Dam1 Complex Research Articles

A communication hub for phosphoregulation of kinetochore-microtubule attachment

The Mps1 and Aurora B kinases regulate and monitor kinetochore attachment to spindle microtubules during cell division, ultimately ensuring accurate chromosome segregation. In yeast, the critical spindle attachment components are the Ndc80 and Dam1 complexes (Ndc80c and DASH/Dam1c, respectively). Ndc80c is a 600-Å-long heterotetramer that binds microtubules through a globular "head" at one end and centromere-proximal kinetochore components through a globular knob at the other end. Dam1c is a heterodecamer that forms a ring of 16-17 protomers around the shaft of the single kinetochore microtubule in point-centromere yeast. The ring coordinates the approximately eight Ndc80c rods per kinetochore. In published work, we showed that a site on the globular "head" of Ndc80c, including residues from both Ndc80 and Nuf2, binds a bipartite segment in the long C-terminal extension of Dam1. Results reported here show, both by invitro binding experiments and by crystal structure determination, that the same site binds a conserved segment in the long N-terminal extension of Mps1. It also binds, less tightly, a conserved segment in the N-terminal extension of Ipl1 (yeast Aurora B). Together with results from experiments in yeast cells and from biochemical assays reported in two accompanying papers, the structures and graded affinities identify a communication hub for ensuring uniform bipolar attachment and for signaling anaphase onset.

A conserved site on Ndc80 complex facilitates dynamic recruitment of Mps1 to yeast kinetochores to promote accurate chromosome segregation

Accurate chromosome segregation relies on kinetochores carrying out multiple functions, including establishing and maintaining microtubule attachments, forming precise bi-oriented attachments between sister chromatids, and activating the spindle assembly checkpoint. Central to these processes is the highly conserved Ndc80 complex. This kinetochore subcomplex interacts directly with microtubules but also serves as a critical platform for recruiting kinetochore-associated factors and as a key substrate for error correction kinases. The precise manner in which these kinetochore factors interact and regulate each other's function remains unknown, considerably hindering our understanding of how Ndc80 complex-dependent processes function together to orchestrate accurate chromosome segregation. Here, we aimed to uncover the role of Nuf2's CH domain, a component of the Ndc80 complex, in ensuring these processes. Through extensive mutational analysis, we identified a conserved interaction domain composed of two segments in Nuf2's CH domain that form the binding site for Mps1 within the yeast Ndc80 complex. Interestingly, this site also associates with the Dam1 complex, suggesting Mps1 recruitment may be subject to regulation by competitive binding with other factors. Mutants disrupting this "interaction hub" exhibit defects in spindle assembly checkpoint function and severe chromosome segregation errors. Significantly, specifically restoring Mps1-Ndc80 complex association rescues these defects. Our findings shed light on the intricate regulation of Ndc80 complex-dependent functions and highlight the essential role of Mps1 in kinetochore bi-orientation and accurate chromosome segregation.

Microtubule end-on attachment maturation regulates Mps1 association with its kinetochore receptor

Faithful chromosome segregation requires that sister chromatids establish bi-oriented kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) prevents premature anaphase onset with incomplete attachments. However, how microtubule attachment and checkpoint signaling are coordinated remains unclear. The conserved kinase Mps1 initiates SAC signaling by localizing transiently to kinetochores in prometaphase and is released upon bi-orientation. Using biochemistry, structure predictions, and cellular assays, we shed light on this dynamic behavior in Saccharomyces cerevisiae. A conserved N-terminal segment of Mps1 binds the neck region of Ndc80:Nuf2, the main microtubule receptor of kinetochores. Mutational disruption of this interface, located at the backside of the paired CH domains and opposite the microtubule-binding site, prevents Mps1 localization, eliminates SAC signaling, and impairs growth. The same interface of Ndc80:Nuf2 binds the microtubule-associated Dam1 complex. We demonstrate that the error correction kinase Ipl1/Aurora B controls the competition between Dam1 and Mps1 for the same binding site. Thus, binding of the Dam1 complex to Ndc80:Nuf2 may release Mps1 from the kinetochore to promote anaphase onset.

Functional plasticity in chromosome-microtubule coupling on the evolutionary time scale.

