White-opaque Switching Research Articles

Differential Regulation of White-Opaque Switching by Individual Subunits of Candida albicans Mediator

The multisubunit eukaryotic Mediator complex integrates diverse positive and negative gene regulatory signals and transmits them to the core transcription machinery. Mutations in individual subunits within the complex can lead to decreased or increased transcription of certain subsets of genes, which are highly specific to the mutated subunit. Recent studies suggest a role for Mediator in epigenetic silencing. Using white-opaque morphological switching in Candida albicans as a model, we have shown that Mediator is required for the stability of both the epigenetic silenced (white) and active (opaque) states of the bistable transcription circuit driven by the master regulator Wor1. Individual deletions of eight C. albicans Mediator subunits have shown that different Mediator subunits have dramatically diverse effects on the directionality, frequency, and environmental induction of epigenetic switching. Among the Mediator deletion mutants analyzed, only Med12 has a steady-state transcriptional effect on the components of the Wor1 circuit that clearly corresponds to its effect on switching. The MED16 and MED9 genes have been found to be among a small subset of genes that are required for the stability of both the white and opaque states. Deletion of the Med3 subunit completely destabilizes the opaque state, even though the Wor1 transcription circuit is intact and can be driven by ectopic expression of Wor1. The highly impaired ability of the med3 deletion mutant to mate, even when Wor1 expression is ectopically induced, reveals that the activation of the Wor1 circuit can be decoupled from the opaque state and one of its primary biological consequences.

Bcr1 plays a central role in the regulation of opaque cell filamentation in Candida albicans

The human fungal pathogen Candida albicans has at least two types of morphological transitions: white to opaque cell transitions and yeast to hyphal transitions. Opaque cells have historically not been known to undergo filamentation under standard filament-inducing conditions. Here we find that Bcr1 and its downstream regulators Cup9, Nrg1 and Czf1 and the cAMP-signalling pathway control opaque cell filamentation in C. albicans. We have shown that deletion of BCR1, CUP9, NRG1 and CZF1 results in opaque cell filamentation under standard culture conditions. Disruption of BCR1 in white cells has no obvious effect on hyphal growth, suggesting that Bcr1 is an opaque-specific regulator of filamentation under the conditions tested. Moreover, inactivation of the cAMP-signalling pathway or disruption of its downstream transcriptional regulators, FLO8 and EFG1, strikingly attenuates filamentation in opaque cells of the bcr1/bcr1 mutant. Deletion of HGC1, a downstream gene of the cAMP-signalling pathway encoding G1 cyclin-related protein, completely blocks opaque cell filamentation induced by inactivation of BCR1. These results demonstrate that Bcr1 regulated opaque cell filamentation is dependent on the cAMP-signalling pathway. This study establishes a link between the white-opaque switch and the yeast-filamentous growth transition in C. albicans.

Regulated proteolysis of Candida albicans Ras1 is involved in morphogenesis and quorum sensing regulation

In Candida albicans, a fungal pathogen, the small G-protein Ras1 regulates many important behaviors including white-opaque switching, biofilm formation, and the induction and maintenance of hyphal growth. Like other Ras proteins, Ras1 is activated upon guanine triphosphate binding, and its activity is further modulated by post-translational lipid modifications. Here, we report that the levels of membrane-associated, full-length Ras1 were higher in hyphae than in yeast, and that yeast contained a shorter, soluble Ras1 species that resulted from cleavage. Deletion of the putative cleavage site led to more rapid induction of hyphal growth and delayed hypha-to-yeast transitions. The cleaved Ras1 species was less able to activate its effector, adenylate cyclase (Cyr1), unless tethered to the membrane by a heterologous membrane-targeting domain. Ras1 cleavage was repressed by cAMP-signalling, indicating the presence of a positive feedback loop in which Cyr1 and cAMP influence Ras1. The C. albicans quorum sensing molecule farnesol, which inhibits Cyr1 and represses filamentation, caused an increase in the fraction of Ras1 in the cleaved form, particularly in nascent yeast formed from hyphae. This newly recognized mode of Ras regulation may control C. albicans Ras1 activity in important ways.

