Hap1 Activation Research Articles

A Unique Mechanism of Chaperone Action: Heme Regulation of Hap1 Activity Involves Separate Control of Repression and Activation

The Hsp90 and Hsp70 molecular chaperones play important roles in the folding and proper functioning of diverse cellular proteins, including transcriptional regulators and protein kinases. In yeast, several transcriptional regulators and protein kinases are known to be substrates for Hsp90 and Hsp70 molecular chaperones. The yeast heme activator protein Hap1 promotes transcription of many genes in response to heme. It requires Hsp90 and Hsp70 molecular chaperones for its activity to be precisely regulated by heme concentration. The mechanism by which molecular chaperones promote heme regulation of Hap1 activity is distinct from the mechanism by which molecular chaperones promote steroid signaling. Hsp70 and Hsp90 molecular chaperones act separately to promote Hap1 repression in heme-deficient cells and heme activation of Hap1 in heme-sufficient cells. Likewise, distinct Hap1 elements or domains act to mediate Hap1 repression and heme activation separately. In this review, we summarize the current knowledge about the molecular mechanism governing heme regulation of Hap1 activity, and we compare this mechanism to the molecular mechanism by which Hsp90 and Hsp70 molecular chaperones promote the regulation of glucocorticoid receptor, the most extensively studied substrate of Hsp90 and Hsp70 molecular chaperones.

Heme levels switch the function of Hap1 of Saccharomyces cerevisiae between transcriptional activator and transcriptional repressor.

Changes in oxygen levels cause widespread changes in gene expression in organisms ranging from bacteria to humans. In Saccharomyces cerevisiae, this response is mediated in part by Hap1, originally identified as a heme-dependent transcriptional activator that functions during aerobic growth. We show here that Hap1 also plays a significant and direct role under hypoxic conditions, not as an activator, but as a repressor. The repressive activity of Hap1 controls several genes, including three ERG genes required for ergosterol biosynthesis. Chromatin immunoprecipitation experiments showed that Hap1 binds to the ERG gene promoters, while additional experiments showed that the corepressor Tup1/Ssn6 is recruited by Hap1 and is also required for repression. Furthermore, mutational analysis demonstrated that conserved Hap1 binding sites in the ERG5 5' regulatory region are required for repression. The switch of Hap1 from acting as a hypoxic repressor to an aerobic activator is determined by heme, which is synthesized only in the presence of oxygen. The ability of Hap1 to function as a ligand-dependent repressor and activator is a property shared with mammalian nuclear hormone receptors and likely allows greater transcriptional control by Hap1 in response to changing oxygen levels.

A nucleosome positioned by α2/Mcm1 prevents Hap1 activator binding in vivo

Nucleosome positioning has been proposed as a mechanism of transcriptional repression. Here, we examined whether nucleosome positioning affects activator binding in living yeast cells. We introduced the cognate Hap1 binding site (UAS1) at a location 24–43bp, 29–48bp, or 61–80bp interior to the edge of a nucleosome positioned by α2/Mcm1 in yeast minichromosomes. Hap1 binding to the UAS1 was severely inhibited, not only at the pseudo-dyad but also in the peripheral region of the positioned nucleosome in α cells, while it was detectable in a cells, in which the nucleosomes were not positioned. Hap1 binding was restored in α cells with tup1 or isw2 mutations, which caused the loss of nucleosome positioning. These results support the mechanism in which α2/Mcm1-dependent nucleosome positioning has a regulatory function to limit the access of transcription factors.

The Heme Activator Protein Hap1 Represses Transcription by a Heme-Independent Mechanism in Saccharomyces cerevisiae

The yeast heme activator protein Hap1 binds to DNA and activates transcription of genes encoding functions required for respiration and for controlling oxidative damage, in response to heme. Hap1 contains a DNA-binding domain with a C6 zinc cluster motif, a coiled-coil dimerization element, typical of the members of the yeast Gal4 family, and an acidic activation domain. The regulation of Hap1 transcription-activating activity is controlled by two classes of Hap1 elements, repression modules (RPM1-3) and heme-responsive motifs (HRM1-7). Previous indirect evidence indicates that Hap1 may repress transcription directly. Here we show, by promoter analysis, by chromatin immunoprecipitation, and by electrophoretic mobility shift assay, that Hap1 binds directly to DNA and represses transcription of its own gene by at least 20-fold. We found that Hap1 repression of the HAP1 gene occurs independently of heme concentrations. While DNA binding is required for transcriptional repression by Hap1, deletion of Hap1 activation domain and heme-regulatory elements has varying effects on repression. Further, we found that repression by Hap1 requires the function of Hsp70 (Ssa), but not Hsp90. These results show that Hap1 binds to its own promoter and represses transcription in a heme-independent but Hsp70-dependent manner.