The Dam1 complex is essential for mitotic progression across evolutionarily divergent fungi. Upon analyzing amino acid (aa) sequences of Dad2, a Dam1 complex subunit, we identified a conserved 10-aa-long Dad2 signature sequence (DSS). An arginine residue (R126) in the DSS is essential for viability in Saccharomyces cerevisiae that possesses point centromeres. The corresponding arginine residues are functionally important but not essential for viability in Candida albicans and Cryptococcus neoformans; both carry several kilobases long regional centromeres. The purified recombinant Dam1 complex containing either Dad2ΔDSS or Dad2R126A failed to bind microtubules (MTs) or form any visible rings like the WT complex. Intriguingly, functional analysis revealed that the requirement of the conserved arginine residue for chromosome biorientation and mitotic progression reduced with increasing centromere length. We propose that plasticity of the invariant arginine of Dad2 in organisms with regional centromeres is achieved by conditional elevation of the kinetochore protein(s) to enable multiple kinetochore MTs to bind to each chromosome. The capacity of a chromosome to bind multiple kinetochore MTs may mask the deleterious effects of such lethal mutations.

Increased Sampling and Intracomplex Homologies Favor Vertical Over Horizontal Inheritance of the Dam1 Complex.

Kinetochores connect chromosomes to spindle microtubules to ensure their correct segregation during cell division. Kinetochores of human and yeasts are largely homologous, their ability to track depolymerizing microtubules, however, is carried out by the nonhomologous complexes Ska1-C and Dam1-C, respectively. We previously reported the unique anti-correlating phylogenetic profiles of Dam1-C and Ska-C found among a wide variety of eukaryotes. Based on these profiles and the limited presence of Dam1-C, we speculated that horizontal gene transfer could have played a role in the evolutionary history of Dam1-C. Here, we present an expanded analysis of Dam1-C evolution, using additional genome as well as transcriptome sequences and recently published 3D structures. This analysis revealed a wider and more complete presence of Dam1-C in Cryptista, Rhizaria, Ichthyosporea, CRuMs, and Colponemidia. The fungal Dam1-C cryo-EM structure supports earlier hypothesized intracomplex homologies, which enables the reconstruction of rooted and unrooted phylogenies. The rooted tree of concatenated Dam1-C subunits is statistically consistent with the species tree of eukaryotes, suggesting that Dam1-C is ancient, and that the present-day phylogenetic distribution is best explained by multiple, independent losses and no horizontal gene transfer was involved. Furthermore, we investigated the ancient origin of Dam1-C via profile-versus-profile searches. Homology among 8 out of the 10 Dam1-C subunits suggests that the complex largely evolved from a single multimerizing subunit that diversified into a hetero-octameric core via stepwise subunit duplication and subfunctionalization of the subunits before the origin of the last eukaryotic common ancestor.

Three interacting regions of the Ndc80 and Dam1 complexes support microtubule tip-coupling under load.

Accurate mitosis requires kinetochores to make persistent, load-bearing attachments to dynamic microtubule tips, thereby coupling chromosome movements to tip growth and shortening. This tip-coupling behavior depends on the conserved Ndc80 complex and, in budding yeast, on the Dam1 complex, which bind each other directly via three distinct interacting regions. The functional relevance of these multiple interactions was mysterious. Here we show that interactions between two of these regions support the high rupture strengths that occur when applied force is rapidly increased and also support the stability of tip-coupling when force is held constant over longer durations. The contribution of either of these two regions to tip-coupling is reduced by phosphorylation by Aurora B kinase. The third interaction region makes no apparent contribution to rupture strength, but its phosphorylation by Aurora B kinase specifically decreases the long-term stability of tip-coupling. The specific reduction of long-term stability relative to short-term strength might have important implications for mitotic error correction.

Phospho‐regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore

Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the Saccharomyces cerevisiae outer kinetochore, assembling into 3 MDa‐sized microtubule‐embracing rings, but how ring assembly is specifically initiated in vivo remains to be understood. Here, we describe a molecular pathway that provides local control of ring assembly during the establishment of sister kinetochore bi‐orientation. We show that Dam1c and the general microtubule plus end‐associated protein (+TIP) Bim1/EB1 form a stable complex depending on a conserved motif in the Duo1 subunit of Dam1c. EM analyses reveal that Bim1 crosslinks protrusion domains of adjacent Dam1c heterodecamers and promotes the formation of oligomers with defined curvature. Disruption of the Dam1c‐Bim1 interaction impairs kinetochore localization of Dam1c in metaphase and delays mitosis. Phosphorylation promotes Dam1c‐Bim1 binding by relieving an intramolecular inhibition of the Dam1 C‐terminus. In addition, Bim1 recruits Bik1/CLIP‐170 to Dam1c and induces formation of full rings even in the absence of microtubules. Our data help to explain how new kinetochore end‐on attachments are formed during the process of attachment error correction.