Identification and characterization of a previously undescribed family of sequence-specific DNA-binding domains

Sequence-specific DNA-binding proteins are among the most important classes of gene regulatory proteins, controlling changes in transcription that underlie many aspects of biology. In this work, we identify a transcriptional regulator from the human fungal pathogen Candida albicans that binds DNA specifically but has no detectable homology with any previously described DNA- or RNA-binding protein. This protein, named White-Opaque Regulator 3 (Wor3), regulates white-opaque switching, the ability of C. albicans to switch between two heritable cell types. We demonstrate that ectopic overexpression of WOR3 results in mass conversion of white cells to opaque cells and that deletion of WOR3 affects the stability of opaque cells at physiological temperatures. Genome-wide chromatin immunoprecipitation of Wor3 and gene expression profiling of a wor3 deletion mutant strain indicate that Wor3 is highly integrated into the previously described circuit regulating white-opaque switching and that it controls a subset of the opaque transcriptional program. We show by biochemical, genetic, and microfluidic experiments that Wor3 binds directly to DNA in a sequence-specific manner, and we identify the set of cis-regulatory sequences recognized by Wor3. Bioinformatic analyses indicate that the Wor3 family arose more recently in evolutionary time than most previously described DNA-binding domains; it is restricted to a small number of fungi that include the major fungal pathogens of humans. These observations show that new families of sequence-specific DNA-binding proteins may be restricted to small clades and suggest that current annotations--which rely on deep conservation--underestimate the fraction of genes coding for transcriptional regulators.

White-Opaque Switching in Natural MTLa/α Isolates of Candida albicans: Evolutionary Implications for Roles in Host Adaptation, Pathogenesis, and Sex

Author SummaryPhenotypic transitions enable fungal pathogens to better adapt to their ever-changing environments. Approximately 10% of natural Candida albicans strains, which are homozygous at the mating type locus (MTL, a/a and α/α), can switch between two distinguishable morphological forms: white and opaque. The two cell types differ in a number of biological aspects including virulence, susceptibility to host immune attacks, and mating competency. Here, we demonstrate that white-opaque switching competency is not restricted to the MTL homozygous strains, but is a general characteristic of all MTL strain types of C. albicans (a/a, α/α, and a/α). Two host environmental cues, N-acetylglucosamine and CO2, promote white-to-opaque switching and stabilize the opaque phenotype. Thus, although switching is normally blocked in a/α cells, this block can be overcome through specific environmental changes. We further show that three transcriptional regulators (Rfg1, Brg1, and Efg1) help to regulate white-opaque switching in MTL heterozygotes of C. albicans. This study generalizes white-opaque switching to strains with all mating-type configurations and emphasizes its importance in host adaptation, pathogenesis, and parasexual reproduction.

MTL–Independent Phenotypic Switching in Candida tropicalis and a Dual Role for Wor1 in Regulating Switching and Filamentation

Phenotypic switching allows for rapid transitions between alternative cell states and is important in pathogenic fungi for colonization and infection of different host niches. In Candida albicans, the white-opaque phenotypic switch plays a central role in regulating the program of sexual mating as well as interactions with the mammalian host. White-opaque switching is controlled by genes encoded at the MTL (mating-type-like) locus that ensures that only a or α cells can switch from the white state to the mating-competent opaque state, while a/α cells are refractory to switching. Here, we show that the related pathogen C. tropicalis undergoes white-opaque switching in all three cell types (a, α, and a/α), and thus switching is independent of MTL control. We also demonstrate that C. tropicalis white cells are themselves mating-competent, albeit at a lower efficiency than opaque cells. Transcriptional profiling of C. tropicalis white and opaque cells reveals significant overlap between switch-regulated genes in MTL homozygous and MTL heterozygous cells, although twice as many genes are white-opaque regulated in a/α cells as in a cells. In C. albicans, the transcription factor Wor1 is the master regulator of the white-opaque switch, and we show that Wor1 also regulates switching in C. tropicalis; deletion of WOR1 locks a, α, and a/α cells in the white state, while WOR1 overexpression induces these cells to adopt the opaque state. Furthermore, we show that WOR1 overexpression promotes both filamentous growth and biofilm formation in C. tropicalis, independent of the white-opaque switch. These results demonstrate an expanded role for C. tropicalis Wor1, including the regulation of processes necessary for infection of the mammalian host. We discuss these findings in light of the ancestral role of Wor1 as a transcriptional regulator of the transition between yeast form and filamentous growth.

