The Dad2 signature sequence (DSS) is a conserved motif at the C-terminus in the Dad2 protein family

The Dad2 protein family is typically defined by the presence of the Dad2 domain (PF08654), a conserved amino acid sequence stretch at the N-terminus (Fig 1A, top). To further understand the domain architecture, we analyzed the primary amino acid sequence of Dad2 from more than 500 fungal species collectively representing the three major fungal phyla—Ascomycota, Basidiomycota, and Mucoromycota (See the Materials and Methods section). We identified a 10-amino acid-long evolutionarily conserved sequence motif towards the C- terminus of Dad2 and named it as the DSS (Fig 1A). The DSS motif remains conserved across species with diverse centromere structures known among fungi. Although the extent of conservation of most amino acid residues is variable, the DSS possesses an almost invariant arginine residue (R′) (Fig 1A). Further analysis of the DSS within specific fungal phyla revealed other features associated with this motif. Among the members of Saccharomycotina, the second position from the invariant arginine was also encoded by a positively charged amino acid such as arginine (R) or lysine (K) in low frequency. However, this position was predominantly represented by proline (P) in other fungal orders such as Pezizomycotina, Taphrinomycotina, and in the fungal phylum of Basidiomycota. Across species, we find the conserved arginine R′ centered on a short hydrophobic patch. Interestingly, in the available three-dimensional structure of the Dam1 complex (PDB:6CFZ) (Jenni & Harrison, 2018), the DSS motif makes multipartite interactions with residues of Dam1, Spc19, and Spc34 subunits within the “central domain,” suggesting its potential contribution to the overall structure and function of the Dam1 complex (Fig 1B). Particularly, the conserved arginine residue makes both hydrophobic and electrostatic interactions with residues of Spc19 which were also conserved across several fungi (Fig S1). Considering the extent of conservation of the DSS across the three major fungal phyla, we sought to understand the functional significance of this domain in Dad2, especially its conserved arginine residue in two fungal systems —S. cerevisiae and C. albicans, diverged from each other by 117 mya (Shen et al, 2018), and known to possess distinct centromere structures and kinetochore architectures.

The essentiality of the DSS is not a conserved feature across centromere lengths

To study the DSS function in S. cerevisiae, we generated constructs expressing mutant versions of Dad2 bearing alanine substitutions of the conserved arginine residues or a truncated version lacking the entire DSS domain (ScDad2-R126A, ScDad2-R128A, and ScDad2-ΔDSS; Fig 1C). To test the function of these mutants, we used a previously reported temperature-sensitive (ts) mutant of DAD2 wherein the genomic allele of DAD2 was deleted and a ts allele was reintegrated (CJY077, dad2::KanMX6 his3Δ200 leu2Δ1::pCJ055 [dad2^ts]) (Janke et al, 2002). This mutant strain was independently transformed with a pRS313-based CEN/ARS plasmid containing either WT or mutant versions of DAD2 (as in Fig 1C) tagged with GFP at the C-terminus. In all these strains, the expression of DAD2-GFP was driven by the native promoter of ScDAD2. The functional significance of each of these residues was tested by a spot dilution assay to determine the ability of the mutants to support growth at an elevated temperature of 37°C (Fig 1D). As expected, the strain YSR01 carrying the empty vector was unable to grow at this temperature, but the strain YSR02 expressing ScDad2-FL grew well. Strikingly, the strains YSR03 expressing ScDad2-R126A and YSR05 expressing ScDad2-ΔDSS failed to grow at 37°C, revealing that R126 makes the DSS essential for Dad2 function in S. cerevisiae. However, the strain YSR04 expressing ScDad2-R128A also supported growth at an elevated temperature, suggestive of a nonessential role of R128 for viability. To rule out the lack of expression of these mutant proteins as a cause for their inability to complement growth at an elevated temperature, we confirmed the localization of the ectopic Dad2 protein (WT, mutant or truncated) at this temperature (Fig 1E). All versions of Dad2 except ScDad2-ΔDSS showed detectable punctate localization signals typical of clustered budding yeast kinetochores and their localization signal intensities were comparable (Fig 1F) (Cheeseman et al, 2001b; Janke et al, 2002). These results strongly indicated that the entire DSS motif is essential for the kinetochore localization of ScDad2. Based on the location of the DSS in the Dam1 complex monomer, we suspect that the deletion of the DSS domain might affect the integrity of the complex and, in turn, the localization of Dad2.