Aurora B switches relative strength of kinetochore-microtubule attachment modes for error correction.

To establish chromosome biorientation, aberrant kinetochore-microtubule interaction must be resolved (error correction) by Aurora B kinase. Aurora B differentially regulates kinetochore attachment to the microtubule plus end and its lateral side (end-on and lateral attachment, respectively). However, it is still unclear how kinetochore-microtubule interactions are exchanged during error correction. Here, we reconstituted the budding yeast kinetochore-microtubule interface in vitro by attaching the Ndc80 complexes to nanobeads. These Ndc80C nanobeads recapitulated in vitro the lateral and end-on attachments of authentic kinetochores on dynamic microtubules loaded with the Dam1 complex. This in vitro assay enabled the direct comparison of lateral and end-on attachment strength and showed that Dam1 phosphorylation by Aurora B makes the end-on attachment weaker than the lateral attachment. Similar reconstitutions with purified kinetochore particles were used for comparison. We suggest the Dam1 phosphorylation weakens interaction with the Ndc80 complex, disrupts the end-on attachment, and promotes the exchange to a new lateral attachment, leading to error correction.

Load-Bearing Interactions between the NDC80 and DAM1 Complexes Differ on Growing and Shortening Microtubule Tips

Interaction between the Ndc80 and Dam1 complexes enhance the load-bearing ability of the kinetochore. We previously found that three different components in the Dam1 complex each binds to a different site in the Ndc80 protein. Mutations in any of these three sites disrupt the ability of the Ndc80 complex to bridge two rings in vitro and disrupt proper Dam1 complex localization to the rest of the kinetochore in vivo. However, which interaction sites between the Ndc80 and Dam1 complexes play a role in load-bearing is still unknown. To test whether regions in the Ndc80p play a role in establishing load-bearing interactions with the Dam1 complex, I generated three lethal insertion mutations along the Ndc80p (ANdc80p, BNdc80p, and CNdc80p) and utilized the optical trap to measure the strength of the Ndc80 complex attachment to an assembling microtubule in the presence of Dam1 complex. Mutation at region ANdc80p conferred a complete defect in the ability of the Ndc80 complex to form a load-bearing interaction with the Dam1 complex, while mutation in region BNdc80p conferred a partial defect and a mutation in region CNdc80p did not confer any defect.I next tested the role of different regions of Ndc80p to form load-bearing interactions on disassembling microtubules in the presence of the Dam1 complex. insertion mutations in either region BNdc80p or region CNdc80p led to a 12-15 fold increase in detachment rate compared to wild-type. in conclusion, region A forms a load-bearing interaction between the Ndc80 and Dam1 complexes on an assembling microtubule tip. Region B forms load-bearing interactions between the Ndc80 and Dam1 complexes on both assembling and disassembling microtubule tips. Region C only forms a load-bearing interaction between the Ndc80 and Dam1 complexes on a disassembling microtubule tip.

Cdk1 Phosphorylation of the Dam1 Complex Strengthens Kinetochore-Microtubule Attachments