Nucleosome Assembly Factors CAF-1 and HIR Modulate Epigenetic Switching Frequencies in an H3K56 Acetylation-Associated Manner in Candida albicans

CAF-1 and HIR are highly conserved histone chaperone protein complexes that function in the assembly of nucleosomes onto chromatin. CAF-1 is characterized as having replication-coupled nucleosome activity, whereas the HIR complex can assemble nucleosomes independent of replication. Histone H3K56 acetylation, controlled by the acetyltransferase Rtt109 and deacetylase Hst3, also plays a significant role in nucleosome assembly. In this study, we generated a set of deletion mutants to genetically characterize pathway-specific and overlapping functions of CAF-1 and HIR in C. albicans. Their roles in epigenetic maintenance of cell type were examined by using the white-opaque switching system in C. albicans. We show that CAF-1 and HIR play conserved roles in UV radiation recovery, repression of histone gene expression, correct chromosome segregation, and stress responses. Unique to C. albicans, the cac2Δ/Δ mutant shows increased sensitivity to the Hst3 inhibitor nicotinamide, while the rtt109Δ/Δ cac2Δ/Δ and hir1Δ/Δ cac2Δ/Δ mutants are resistant to nicotinamide. CAF-1 plays a major role in maintaining cell types, as the cac2Δ/Δ mutant exhibited increased switching frequencies in both directions and switched at a high frequency to opaque in response to nicotinamide. Like the rtt109Δ/Δ mutant, the hir1Δ/Δ cac2Δ/Δ double mutant is defective in maintaining the opaque cell fate and blocks nicotinamide-induced opaque formation, and the defects are suppressed by ectopic expression of the master white-opaque regulator Wor1. Our data suggest an overlapping function of CAF-1 and HIR in epigenetic regulation of cell fate determination in an H3K56 acetylation-associated manner.

The ‘obligate diploid’ Candida albicans forms mating-competent haploids

Candida albicans, the most prevalent human fungal pathogen, is considered to be an obligate diploid that carries recessive lethal mutations throughout the genome. Here we demonstrate that C. albicans has a viable haploid state that can be derived from diploid cells under in vitro and in vivo conditions, and that seems to arise through a concerted chromosome loss mechanism. Haploids undergo morphogenetic changes like those of diploids, including the yeast-hyphal transition, chlamydospore formation and a white-opaque switch that facilitates mating. Haploid opaque cells of opposite mating type mate efficiently to regenerate the diploid form, restoring heterozygosity and fitness. Homozygous diploids arise spontaneously by auto-diploidization, and both haploids and auto-diploids show a similar reduction in fitness, in vitro and in vivo, relative to heterozygous diploids, indicating that homozygous cell types are transient in mixed populations. Finally, we constructed stable haploid strains with multiple auxotrophies that will facilitate molecular and genetic analyses of this important pathogen.

White-Opaque Switching of Candida albicans Allows Immune Evasion in an Environment-Dependent Fashion

Candida albicans strains that are homozygous at the mating type locus can spontaneously and reversibly switch from the normal yeast morphology (white) to an elongated cell type (opaque), which is the mating-competent form of the fungus. White-opaque switching also influences the ability of C. albicans to colonize and proliferate in specific host niches and its susceptibility to host defense mechanisms. We used live imaging to observe the interaction of white and opaque cells with host phagocytic cells. For this purpose, we generated derivatives of the switching-competent strain WO-1 that express green fluorescent protein from a white-specific promoter and red fluorescent protein from an opaque-specific promoter or vice versa. When mixed populations of these differentially labeled white and opaque cells were incubated with human polymorphonuclear neutrophils (PMNs) on a glass slide, the neutrophils selectively phagocytosed and killed white cells, despite frequent physical interaction with opaque cells. White cells were attacked only after they started to form a germ tube, indicating that the suppression of filamentation in opaque cells saved them from recognition by the PMNs. In contrast to neutrophils, dendritic cells internalized white as well as opaque cells. However, when embedded in a collagen matrix, the PMNs also phagocytosed both white and opaque cells with similar efficiency. These results suggest that, depending on the environment, white-opaque switching enables C. albicans to escape from specific host defense mechanisms.

Candida albicans.

Candida albicans.