To rule out the temperature sensitivity of mutant cells expressing ScDad2-R126A and ScDad2-ΔDSS observed in the above assay, we tested the essentiality of this residue at an ambient growth temperature (30°C) as well. A tester strain was engineered such that the genomic allele of Dad2 was deleted after expressing Dad2 from an ectopic allele cloned in a centromeric plasmid carrying the URA3 gene (Fig S2A). This strain was then transformed with pRS313-based plasmids containing WT or mutant versions of DAD2. The ability of strains to lose the protection allele present in the plasmid and support subsequent growth on media containing 5′FOA was used to test for the essentiality of the mutant Dad2 protein for viability. The inability of YSR10 expressing ScDad2-R126A and YSR12 expressing ScDad2-ΔDSS to grow on the Ura⁺ counter-selection media containing 5′FOA at various dilutions tested in the spot dilution assay confirmed the essentiality of R126 and thereby the role of the DSS in cell viability (Fig S2A). These results confirmed that the highly conserved R126 residue is a critical amino acid for the essential function performed by Dad2 in S. cerevisiae.

Because ScDad2-R126A and ScDad2-ΔDSS mutants were inviable in S. cerevisiae, we sought to test the importance of the corresponding arginine residues in Dad2 that is essential for viability in C. albicans. To test this in C. albicans, we used a previously reported conditional mutant of dad2 (J108, dad2/PCK1pr-DAD2) (Thakur & Sanyal, 2011). In this conditional mutant, the only genomic allele of CaDAD2 is expressed under the PCK1 promoter that shuts down its expression in the presence of dextrose in growth media (Fig S2B). Whereas the conditional mutant J108 was unable to grow in media supplemented with dextrose, the complemented strains J108A and J108B expressing CaDad2-FL and CaDad2-ΔDSS, respectively, supported growth in this media, suggesting that unlike in S. cerevisiae, the deletion of the DSS motif does not make C. albicans cells inviable (Fig S2C). We further validated this observation by engineering the only allele of DAD2 in ASR01 (dad2/DAD2) to express GFP-tagged versions of CaDad2-FL, -R92A, and -ΔDSS in strains ASR02, ASR03, and ASR04, respectively (Fig 1G). Spot dilution assays were performed to assess if any of the CaDad2 derivatives exhibited growth defects as compared with the WT strain. Whereas we could observe a mild growth retardation at 30°C, the growth of mutant strains ASR03 and ASR04 expressing CaDad2-R92A and CaDad2-ΔDSS, respectively, was significantly compromised at a lower temperature of 18°C when compared with CaDad2-FL-expressing strain ASR02 or the WT strain SN148 grown under similar conditions (Fig 1H). We could detect punctate localization signals in strains expressing not only CaDad2-R92A but also CaDad2-ΔDSS, suggesting that kinetochore localization was neither affected by alanine substitution of the critical arginine residue nor when the entire DSS was deleted in C. albicans (Fig 1I and J). These experiments reveal that the presence of the evolutionarily conserved arginine residue in Dad2 is dispensable for the viability of C. albicans under normal growth conditions. Having observed such phenotypic differences in mutants of Dad2 in S. cerevisiae and C. albicans, we sought to dissect the mutant phenotype further to study the extent of functional conservation of the DSS between them.

The arginine residue R126 in the conserved DSS motif is critical for proper spindle dynamics and bipolar attachment of kinetochores in S. cerevisiae

To probe deeper into the role of the DSS and its conserved arginine R126 in chromosome segregation in S. cerevisiae, we generated conditional mutants of DAD2 by replacing the endogenous DAD2 promoter with the GAL_1–10 promoter in the strain SBY12503 where spindle pole bodies (SPBs) (Spc110 is tagged with mCherry) and CEN3 are marked (by the binding of GFP-LacI to LacO arrays integrated adjacent to CEN3) (Umbreit et al, 2014). The resulting strain YSR13 (GALpr-DAD2) fails to grow when dextrose is the sole carbon source in the growth media (Fig S3A and B). This conditional mutant strain YSR13 was then independently transformed with vectors that reintegrate WT or mutant versions of DAD2 expressed from the DAD2 promoter at the native locus. In line with our previous observations, the conditional mutant complemented with ScDad2-FL in YSR14 and ScDad2-R128A in YSR16 supported growth on dextrose-containing media. On the other hand, the strains YSR15 and YSR17 expressing ScDad2-R126A and ScDad2-ΔDSS, respectively, could not complement function as they were unable to support the growth of the conditional mutant on this media (Fig S3B).