To ensure the faithful inheritance of DNA, a macromolecular protein complex called the kinetochore sustains the connection between chromosomes and force-generating dynamic microtubules during cell division. Defects in this process lead to aneuploidy, a common feature of cancer cells and the cause of many developmental diseases [1, 2, 3, 4]. One of the major microtubule-binding activities in the kinetochore is mediated by the conserved Ndc80 complex (Ndc80c) [5, 6, 7]. In budding yeast, the retention of kinetochores on dynamic microtubule tips also depends on the essential heterodecameric Dam1 complex (Dam1c) [8, 9, 10, 11, 12, 13, 14, 15], which binds to the Ndc80c and is proposed to be a functional ortholog of the metazoan Ska complex [16, 17]. The load-bearing activity of the Dam1c depends on its ability to oligomerize, and the purified complex spontaneously self-assembles into microtubule-encircling oligomeric rings, which are proposed to function as collars that allow kinetochores to processively track the plus-end tips of microtubules and harness the forces generated by disassembling microtubules [10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22]. However, it is unknown whether there are specific regulatory events that promote Dam1c oligomerization to ensure accurate segregation. Here, we used a reconstitution system to discover that Cdk1, the major mitotic kinase that drives the cell cycle, phosphorylates the Ask1 component of the Dam1c to increase its residence time on microtubules and enhance kinetochore-microtubule attachment strength. We propose that Cdk1 activity promotes Dam1c oligomerization to ensure that kinetochore-microtubule attachments are stabilized as kinetochores come under tension in mitosis.

Structural view of the yeast Dam1 complex, a ring-shaped molecular coupler for the dynamic microtubule end.

In a dividing eukaryotic cell, proper chromosome segregation requires the dynamic yet persistent attachment of kinetochores to spindle microtubules. In the budding yeast Saccharomyces cerevisiae, this function is especially crucial because each kinetochore is attached to a single microtubule; consequently, loss of attachment could lead to unrecoverable chromosome loss. The highly specialized heterodecameric Dam1 protein complex achieves this coupling by assembling into a microtubule-encircling ring that glides near the end of the dynamic microtubule to mediate chromosome motion. In recent years, we have learned a great deal about the structural properties of the Dam1 heterodecamer, its mechanism of self-assembly into rings, and its tethering to the kinetochore by the elongated Ndc80 complex. The most remarkable progress has resulted from defining the fine structures of helical bundles within Dam1 heterodecamer. In this review, we critically analyze structural observations collected by diverse approaches with the goal of obtaining a unified view of Dam1 ring architecture. A considerable consistency between different studies supports a coherent model of the circular core of the Dam1 ring. However, there are persistent uncertainties about the composition of ring protrusions and flexible extensions, as well as their roles in mediating ring core assembly and interactions with the Ndc80 complex and microtubule.

Ultrafast Force-Clamp Studies of the Diffusing Microtubule-Binding Proteins

Chromosome segregation during cell division crucially depends on the activities of the microtubule associated proteins (MAPs) that couple chromosomal kinetochores to spindle microtubules. When a kinetochore-bound microtubule shortens, it pulls the coupled kinetochore toward the spindle pole-associated microtubule minus-end, whereas the sister kinetochore is dragged toward the microtubule plus-end. However, there is little understanding of how the coupling kinetochore MAPs translocate along microtubule in different directions under the dragging force. To bridge this gap, we applied a highly sensitive ultrafast force-clamp spectroscopy to investigate directional mobility of key kinetochore components, including human Ndc80 and Ska complexes, and the ring-forming Dam1 complex from yeast. Single molecules of these proteins were conjugated to the coverslip-immobilized bead pedestals and the microtubule “dumbbell” was oscillated near the pedestal's surface under a continuously operating force clamp. Surprisingly, the character of the force-induced translocation was dramatically different for these MAPs, even though they undergo thermal diffusion along microtubules at highly similar rates. Under the dragging force, the Ska complex glides smoothly in both microtubule directions, whereas the Ndc80 exhibits differential gliding and the Dam1 shows asymmetric force-induced static bindings. These results strongly support the model in which the translocating kinetochore MAPs function as integral components of the molecular machine powered by microtubule depolymerization, rather than by biased diffusion. We propose that the asymmetric behavior of Ndc80 and Dam1 complexes underlies differential internal friction at moving kinetochores, which enables their poleward-directed gliding, while reducing the microtubule end slippage under tension.

Dual role for fungal-specific outer kinetochore proteins during cell cycle and development in Magnaporthe oryzae.