A Novel Mode of Chaperone Action: HEME ACTIVATION OF Hap1 BY ENHANCED ASSOCIATION OF Hsp90 WITH THE REPRESSED Hsp70-Hap1 COMPLEX

Molecular chaperones Hsp90 and Hsp70 control many signal transducers, including cyclin-dependent kinases and steroid receptors. The yeast heme-responsive transcriptional activator Hap1 is a native substrate of both Hsp90 and Hsp70. Hsp90 and Hsp70 are critical for the precise regulation of Hap1 activity by heme. Here, to decipher the molecular events underlying the actions of Hsp90 and Hsp70 in heme regulation, we purified various multichaperone-Hap1 complexes and characterized the complexes linked to Hap1 repression and activation by two-dimensional electrophoresis analysis. Notably, we found that in vitro Hap1 is associated continuously with Ssa and its co-chaperones, and this association is not weakened by heme. Heme enhances the interaction between Hap1 and Hsp90. In vivo, defective Ssa, Ydj1, or Sro9 function causes Hap1 derepression in the absence of heme, whereas defective Hsp90 function causes reduced Hap1 activity at high heme concentrations. These results show that continuous association of Hap1 with Ssa, Ydj1, and Sro9 confers Hap1 repression, whereas enhanced association of Hsp90 with the repressed Hap1-Ssa-Ydj1-Sro9 complex by heme causes Hap1 activation. This novel mechanism of chaperone action may operate to control the activity of other important signal transducers.

A Mechanism of Oxygen Sensing in Yeast: MULTIPLE OXYGEN-RESPONSIVE STEPS IN THE HEME BIOSYNTHETIC PATHWAY AFFECT Hap1 ACTIVITY

Heme plays central roles in oxygen sensing and utilization in many living organisms. In yeast, heme mediates the effect of oxygen on the expression of many genes involved in using or detoxifying oxygen. However, a direct link between intracellular heme level and oxygen concentration has not been vigorously established. In this report, we have examined the relationships among oxygen levels, heme levels, Hap1 activity, and HAP1 expression. We found that Hap1 activity is controlled in vivo by heme and not by its precursors and that heme activates Hap1 even in anoxic cells. We also found that Hap1 activity exhibits the same oxygen dose-response curves as Hap1-dependent aerobic genes and that these dose-response curves have a sharp break at approximately 1 microM O2. The results show that the intracellular signaling heme level, reflected as Hap1 activity, is closely correlated with oxygen concentration. Furthermore, we found that bypass of all heme synthetic steps but ferrochelatase by deuteroporphyrin IX does not circumvent the need for oxygen in Hap1 full activation by heme, suggesting that the last step of heme synthesis, catalyzed by ferrochelatase, is also subjected to oxygen control. Our results show that multiple heme synthetic steps can sense oxygen concentration and provide significant insights into the mechanism of oxygen sensing in yeast.

Structural environment dictates the biological significance of heme-responsive motifs and the role of Hsp90 in the activation of the heme activator protein Hap1.

Heme-responsive motifs (HRMs) mediate heme regulation of diverse regulatory proteins. The heme activator protein Hap1 contains seven HRMs, but only one of them, HRM7, is essential for heme activation of Hap1. To better understand the molecular basis underlying the biological significance of HRMs, we examined the effects of various mutations of HRM7 on Hap1. We found that diverse mutations of HRM7 significantly diminished the extent of Hap1 activation by heme and moderately enhanced the interaction of Hap1 with Hsp90. Furthermore, deletions of nonregulatory sequences completely abolished heme activation of Hap1 and greatly enhanced the interaction of Hap1 with Hsp90. These results show that the biological functions of HRMs and Hsp90 are highly sensitive to structural changes. The unique role of HRM7 in heme activation stems from its specific structural environment, not its mere presence. Likewise, the role of Hsp90 in Hap1 activation is dictated by the conformational or structural state of Hap1, not by the mere strength of Hap1-Hsp90 interaction. It appears likely that HRM7 and Hsp90 act together to promote the Hap1 conformational changes that are necessary for Hap1 activation. Such fundamental mechanisms of HRM-Hsp90 cooperation may operate in diverse regulatory systems to mediate signal transduction.

