, the bromodomain mutant SWI/SNF complex is less efficient in SAGA displacement. Thus, the Swi2/Snf2 bromodomain is required for the full functional activity of SWI/SNF on acetylated nucleosomes and is important for the displacement of SAGA from acetylated promoter nucleosomes.

We have previously shown that the yeast SWI/SNF complex stimulates in vitro transcription from chromatin templates in an ATP-dependent manner. SWI/SNF function in this regard requires the presence of an activator with which it can interact directly, linking activator recruitment of SWI/SNF to transcriptional stimulation. In this study, we determine the SWI/SNF subunits that mediate its interaction with activators. Using a photo-cross-linking label transfer strategy, we show that the Snf5, Swi1, and Swi2/Snf2 subunits are contacted by the yeast acidic activators, Gcn4 and Hap4, in the context of the intact native SWI/SNF complex. In addition, we show that the same three subunits can interact individually with acidic activation domains, indicating that each subunit contributes to binding activators. Furthermore, mutations that reduce the activation potential of these activators also diminish its interaction with each of these SWI/SNF subunits. Thus, three distinct subunits of the SWI/SNF complex contribute to its interactions with activation domains.

The genome of the cell is often exposed to DNA damaging agents and therefore requires an intricate well-regulated DNA damage response (DDR) to overcome its deleterious effects. The DDR needs proper regulation for its timely activation, repression, as well as appropriate choice of repair pathway. Studies in Saccharomyces cerevisiae have advanced our understanding of the DNA damage response, as well as the mechanisms the cell employs to maintain genome stability and how these mechanisms are regulated. Eukaryotic cells utilize post-translational modifications as a means for fine-tuning protein functions. Ubiquitylation and SUMOylation involve the attachment of small protein molecules onto proteins to modulate function or protein–protein interactions. SUMO in particular, was shown to act as a molecular glue when DNA damage occurs, facilitating the assembly of large protein complexes in repair foci. In other instances, SUMOylation alters a protein's biochemical activities, and interactions. SUMO-targeted ubiquitin ligases (STUbLs) are enzymes that target SUMOylated proteins for ubiquitylation and subsequent degradation, providing a function for the SUMO modification in the regulation and disassembly of repair complexes. Here, we discuss the major contributions of SUMO and STUbLs in the regulation of DNA damage repair pathways as well as in the maintenance of critical regions of the genome, namely rDNA regions, telomeres and the 2 μm circle in budding yeast.

To investigate the function of SWI/SNF in site-specific chromatin remodeling at promoters, we have used a purified system to analyze its distribution, function, and retention following recruitment by a sequence-specific transcription activator. Activator recruitment of SWI/SNF bound the complex to promoter proximal nucleosomes and led to localized nucleosome disruption. However, retention of SWI/SNF on the promoter required either the continued binding of the transcription activator or acetylated histones. Histone acetylation by either the SAGA or NuA4 HAT complexes increased the retention of SWI/SNF on the promoter. These data illustrate a functional link between HAT complexes and the SWI/SNF chromatin remodeling complex and provide a mechanistic basis for the ordered recruitment of these complexes.

The importance of histones and chromatin structure in the regulation of eukaryotic gene transcription has become much more widely accepted over the past few years. It has been clear for a decade that histones contribute to the regulation of transcription both in vitro and in vivo (reviewed in references 14, 34, 50, 64, and 120). More recent studies have led to the striking observation that several protein complexes involved in transcription regulation can function, at least in part, by modifying histones or altering chromatin structure (for recent reviews, see references 3, 44, 49, 51, 52, 87, 100, and 119). While it is clear that many of these protein complexes have functions in addition to chromatin modification, they illustrate the importance of chromatin structure as a part of transcription regulation mechanisms. The most widely characterized chromatin-modifying complexes studied to date can be classified into two major groups, based on their modes of action, as follows: (i) ATP-dependent complexes, which use the energy of ATP hydrolysis to locally disrupt or alter the association of histones with DNA, and (ii) histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes, which regulate the transcriptional activity of genes by determining the level of acetylation of the amino-terminal domains of nucleosomal histones associated with them. This review will focus primarily on the ATP-dependent remodeling complexes. For recent reviews of HAT and HDAC complexes, see references 5, 20, 31, and 55. Here we provide an organized listing of the ATP-dependent chromatin-remodeling complexes described to date and illustrate the relationships between their subunits. We also review the data available with regard to their mechanisms of action and promoter targeting as well as regulation of their activity. Finally, we examine the relationship between these complexes and the HAT complexes.