A Conserved Transcriptional Regulator Governs Fungal Morphology in Widely Diverged Species

Fungi exhibit a large variety of morphological forms. Here, we examine the functions of a deeply conserved regulator of morphology in three fungal species: Saccharomyces cerevisiae, Candida albicans, and Histoplasma capsulatum. We show that, despite an estimated 600 million years since those species diverged from a common ancestor, Wor1 in C. albicans, Ryp1 in H. capsulatum, and Mit1 in S. cerevisiae are transcriptional regulators that recognize the same DNA sequence. Previous work established that Wor1 regulates white-opaque switching in C. albicans and that its ortholog Ryp1 regulates the yeast to mycelial transition in H. capsulatum. Here we show that the ortholog Mit1 in S. cerevisiae is also a master regulator of a morphological transition, in this case pseudohyphal growth. Full-genome chromatin immunoprecipitation experiments show that Mit1 binds to the control regions of the previously known regulators of pseudohyphal growth as well as those of many additional genes. Through a comparison of binding sites for Mit1 in S. cerevisiae, Wor1 in C. albicans, and Wor1 ectopically expressed in S. cerevisiae, we conclude that the genes controlled by the orthologous regulators overlap only slightly between these two species despite the fact that the DNA binding specificity of the regulators has remained largely unchanged. We suggest that the ancestral Wor1/Mit1/Ryp1 protein controlled aspects of cell morphology and that movement of genes in and out of the Wor1/Mit1/Ryp1 regulon is responsible, in part, for the differences of morphological forms among these species.

Defining pheromone-receptor signaling inCandida albicansand related asexualCandidaspecies

Candida albicans is an important human fungal pathogen in which sexual reproduction is under the control of the novel white-opaque switch. Opaque cells are the mating-competent form, whereas white cells do not mate but can still respond to pheromones, resulting in biofilm formation. In this study, we first define the domains of the α-pheromone receptor Ste2 that are necessary for signaling in both white and opaque forms. Both cell states require the IC loop 3 (IC3) and the C-terminal tail of Ste2 for the cellular response, whereas the first IC loop (IC1) of Ste2 is dispensable for signaling. To also address pheromone-receptor interactions in related species, including apparently asexual Candida species, Ste2 orthologues were heterologously expressed in Candida albicans. Ste2 receptors from multiple Candida clade species were functional when expressed in C. albicans, whereas the Ste2 receptor of Candida lusitaniae was nonfunctional. Significantly, however, expression of a chimeric C. lusitaniae Ste2 receptor containing the C-terminal tail of Ste2 from C. albicans generated a productive response to C. lusitaniae pheromone. This system has allowed us to characterize pheromones from multiple Candida species and indicates that functional pheromone-receptor couples exist in fungal species that have yet to be shown to undergo sexual mating.

Regulation of white and opaque cell‐type formation in Candida albicans by Rtt109 and Hst3

How different cell types with the same genotype are formed and heritability maintained is a fundamental question in biology. We utilized white-opaque switching in Candida albicans as a system to study mechanisms of cell-type formation and maintenance. Each cell type has tractable characters, which are maintained over many cell divisions. Cell-type specification is under the control of interlocking transcriptional feedback loops, with Wor1 being the master regulator of the opaque cell type. Here we show that deletion of RTT109, encoding the acetyltransferase for histone H3K56, impairs stochastic and environmentally stimulated white-opaque switching. Ectopic expression of WOR1 mostly bypasses the requirement for RTT109, but opaque cells lacking RTT109 cannot be maintained. We have also discovered that nicotinamide induces opaque cell formation, and this activity of nicotinamide requires RTT109. Reducing the copy number of HST3, which encodes the H3K56 deacetylase, also leads to increased opaque formation. We further show that the Hst3 level is downregulated in the presence of genotoxins and ectopic expression of HST3 blocks genotoxin induced switching. This finding links genotoxin induced switching to Hst3 regulation. Together, these findings suggest RTT109 and HST3 genes play an important role in the regulation of white-opaque switching in C. albicans.

Candida albicans Zcf37, a zinc finger protein, is required for stabilization of the white state

Candida albicans, the most prevalent human fungal pathogen, can switch stochastically between white and opaque phases. In this study, we identified Zcf37, a zinc finger protein, as a new regulator of white-opaque switching. Deletion of ZCF37 increased white-to-opaque switching frequency and stabilized the opaque state. Overexpression of ZCF37 promoted conversion of opaque cells to white phase, but needed existence of Efg1, a key regulator required for maintenance of the white state. Deletion of EFG1 abolished the effect of ectopically expressed Zcf37 on opaque-to-white switching, whereas ectopic expression of EFG1 promoted white cell formation without presence of Zcf37. Our results suggest that Zcf37 acts as an activator of white cell formation and a repressor of opaque state and functions upstream of Efg1.