Reconstitution of the base excision repair pathway for 7,8-dihydro-8-oxoguanine with purified human proteins.

In mammalian cells, repair of the most abundant endogenous premutagenic lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is initiated by the bifunctional DNA glycosylase OGG1. By using purified human proteins, we have reconstituted repair of 8-oxoG lesions in DNA in vitro on a plasmid DNA substrate containing a single 8-oxoG residue. It is shown that efficient and complete repair requires only hOGG1, the AP endonuclease HAP1, DNA polymerase (Pol) beta and DNA ligase I. After glycosylase base removal, repair occurred through the AP lyase step of hOGG1 followed by removal of the 3'-terminal sugar phosphate by the 3'-diesterase activity of HAP1. Addition of PCNA had a slight stimulatory effect on repair. Fen1 or high concentrations of Pol beta were required to induce strand displacement DNA synthesis at incised 8-oxoG in the absence of DNA ligase. Fen1 induced Pol beta strand displacement DNA synthesis at HAP1-cleaved AP sites differently from that at gaps introduced by hOGG1/HAP1 at 8-oxoG sites. In the presence of DNA ligase I, the repair reaction at 8-oxoG was confined to 1 nt replacement, even in the presence of high levels of Pol beta and Fen1. Thus, the assembly of all the core proteins for 8-oxoG repair catalyses one major pathway that involves single nucleotide repair patches.

The Molecular Chaperone Hsp90 Mediates Heme Activation of the Yeast Transcriptional Activator Hap1

Hsp90 plays critical roles in the proper functioning of a wide array of eukaryotic signal transducers such as steroid receptors and tyrosine kinases. Hap1 is a naturally occurring substrate of Hsp90 in Saccharomyces cerevisiae. Hap1 transcriptional activity is precisely and stringently controlled by heme. Previous biochemical studies suggest that in the absence of heme, Hap1 is bound to Hsp90 and other proteins, forming a higher order complex termed HMC (high molecular weight complex), and is repressed. Heme promotes the disruption of the HMC and activates Hap1, permitting Hap1 to bind to DNA with high affinity and to stimulate transcription. By lowering the expression levels of wild-type Hsp90, using a highly specific Hsp90 inhibitor, and by examining the effects of various Hsp90 mutants on Hap1, we show that Hsp90 is critical for Hap1 activation by heme. Furthermore, we show that many Hsp90 mutants exert differential effects on Hap1 and steroid receptors. Notably, mutant G313N weakens Hsp90 steroid receptor interaction but strongly enhances Hsp90-Hap1 interaction and increases Hap1 resistance to protease digestion. Additionally, we found that a heme-independent Hap1 mutant still depends on Hsp90 for high activity. These experiments together suggest that Hsp90 promotes Hap1 activation by inducing or maintaining Hap1 in a transcriptionally active conformation.

The Hsp70-Ydj1 molecular chaperone represses the activity of the heme activator protein Hap1 in the absence of heme.

In Saccharomyces cerevisiae, heme directly mediates the effects of oxygen on transcription through the heme activator protein Hap1. In the absence of heme, Hap1 is bound by at least four cellular proteins, including Hsp90 and Ydj1, forming a higher-order complex, termed HMC, and its activity is repressed. Here we purified the HMC and showed by mass spectrometry that two previously unidentified major components of the HMC are the Ssa-type Hsp70 molecular chaperone and Sro9 proteins. In vivo functional analysis, combined with biochemical analysis, strongly suggests that Ssa proteins are critical for Hap1 repression in the absence of heme. Ssa may repress the activities of both Hap1 DNA-binding and activation domains. The Ssa cochaperones Ydj1 and Sro9 appear to assist Ssa in Hap1 repression, and only Ydj1 residues 1 to 172 containing the J domain are required for Hap1 repression. Our results suggest that Ssa-Ydj1 and Sro9 act together to mediate Hap1 repression in the absence of heme and that molecular chaperones promote heme regulation of Hap1 by a mechanism distinct from the mechanism of steroid signaling.