Summary regions in chromatin. The possibility that SWI/SNF is targeted by transcription activators is consistent with The yeast SWI/SNF complex is required for the tran- several results. Interactions between the SWI/SNF comscription of several yeast genes and has been shown plex and the glucocorticoid receptor (Yoshinaga et al., to alter nucleosome structure in an ATP-dependent 1992; Fryer and Archer, 1998) and the lymphoid-specific reaction. In this study, we show that the complex stimIkaros DNA-binding proteins (Kim et al., 1999) have been ulated in vitro transcription from nucleosome tem- reported in cell extracts. A SWI/SNF-related complex plates in an activation domain‐dependent manner. was purified as an essential cofactor for the erythroid Transcription stimulation by SWI/SNF required an acti- transcription activator EKLF (Armstrong et al., 1998). vation domain with which it directly interacts. The Moreover, recent chromatin immunoprecipitation studacidic activation domains of VP16, Gcn4, Swi5, and ies in yeast suggest that interaction of the SWI/SNF Hap4 interacted directly with the purified SWI/SNF complex with the HO endonuclease gene promoter folcomplex and with the SWI/SNF complex in whole-cell lows the binding of the Swi5 transcription activator

We have identified two Gcn5-dependent histone acetyltransferase (HAT) complexes from Saccharomyces cerevisiae, the 0.8-MDa ADA complex and the 1.8-MDa SAGA complex. The SAGA (Spt-Ada-Gcn5-acetyltransferase) complex contains several subunits which also function as part of other protein complexes, including a subset of TATA box binding protein-associated factors (TAFIIs) and Tra1. These observations raise the question of whether the 0.8-MDa ADA complex is a subcomplex of SAGA or whether it is a distinct HAT complex that also shares subunits with SAGA. To address this issue, we sought to determine if the ADA complex contained subunits that are not present in the SAGA complex. In this study, we report the purification of the ADA complex over 10 chromatographic steps. By a combination of mass spectrometry analysis and immunoblotting, we demonstrate that the adapter proteins Ada2, Ada3, and Gcn5 are indeed integral components of ADA. Furthermore, we identify the product of the S. cerevisiae gene YOR023C as a novel subunit of the ADA complex and name it Ahc1 for ADA HAT complex component 1. Biochemical functions of YOR023C have not been reported. However, AHC1 in high copy numbers suppresses the cold sensitivity caused by particular mutations in HTA1 (I. Pinto and F. Winston, personal communication), which encodes histone H2A (J. N. Hirschhorn et al., Mol. Cell. Biol. 15:1999-2009, 1995). Deletion of AHC1 disrupted the integrity of the ADA complex but did not affect SAGA or give rise to classic Ada(-) phenotypes. These results indicate that Gcn5, Ada2, and Ada3 function as part of a unique HAT complex (ADA) and represent shared subunits between this complex and SAGA.

The preference of the linker histones to bind to superhelical DNA in comparison with linear or relaxed molecules suggests that these proteins might, in turn, change the twist and/or writhe of DNA molecules upon binding. In order to explore such a possibility, we looked for changes in the linking number of plasmid pBR322 caused by H1 binding, using assays that involve nicking and resealing of DNA strands. Two types of enzymes were used, eukaryotic topoisomerase I and prokaryotic DNA ligase. The results revealed that H1 binding causes unwinding of the DNA, with the unwinding angle being approximately 10°. The globular domain of histone H1 is also capable of unwinding DNA, but to a lesser degree.