The outer kinetochore DASH complex (also known as the Dam1 complex) ensures proper spindle structure and chromosome segregation. While DASH complex protein requirement diverges among different yeasts, its role in filamentous fungi has not yet been investigated. We studied the dynamics and role of middle (Mis12) and outer (Dam1 and Ask1) kinetochore proteins in the filamentous fungal pathogen, Magnaporthe oryzae, which undergoes multiple cell cycle-linked developmental transitions. While Mis12 was constitutively present in the nucleus, Dam1 and Ask1 were recruited only during mitosis. Although Dam1 was not required for viability, loss of its function (dam1Δ) delayed mitotic progression, resulting in impaired conidial and hyphal development. Both Dam1 and Ask1 also localised to the hyphal tips, in the form of punctae oscillating back and forth from the growing ends, suggesting that Magnaporthe DASH complex proteins may play a non-canonical role in polarised growth during interphase, in addition to their function in nuclear segregation during mitosis. Impaired appressorial (infection structure) development and host penetration in the dam1Δ mutant suggest that fungus-specific Dam1 complex proteins could be an attractive target for a novel anti-fungal strategy.This article has an associated First Person interview with the first author of the paper.

Glucose Signaling Is Connected to Chromosome Segregation Through Protein Kinase A Phosphorylation of the Dam1 Kinetochore Subunit in Saccharomycescerevisiae.

The Dam1 complex is an essential component of the outer kinetochore that mediates attachments between spindle microtubules and chromosomes. Dam1p, a subunit of the Dam1 complex, binds to microtubules and is regulated by Aurora B/Ipl1p phosphorylation. We find that overexpression of cAMP-dependent protein kinase (PKA) catalytic subunits (i.e., TPK1, TPK2, TPK3) is lethal in DAM1 mutants and increases the rate of chromosome loss in wild-type cells. Replacing an evolutionarily conserved PKA site (S31) in Dam1p with a nonphosphorylatable alanine suppressed the high-copy PKA dosage lethality in dam1-1 Consistent with Dam1p as a target of PKA, we find that in vitro PKA can directly phosphorylate S31 in Dam1p and we observed phosphorylation of S31 in Dam1p purified from asynchronously growing yeast cells. Cells carrying high-copy TPK2 or a Dam1p phospho-mimetic S31D mutant displayed a reduction in Dam1p localization at the kinetochore, suggesting that PKA phosphorylation plays a role in assembly and/or stability of the Dam1 complex. Furthermore, we observed spindle defects associated with S31 phosphorylation. Finally, we find that phosphorylation of Dam1p on S31 is reduced when glucose is limiting as well as during α-factor arrest, conditions that inhibit PKA activity. These observations suggest that the PKA site of Dam1p participates in regulating kinetochore activity. While PKA is a well-established effector of glucose signaling, our work shows for the first time that glucose-dependent PKA activity has an important function in chromosome segregation.

A Kinesin-5, Cin8, Recruits Protein Phosphatase 1 to Kinetochores and Regulates Chromosome Segregation

Kinesin-5 is a highly conserved homo-tetrameric protein complex responsible for crosslinking microtubules and pushing spindle poles apart. The budding yeast Kinesin-5, Cin8, is highly concentrated at kinetochores in mitosis before anaphase, but its functions there are largely unsolved. Here, we show that Cin8 localizes to kinetochores in a cell-cycle-dependent manner and concentrates near the microtubule binding domains of Ndc80 at metaphase. Cin8's kinetochore localization depends on the Ndc80 complex, kinetochore microtubules, and the Dam1 complex. Consistent with its kinetochore localization, a Cin8 deletion induces a loss of tension at the Ndc80 microtubule binding domains and a major delay in mitotic progression. Cin8 associates with Protein Phosphatase 1 (PP1), and mutants that inhibit its PP1 binding also induce a loss of tension at the Ndc80 microtubule binding domains and delay mitotic progression. Taken together, our results suggest that Cin8-PP1 plays a critical role at kinetochores to promote accurate chromosome segregation by controlling Ndc80 attachment to microtubules.

Identification of potential inhibitors against nuclear Dam1 complex subunit Ask1 of Candida albicans using virtual screening and MD simulations

Identification of hit compounds against specific target form the starting point for a drug discovery program. A consistent decline of new chemical entities (NCEs) in recent years prompted a challenge to explore newer approaches to discover potential hit compounds that in turn can be converted into leads, and ultimately drug with desired therapeutic efficacy. The vast amount of omics and activity data available in public databases offers an opportunity to identify novel targets and their potential inhibitors. State of the art in silico methods viz., clustering of compounds, virtual screening, molecular docking, MD simulations and MMPBSA calculations were employed in a pipeline to identify potential ‘hits’ against those targets as well whose structures, as of now, could only predict through threading approaches. In the present work, we have started from scratch, amino acid sequence of target and compounds retrieved from PubChem compound database, modeled it in such a way that led to the identification of possible inhibitors of Dam1 complex subunit Ask1 of Candida albicans. We also propose a ligand based binding site determination approach. We have identified potential inhibitors of Ask1 subunit of a Dam1 complex of C. albicans, which is required to prevent precocious spindle elongation in pre-mitotic phases. The proposed scheme may aid to find virtually potential inhibitors of other unique targets against candida.