Chaperone-Mediated Repression

Chaperone proteins are implicated not only in aiding the formation of properly folded and functional signaling proteins, but also in subsequent control of the activity of their protein targets. In particular the Hsp90- and Hsp70-type chaperone proteins take part in assembly of nuclear receptor complexes and control of transcription by those receptors (see STKE Perspective by DeFranco and Csermely ). Hon et al. now present evidence for similar, yet distinct, roles for chaperone proteins of these types in control of the heme activator protein (Hap1) in the yeast Saccharomyces cerevisiae . Yeast respond to oxygen with increased production of heme protein, which activates Hap1 transcription factor to promote transcription of genes whose products function in respiration and protection from oxygen damage. In the absence of heme, the transcriptional activity of Hap1 is normally repressed by interactions with cellular proteins, including Hsp90 and now the Hsp70 proteins designated Ssa and Sro9. Decreased expression of the Hsp70 Ssa (and, to a lesser extent, decreased expression of Sro or its partner Ydj1) caused enhanced transcriptional and DNA binding activity of Hap1 in the absence of heme, but did not affect Hap1 activity in the presence of high concentrations of heme. This contrasts with the effect of Hsp90 on Hap1 or on nuclear receptors. Loss of Hsp90 does not lead to activation, but rather reduces transcriptional activity of its partners even in the presence of activators (steroid hormones or heme). The authors note that distinct modes of regulation of Hap1 activity by the Hsp70 and Hsp90 chaperones provide a means for precise regulation of Hap1 that reveals previously unrecognized versatility in the control of transcription in complexes with multiple chaperones. T. Hon, H. C. Lee, A. Hach, J. L. Johnson, E. A. Craig, H. Erdjument-Bromage, P. Tempst, L. Zhang, The Hsp70-Ydj1 molecular chaperone represses the activity of the heme activator protein Hap1 in the absence of heme. Mol. Cell. Biol . 21 , 7923-7932 (2001). [Abstract] [Full Text] D. B. DeFranco, P. Csermely, Steroid receptor and molecular chaperone encounters in the nucleus. Science's STKE (2000), http://www.stke.org/cgi/content/full/OC_sigtrans;2000/42/pe1 [Summary] [Full Text]

Functional Analysis of Heme Regulatory Elements of the Transcriptional Activator Hap1

Heme regulation of the activity of diverse proteins was thought to be mediated by heme-responsive motifs (HRMs). The yeast transcriptional activator Hap1 contains seven HRMs: HRM1–7. Three copies of a 17-amino-acid repeat are also located in the region encompassing HRM1 to -6. We examined the effects of these HRMs and repeats on heme regulation of Hap1 activity by deletion analysis and by Ala substitutions of key residues. We found that the effect of mutation or deletion of one HRM or 17-amino-acid repeat on Hap1 heme responsiveness is different from the effect of mutation or deletion of another HRM or repeat. Our data suggest that HRM7 plays a dominant role in mediating heme activation of Hap1 in heme-sufficient cells while HRM1–6 may scavenge heme and cause a low level of Hap1 activation in heme-deficient cells. These results may help in understanding the roles of HRMs in other hemoproteins.

Substitution of Asp-210 in HAP1 (APE/Ref-1) eliminates endonuclease activity but stabilises substrate binding.

HAP1, also known as APE/Ref-1, is the major apurinic/apyrimidinic (AP) endonuclease in human cells. Previous structural studies have suggested a possible role for the Asp-210 residue of HAP1 in the enzymatic function of this enzyme. Here, we demonstrate that substitution of Asp-210 by Asn or Ala eliminates the AP endonuclease activity of HAP1, while substitution by Glu reduces specific activity approximately 500-fold. Nevertheless, these mutant proteins still bind efficiently to oligonucleotides containing either AP sites or the chemically unrelated bulky p-benzoquinone (pBQ) derivatives of dC, dA and dG, all of which are substrates for HAP1. These results indicate that Asp-210 is required for catalysis, but not substrate recognition, consistent with enzyme kinetic data indicating that the HAP1-D210E protein has a 3000-fold reduced K(cat )for AP site cleavage, but an unchanged K(m). Through analysis of the binding of Asp-210 substitution mutants to oligonucleotides containing either an AP site or a pBQ adduct, we conclude that the absence of Asp-210 allows the formation of a stable HAP1-substrate complex that exists only transiently during the catalytic cycle of wild-type HAP1 protein. We interpret these data in the context of the structure of the HAP1 active site and the recently determined co-crystal structure of HAP1 bound to DNA substrates.