Deletion of <italic>EFG1</italic> promotes <italic>Candida albicans</italic> opaque formation responding to pH via Rim101

Phenotypic switching in Candida albicans spontaneously generates different cellular morphologies. The reversible switching between white and opaque phenotypes is regulated by multiple regulators including Efg1 and Wor1. In mating-type-like locus (MTL) homozygous cells, the Efg1 functions as a repressor, whereas the Wor1 acts as an activator in white-opaque switching. We presented evidence that switching between white and opaque in efg1/efg1 mutant is regulated by ambient pH. In pH 6.8 media, the efg1/efg1 mutant cells exhibited opaque form, but shifted to white form in pH 4.5 media. The pH-dependent morphological switching is not blocked by further deletion of WOR1 in the efg1/efg1 mutant. Correlated with the phenotype, the opaque-phase-specific gene OP4 was induced in efg1/efg1 mutant cells when cultured in pH 6.8 media, and was repressed in pH 4.5 media. Consistently, the MTLa efg1/efg1 mutant cells could mate efficiently with MTLα cells in pH 6.8 media, but poorly in pH 4.5 media. Ectopic expression of the Rim101-405 allele in the efg1/efg1 mutant helped to bypass the pH restriction on white-opaque switching and show opaque form in both neutral and acidic media. We proposed that relief of the Efg1 repression enables C. albicans to undergo white-opaque switching in pH-dependent regulation mediated by Rim101-signaling pathway.

Identification of a Cell Death Pathway in Candida albicans during the Response to Pheromone

Mating in hemiascomycete yeasts involves the secretion of pheromones that induce sexual differentiation in cells of the opposite mating type. Studies in Saccharomyces cerevisiae have revealed that a subpopulation of cells experiences cell death during exposure to pheromone. In this work, we tested whether the phenomenon of pheromone-induced death (PID) also occurs in the opportunistic pathogen Candida albicans. Mating in C. albicans is uniquely regulated by white-opaque phenotypic switching; both cell types respond to pheromone, but only opaque cells undergo the morphological transition and cell conjugation. We show that approximately 20% of opaque cells, but not white cells, of laboratory strain SC5314 experience pheromone-induced death. Furthermore, analysis of mutant strains revealed that PID was significantly reduced in strains lacking Fig1 or Fus1 transmembrane proteins that are induced during the mating process and, we now show, are necessary for efficient mating in C. albicans. The level of PID was also Ca(2+) dependent, as chelation of Ca(2+) ions increased cell death to almost 50% of the population. However, in contrast to S. cerevisiae PID, pheromone-induced killing of C. albicans cells was largely independent of signaling via the Ca(2+)-dependent protein phosphatase calcineurin, even when combined with the loss of Cmk1 and Cmk2 proteins. Finally, we demonstrate that levels of PID vary widely between clinical isolates of C. albicans, with some strains experiencing close to 70% cell death. We discuss these findings in light of the role of prodeath and prosurvival pathways operating in yeast cells undergoing the morphological response to pheromone.

The Transcriptomes of Two Heritable Cell Types Illuminate the Circuit Governing Their Differentiation

The differentiation of cells into distinct cell types, each of which is heritable for many generations, underlies many biological phenomena. White and opaque cells of the fungal pathogen Candida albicans are two such heritable cell types, each thought to be adapted to unique niches within their human host. To systematically investigate their differences, we performed strand-specific, massively-parallel sequencing of RNA from C. albicans white and opaque cells. With these data we first annotated the C. albicans transcriptome, finding hundreds of novel differentially-expressed transcripts. Using the new annotation, we compared differences in transcript abundance between the two cell types with the genomic regions bound by a master regulator of the white-opaque switch (Wor1). We found that the revised transcriptional landscape considerably alters our understanding of the circuit governing differentiation. In particular, we can now resolve the poor concordance between binding of a master regulator and the differential expression of adjacent genes, a discrepancy observed in several other studies of cell differentiation. More than one third of the Wor1-bound differentially-expressed transcripts were previously unannotated, which explains the formerly puzzling presence of Wor1 at these positions along the genome. Many of these newly identified Wor1-regulated genes are non-coding and transcribed antisense to coding transcripts. We also find that 5′ and 3′ UTRs of mRNAs in the circuit are unusually long and that 5′ UTRs often differ in length between cell-types, suggesting UTRs encode important regulatory information and that use of alternative promoters is widespread. Further analysis revealed that the revised Wor1 circuit bears several striking similarities to the Oct4 circuit that specifies the pluripotency of mammalian embryonic stem cells. Additional characteristics shared with the Oct4 circuit suggest a set of general hallmarks characteristic of heritable differentiation states in eukaryotes.