Unique Phylogenetic Distributions of the Ska and Dam1 Complexes Support Functional Analogy and Suggest Multiple Parallel Displacements of Ska by Dam1.

Faithful chromosome segregation relies on kinetochores, the large protein complexes that connect chromatin to spindle microtubules. Although human and yeast kinetochores are largely homologous, they track microtubules with the unrelated protein complexes Ska (Ska-C, human) and Dam1 (Dam1-C, yeast). We here uncovered that Ska-C and Dam1-C are both widespread among eukaryotes, but in an exceptionally inverse manner, supporting their functional analogy. Within the complexes, all Ska-C and various Dam1-C subunits are ancient paralogs, showing that gene duplication shaped these complexes. We examined various evolutionary scenarios to explain the nearly mutually exclusive patterns of Ska-C and Dam1-C in present-day species. We propose that Ska-C was present in the last eukaryotic common ancestor, that subsequently Dam1-C displaced Ska-C in an early fungus and was horizontally transferred to diverse non-fungal lineages, displacing Ska-C in these lineages too.

Mechanism of Ska Recruitment by Ndc80 Complexes to Kinetochores

Yeast use the ring-shaped Dam1 complex to slide down depolymerizing microtubules to move chromosomes, but current models suggest that other eukaryotes do not have a sliding ring. We visualized Ndc80 and Ska complexes on microtubules by electron microscopic tomography to identify the structure of the human kinetochore-microtubule attachment. Ndc80 recruits the Ska complex so that the V shape of the Ska dimer interacts along protofilaments. We identify a mutant of the Ndc80 tail that isdeficient in Ska recruitment to kinetochores and in orienting Ska along protofilaments invitro. This mutant Ndc80 binds microtubules with normal affinity but is deficient in clustering along protofilaments. We propose that Ska is recruited to kinetochores by clusters of Ndc80 proteins and that our structure ofNdc80 and Ska complexes on microtubules suggests a mechanism for metazoan kinetochores to couple the depolymerization of microtubules to power the movement of chromosomes.

The Ndc80 complex bridges two Dam1 complex rings.

Strong kinetochore-microtubule attachments are essential for faithful segregation of sister chromatids during mitosis. The Dam1 and Ndc80 complexes are the main microtubule binding components of the Saccharomyces cerevisiae kinetochore. Cooperation between these two complexes enhances kinetochore-microtubule coupling and is regulated by Aurora B kinase. We show that the Ndc80 complex can simultaneously bind and bridge across two Dam1 complex rings through a tripartite interaction, each component of which is regulated by Aurora B kinase. Mutations in any one of the Ndc80p interaction regions abrogates the Ndc80 complex's ability to bind two Dam1 rings in vitro, and results in kinetochore biorientation and microtubule attachment defects in vivo. We also show that an extra-long Ndc80 complex, engineered to space the two Dam1 rings further apart, does not support growth. Taken together, our work suggests that each kinetochore in vivo contains two Dam1 rings and that proper spacing between the rings is vital.

Divalent Cations Affect the Stability and Structure of Dad2p, a Subunit of the Candida Albicans Kinetochore Dam1 Complex

<p class="1Body">The heterodecameric Dam1 complex is involved in establishing and maintaining the connection between the kinetochore and the mitotic spindle during mitosis. Biochemical studies of the reconstituted complex have shed light upon how it interacts with microtubules. However, little information about the biochemical properties of the isolated subunits has been available. This report examines the stability and structure of Dad2p, one of the Dam1 complex subunits isolated from <em>Candida albicans</em>. By employing differential scanning fluorimetry, protease protection and hydrodynamic analyses, we show that Dad2p is specifically responsive to the presence of divalent cations. This observation may be important for understanding the dynamic structure and regulation of the Dam1 complex in fungal cells.</p>