Fusions with histone H3 result in highly specific alteration of gene expression

Hap1 is a yeast transcriptional activator which controls expression of genes such as CYC1 and CYC7. Our results show that Hap1 activity is dependent on a functional chromatin remodeling complex SWI/SNF. Using a modified two-hybrid screen with Hap1 as bait, we recovered expression vectors encoding the Gal4 activation domain fused to histone H3 [Gal4(AD)-H3]. Hap1 activity at CYC1 or CYC7 was increased by Gal4(AD)-H3 and the effect was dependent on the presence of the activation domain of Hap1 and a functional SWI complex. Importantly, overexpression of H3 alone had no effect on Hap1 activity. Analysis of Gal4(AD)-H3 revealed that the fusion is not incorporated into the nucleosome while a functional Gal4 activation domain is dispensable. Activity of many other transcriptional activators was unchanged or slightly affected in the presence of Gal4(AD)-H3. Thus, our results identify a new class of histone H3 variants that cause highly specific alteration of gene expression. Hap1 may interact directly with H3 favoring chromatin remodeling by the SWI/SNF complex.

Molecular mechanism of heme signaling in yeast: the transcriptional activator Hap1 serves as the key mediator.

Heme is a key molecule in mediating the effects of oxygen on various molecular and cellular processes in many living organisms. In the yeast Saccharomyces cerevisiae, heme serves as a secondary signal for oxygen; intracellular heme synthesis directly correlates with oxygen tension in the environment. In yeast, oxygen sensing and heme signaling are primarily mediated by the heme activator protein Hap1, which, in response to heme, activates the transcription of genes required for respiration and for controlling oxidative damage. Heme regulation of many genes required for anaerobic growth is mediated by the aerobic repressor Rox1, whose expression is controlled by heme. In this review, we summarize recent knowledge about (i) how heme synthesis may be controlled by oxygen tension, (ii) how heme precisely and stringently controls Hap1 activity and (iii) whether other transcriptional activators can also mediate heme action.

The Yeast Heme-responsive Transcriptional Activator Hap1 Is a Preexisting Dimer in the Absence of Heme

In the absence of heme, Hap1 is associated with molecular chaperones such as Hsp90 and Ydj1 and forms a higher order complex termed HMC. Heme disrupts this complex and permits Hap1 to bind to DNA with high affinity, thereby activating transcription. Heme regulation of Hap1 activity is analogous to the regulation of steroid receptors by steroids, which involves molecular chaperones. Steroid receptors often exist as monomers when associated with molecular chaperones in the absence of ligand but as dimers when activated by steroids. Furthermore, previous studies indicate that dimerization might be important for heme activation of Hap1. We therefore determined whether Hap1 is a monomer or oligomer in the absence of heme. By coeluting two Hap1 size variants and by comparing DNA binding properties of the HMC and Hap1 dimer, we show that Hap1 is a preexisting dimer in the HMC. Further, increasing overexpression of Hap1 caused progressive increases in Hap1 DNA binding and transcriptional activities. Our data suggest that in the absence of heme, Hap1 exists as a dimer, and the two subunits act cooperatively in DNA binding. Hap1 repression is caused, at least in part, by inhibition of the DNA binding activity of the preexisting dimer.

A new class of repression modules is critical for heme regulation of the yeast transcriptional activator Hap1.

Heme plays key regulatory roles in numerous molecular and cellular processes for systems that sense or use oxygen. In the yeast Saccharomyces cerevisiae, oxygen sensing and heme signaling are mediated by heme activator protein 1 (Hap1). Hap1 contains seven heme-responsive motifs (HRMs): six are clustered in the heme domain, and a seventh is near the activation domain. To determine the functional role of HRMs and to define which parts of Hap1 mediate heme regulation, we carried out a systematic analysis of Hap1 mutants with various regions deleted or mutated. Strikingly, the data show that HRM1 to -6, located in the previously designated Hap1 heme domain, have little impact on heme regulation. All seven HRMs are dispensable for Hap1 repression in the absence of heme, but HRM7 is required for Hap1 activation by heme. More importantly, we show that a novel class of repression modules-RPM1, encompassing residues 245 to 278; RPM2, encompassing residues 1061 to 1185; and RPM3, encompassing residues 203 to 244-is critical for Hap1 repression in the absence of heme. Biochemical analysis indicates that RPMs mediate Hap1 repression, at least partly, by the formation of a previously identified higher-order complex termed the high-molecular-weight complex (HMC), while HRMs mediate heme activation by permitting heme binding and the disassembly of the HMC. These findings provide significant new insights into the molecular interactions critical for Hap1 repression in the absence of heme and Hap1 activation by heme.

Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate.

Human DNA polymerase and DNA ligase utilization for the repair of a major class of ionizing radiation-induced DNA lesion [DNA single-strand breaks containing 3'-phosphoglycolate (3'-PG)] was examined using a novel, chemically defined vector substrate containing a single, site-specific 3'-PG single-strand break lesion. In addition, the major human AP endonuclease, HAP1 (also known as APE1, APEX, Ref-1), was tested to determine if it was involved in initiating repair of 3'-PG-containing single-strand break lesions. DNA polymerase beta was found to be the primary polymerase responsible for nucleotide incorporation at the lesion site following excision of the 3'-PG blocking group. However, DNA polymerase delta/straightepsilon was also capable of nucleotide incorporation at the lesion site following 3'-PG excision. In addition, repair reactions catalyzed by DNA polymerase beta were found to be most effective in the presence of DNA ligase III, while those catalyzed by DNA polymerase delta/straightepsilon appeared to be more effective in the presence of DNA ligase I. Also, it was demonstrated that the repair initiating 3'-PG excision reaction was not dependent upon HAP1 activity, as judged by inhibition of HAP1 with neutralizing HAP1-specific polyclonal antibody.

Molecular mechanism governing heme signaling in yeast: a higher-order complex mediates heme regulation of the transcriptional activator HAP1.

Apart from serving as a prosthetic group in globins and enzymes, heme is a key regulator controlling a wide range of molecular and cellular processes involved in oxygen sensing and utilization. To gain insights into molecular mechanisms of heme signaling and oxygen sensing in eukaryotes, we investigated the yeast heme-responsive transcriptional activator HAP1. HAP1 activity is regulated precisely and tightly by heme. Here we show that in the absence of heme, HAP1 forms a biochemically distinctive higher-order complex. Our data suggest that this complex contains HAP1 and four other cellular proteins including Hsp82 and Ydj1. The formation of this complex is directly correlated with HAP1 repression in the absence of heme, and mutational or heme disruption of the complex correlates with HAP1 activation, suggesting that this complex is responsible for heme regulation of HAP1 activity. Further, we determined HAP1 domains required for heme regulation: three domains-the dimerization domain, the heme domain, and the HRM7 (heme-responsive motif 7) domain-cooperate to form the higher-order complex and mediate heme regulation. Strikingly, we uncovered a novel function for the HAP1 dimerization domain: it not only allows dimerization but also provides critical functions in heme regulation and transcriptional activation. Our studies provide significant insights into the molecular events leading to heme activation of HAP1 and may shed light on molecular mechanisms of various heme-controlled biological processes in diverse organisms.

Mutations in target DNA elements of yeast HAP1 modulate its transcriptional activity without affecting DNA binding.

The yeast zinc cluster protein HAP1, a member of the GAL4 family, is a transcriptional activator that binds as a homodimer to target DNA sequences. These targets include the upstream activating sequences of the CYC1 and CYC7 genes, which have no obvious sequence similarity. Even though both sites have the same affinity for HAP1, activation differs at these two sites, even when the sequences are placed in an identical promoter context. In addition, mutants of HAP1 that can bind to both sites but are specifically transcriptionally inactive at CYC7 have been previously isolated. In order to identify nucleotides that are responsible for this differential activity, we have performed random and site-directed mutagenesis of these target sites and assayed their binding to HAP1 in vitro and their activity in vivo in reporter plasmids. Our results show that HAP1 binding sites are degenerate forms of the direct repeat CGG N3 TA N CGG N3 TA. Moreover, we show that activity of HAP1 mutants defective for activation of the CYC7gene is restored by specific mutations in the CYC7 binding site. Conversely, other mutations of the target sites prevent activation by HAP1, without interfering with DNA binding. The results suggest that the sequence of the target sites influences the conformation and, hence, the activity of DNA-bound HAP1.