HOS2 and HDA1 Encode Histone Deacetylases with Opposing Roles in Candida albicans Morphogenesis

Epigenetic mechanisms regulate the expression of virulence traits in diverse pathogens, including protozoan and fungi. In the human fungal pathogen Candida albicans, virulence traits such as antifungal resistance, white-opaque switching, and adhesion to lung cells are regulated by histone deacetylases (HDACs). However, the role of HDACs in the regulation of the yeast-hyphal morphogenetic transitions, a critical virulence attribute of C. albicans, remains poorly explored. In this study, we wished to determine the relevance of other HDACs on C. albicans morphogenesis. We generated mutants in the HDACs HOS1, HOS2, RPD31, and HDA1 and determined their ability to filament in response to different environmental stimuli. We found that while HOS1 and RPD31 have no or a more limited role in morphogenesis, the HDACs HOS2 and HDA1 have opposite roles in the regulation of hyphal formation. Our results demonstrate an important role for HDACs on the regulation of yeast-hyphal transitions in the human pathogen C. albicans.

Temporal anatomy of an epigenetic switch in cell programming: the white‐opaque transition of C. albicans

The human pathogen Candida albicans undergoes a well-defined switch between two distinct cell types, named 'white' and 'opaque'. White and opaque cells differ in metabolic preferences, mating behaviours, cellular morphologies and host interactions. Each cell type is stable through many generations; switching between them is rare, stochastic and occurs without any known changes in the primary sequence of the genome; thus the switch is epigenetic. The white-opaque switch is regulated by a transcriptional circuit, composed of four regulators arranged in a series of interlocking feedback loops. To understand how switching occurs, we investigated the order of regulatory changes that occur during the switch from the opaque to the white cell type. Surprisingly, changes in key transcriptional regulators occur gradually, extending over several cell divisions with little cell-to-cell variation. Additional experiments, including perturbations to regulator concentrations, refine the signature of the commitment point. Transcriptome analysis reveals that opaque cells begin to globally resemble white cells well before they irreversibly commit to switching. We propose that these characteristics of the switching process permit C. albicans to 'test the waters' before making an all-or-none decision.

Distinct class of DNA-binding domains is exemplified by a master regulator of phenotypic switching in Candida albicans.

Among the most important classes of regulatory proteins are the sequence-specific DNA-binding proteins that control transcription through the occupancy of discrete DNA sequences within genomes. Currently, this class of proteins encompasses at least 37 distinct structural superfamilies and more than 100 distinct structural motifs. In this paper, we examine the transcriptional regulator Wor1, a master regulator of white-opaque switching in the human fungal pathogen Candida albicans. As assessed by a variety of algorithms, this protein has no sequence or structural similarity to any known DNA-binding protein. It is, however, conserved across the vast fungal lineage, with a 300aa region of sequence conservation. Here, we show that this 300aa region of Wor1 exhibits sequence-specific DNA binding and therefore represents a new superfamily of DNA-binding proteins. We identify the 14-nucleotide-pair DNA sequence recognized by Wor1, characterize the site through mutational analysis, and demonstrate that this sequence is sufficient for the Wor1-dependent activation of transcription in vivo. Within the 300aa DNA-binding conserved region, which we have termed the WOPR box, are two domains (WOPRa and WOPRb), dissimilar to each other but especially well-conserved across the fungal lineage. We show that the WOPR box binds DNA as a monomer and that neither domain, when expressed and purified separately, exhibits sequence-specific binding. DNA binding is restored, however, when the two isolated domains are added together. These results indicate that the WOPR family of DNA-binding proteins involves an unusual coupling between two dissimilar, covalently linked domains.