To identify the defects that led to viability loss, the dad2 conditional mutant YSR13 along with the reintegrant strains YSR14 through YSR17 were grown for 8 h in dextrose-containing media to deplete Dad2 expressed under the GAL promoter. Cells were harvested and analyzed for cell cycle progression, spindle dynamics, and kinetochore–MT orientation post depletion (Fig 2A). Upon 8 h of growth in repressive media, flow cytometric analysis of propidium iodide-stained cells revealed the parent strain YSR13 (GALpr-DAD2) was arrested at the G2/M stage in line with previous observations (Fig 2B). The impaired MT-binding because of the lack of any Dam1 complex subunits is known to activate the spindle assembly checkpoint (SAC) (Hofmann et al, 1998; Janke et al, 2002; Thakur & Sanyal, 2011), resulting in the observed G2/M arrest. This arrest was rescued when the mutant was complemented with ScDad2-FL or ScDad2-R128A as observed in YSR14 and YSR16, respectively, (Figs 2B and S3C). However, no rescue in the arrest was observed in strains YSR15 and YSR17 expressing ScDad2-R126A and ScDad2-ΔDSS, respectively, suggesting the conserved arginine R126 in the DSS motif is essential for mitotic progression in S. cerevisiae.

At the same hour of Dad2 protein depletion, we examined the spindle dynamics using the fluorescently tagged spindle pole body protein, Spc110-mCherry, as the marker. In line with the metaphase arrest observed upon depletion of Dad2 in YSR13, we found these cells to have a short mitotic spindle (<2 μm) (Fig 2C). The conditional mutant strain, when complemented with ScDad2-FL as in YSR14 or with ScDad2-R128A as in YSR16, was able to transit metaphase and enter anaphase, as suggested by an increased average spindle length of >2 μm (Fig 2C). We did not observe the rescue of the mitotic arrest and a corresponding increase in the spindle length when the same conditional mutant was complemented with ScDad2-R126A as in YSR15 or ScDad2-ΔDSS as in YSR17 (Fig 2C).

To validate if the observed defects were consequential of improper kinetochore–MT orientation at metaphase, we studied localization of centromeres (marked by CEN3-GFP) relative to the SPBs (marked by Spc110-mCherry) to monitor the nature of these attachments (Umbreit et al, 2014). Sister kinetochores were bioriented in most of the cells in YSR14 expressing ScDad2-FL or YSR16 expressing ScDad2-R128A (Fig 2D and E). By contrast, a significant proportion of YSR15 cells expressing ScDad2-R126A or YSR17 cells expressing ScDad2-ΔDSS exhibited monooriented kinetochores as in the case in YSR13 upon depletion of Dad2. Together, these observations suggest that the conserved arginine residue R126 in the DSS motif plays a critical role in facilitating biorientation of the kinetochores, lacking which, cells remain arrested at metaphase. The observation that the phenotype of mutants expressing ScDad2-R126A or ScDad2-ΔDSS are comparable with the depletion of Dad2 suggests the DSS region, particularly its R126 residue, plays a significant role in the proper functioning of the Dam1 complex in S. cerevisiae.

DSS is critical for oligomerization and MT binding of Dam1 complex

Our genetic studies clearly suggest that the kinetochores bearing mutant versions of Dad2 are unable to attach or remain attached with the MT ends resulting in an increased proportion of cells with monooriented kinetochores. We wondered if the observed phenotype in DSS mutants is indicative of structural changes in the Dam1 complex.

To understand this further, we modified the sequenced encoding for Dad2 in the previously reported polycistronic expression vector (Miranda et al, 2005) encoding all 10 subunits of the ScDam1 complex to express ScDad2-R126A or ScDad2-ΔDSS instead of WT Dad2. We then expressed and purified three versions of the Dam1 complex from E. coli containing Dad2-FL (DASH^WT), Dad2-R126A (DASH^R126A), or Dad2-ΔDSS (DASH^ΔDSS), respectively. During the purification process, the DASH^WT eluted as a single peak upon gel filtration (Fig 3A, peak fraction highlighted in gray). On the other hand, both DASH^R126A and DASH^ΔDSS eluted later than the DASH^WT. Furthermore, we also observed a second peak around Ve = 18 ml in the mutants that were more pronounced than that observed in the DASH^WT profile. These are suggestive of structural or/and compositional changes in the complex. We analyzed the copurifying subunits from each of the mutant peak fractions (b for DASH^R126A and d for DASH^ΔDSS) to that of DASH^WT by SDS–PAGE. We also included the fractions marked “a” and “c” in the elution profile of the mutants that corresponded to the peak fraction from the DASH^WT purification. We find that the DASH^R126A complex does not contain the subunit Spc19 indicative of an incomplete complex in the presence of this mutation (Fig 3B). Intriguingly, this was not the case with DASH^ΔDSS which contained a band corresponding to Spc19 despite eluting at a higher volume as compared with DASH^WT. These observations clearly suggest that disruptions in the DSS affect the structure of the complex.