The Saccharomyces cerevisiae Fun30 (Function unknown now 30) protein shares homology with an extended family of Snf2-related ATPases. Here we report the purification of Fun30 principally as a homodimer with a molecular mass of about 250 kDa. Biochemical characterization of this complex reveals that it has ATPase activity stimulated by both DNA and chromatin. Consistent with this, it also binds to both DNA and chromatin. The Fun30 complex also exhibits activity in ATP-dependent chromatin remodeling assays. Interestingly, its activity in histone dimer exchange is high relative to the ability to reposition nucleosomes. Fun30 also possesses a weakly conserved CUE motif suggesting that it may interact specifically with ubiquitinylated proteins. However, in vitro Fun30 was found to have no specificity in its interaction with ubiquitinylated histones.

Bromodomains are present in many chromatin-associated proteins such as the SWI/SNF and RSC chromatin remodelling and the SAGA HAT (histone acetyltransferase) complexes, and can bind to acetylated lysine residues in the N-terminal tails of the histones. Lysine acetylation is a histone modification that forms a stable epigenetic mark on chromatin for bromodomain-containing proteins to dock and in turn regulate gene expression. In order to better understand how bromodomains read the ‘histone code’ and interact with acetylated histones, we have tested the interactions of several bromodomains within transcriptional co-activators with differentially acetylated histone tail peptides and HAT-acetylated histones. Using GST (glutathione S-transferase) pull-down assays, we show specificity of binding of some bromodomains to differentially acetylated H3 and H4 peptides as well as HAT-acetylated histones. Our results reveal that the Swi2/Snf2 bromodomain interacts with various acetylated H3 and H4 peptides, whereas the Gcn5 bromodomain interacts only with acetylated H3 peptides and tetra-acetylated H4 peptides. Additionally we show that the Spt7 bromodomain interacts with acetylated H3 peptides weakly, but not with acetylated H4 peptides. Some bromodomains such as the Bdf1-2 do not interact with most of the acetylated peptides tested. Results of the peptide experiments are confirmed with tests of interactions between these bromodomains and HAT-acetylated histones. Furthermore, we demonstrate that the Swi2/Snf2 bromodomain is important for the binding and the remodelling activity of the SWI/SNF complex on hyperacetylated nucleosomes. The selective recognition of the bromodomains observed in the present study accounts for the broad effects of bromodomain-containing proteins observed on binding to histones.

Publisher Summary This chapter describes an in vitro assay for analyzing activator recruitment of chromatin-modifying complexes to a nucleosomal promoter. The template used in this assay contains five Gal4 binding sites upstream of the Adenovirus E4 core promoter and five sea urchin 5S nucleosome positioning sequences flanking each at end of the 5XGal4-E4 promoter. These positioning sequences result in twelve nucleosomes, with two over the 5XGal-E4 promoter. The use of a nucleosomal template immobilized on paramagnetic beads is central to this assay. The immobilization of the template allows removal and addition of soluble components in the recruitment reactions by collecting templates on a magnetic concentrator and washing the beads. For example, chromatin-modifying complexes, such as the SAGA histone acetyltransferase and SWI/SNF ATP-dependent chromatin-remodeling complexes can be targeted to a nucleosomal promoter by a transcription activator protein. Afterwards, the activator protein is competed from the template using sequence-specific competitor DNA and the template is washed. The ability of the complexes to remain bound to the promoter in the absence of an activator can then be assessed by western analysis for different components of the reaction, such as acetylated histones, the activator protein, and chromatin-modifying complex components.

The SWI-SNF complex has been shown to alter nucleosome conformation in an ATP-dependent manner, leading to increased accessibility of nucleosomal DNA to transcription factors. In this study, we show that the SWI-SNF complex can potentiate the activity of the glucocorticoid receptor (GR) through the N-terminal transactivation domain, tau1, in both yeast and mammalian cells. GR-tau1 can directly interact with purified SWI-SNF complex, and mutations in tau1 that affect the transactivation activity in vivo also directly affect tau1 interaction with SWI-SNF. Furthermore, the SWI-SNF complex can stimulate tau1-driven transcription from chromatin templates in vitro. Taken together, these results support a model in which the GR can directly recruit the SWI-SNF complex to target promoters during glucocorticoid-dependent gene activation. We also provide evidence that the SWI-SNF and SAGA complexes represent independent pathways of tau1-mediated activation but play overlapping roles that are able to compensate for one another under some conditions.