We next tested if the changes described above affected their ability to oligomerize into rings around MTs, a defining feature of the Dam1 complex. The proteins derived from both the peaks for DASH^R126A and DASH^ΔDSS were used in these experiments. In the presence of MTs, we could detect the rings formed by the DASH^WT complex by negative stain electron microscopy (Fig 3C). However, the mutant complexes DASH^R126A and DASH^ΔDSS failed to form such structures indicating that the DSS region is essential for oligomerization of the Dam1 complex to form higher assemblies like the ring (Fig 3C).

Previous studies on the Dam1 complex have identified oligomerization-deficient mutants that still retained the ability to bind MTs. We tested this property in our mutant complexes by MT-cosedimentation assay. The DASH^WT was consistently detected in the pellet fraction in all the conditions we tested (0.5 μM–2 μM MTs in twofold increments) suggesting that they physically bound to MTs as reported previously (Figs 3D and S4). In contrast, the binding of both DASH^R126A and DASH^ΔDSS to the MTs was severely compromised across the conditions tested. Our results suggest that the DSS domain plays a significant role in both the structure (oligomerization) and function (MT binding) of the Dam1 complex in S. cerevisiae. These defects are consistent with our observations in vivo and can be correlated with the increased frequency of monooriented kinetochores in DSS mutants.

It is known that Dad2 is essential for viability in both C. albicans and S. cerevisiae (Hofmann et al, 1998; Janke et al, 2002; Thakur & Sanyal, 2011). Whereas the DSS mutants were viable in C. albicans, they were found lethal S. cerevisiae. Considering our observations described above, it is possible that CaDam1 complex is structurally divergent from the budding yeast version, or the kinetochore–MT interface in C. albicans shows sufficient plasticity to tolerate variations in the Dam1 complex. To better understand the observation in C. albicans, we next studied the kinetochore integrity and chromosome segregation fidelity of these mutants in further detail.

The conserved arginine residue in the DSS is essential for faithful chromosome segregation in C. albicans

To test for functional conservation of the DSS in C. albicans, we used the strains ASR02, ASR03, and ASR04 expressing CaDad2-FL, CaDad2-R92A, and CaDad2-ΔDSS, respectively, from the native genomic locus. A typical feature of C. albicans kinetochore is that any defect/mutation compromising the structural integrity of the kinetochore results in the disintegration of the kinetochore ensemble followed by proteasomal degradation of CENPA (Thakur & Sanyal, 2012). We used this property as a test to assess the integrity of the kinetochore upon mutations in the DSS. We engineered the strains ASR02 through ASR04 to express protein-A tagged CENPA. Immunoblotting revealed comparable levels of CENPA between ASR07 cells expressing CaDad2-FL, and the mutant strains ASR08 and ASR09, expressing CaDad2-R92A and CaDad2-ΔDSS, respectively (Fig 4A). The absence of CENPA degradation in this species unlike the case with depletion of Dad2 or other Dam1 complex subunits (Thakur & Sanyal, 2012), is a strong indicator of an intact kinetochore ensemble. This is further supported by the detection of punctate localization signals of Dad2 mutants with similar fluorescence intensities as compared with WT Dad2 (Fig 1J). Together, our observations suggest that neither the deletion of the DSS nor substitution in the conserved arginine residue in the DSS affects the kinetochore assembly in C. albicans.

To understand the growth defects of dad2 mutants observed at a lower temperature, we grew these strains overnight at 30°C, reinoculated them to fresh media in duplicates, and allowed them to complete two generations each at 30°C and 18°C, respectively. Analysis of cell cycle progression of strains grown at 30°C revealed a modest increase in the proportion of cells at the G2/M stage when they expressed CaDad2-R92A as in ASR03 or CaDad2-ΔDSS as in ASR04 when compared with the control strain ASR02 that expressed CaDad2-FL (Figs 4B and S5A). The frequency of G2/M arrested cells was further amplified when the DSS mutants were grown at 18°C wherein most of the cells displayed this phenotype (Fig 4B). Given that the kinetochore assembly remained unaffected in the DSS mutants (Figs 1J and 4A), we suspected the increase in cells at G2/M and cold-sensitive phenotype of these mutants to be consequential of aberrant kinetochore–MT interactions.

When cells were collected for flow cytometry analysis, additional aliquots of similarly grown cells were examined to analyze their nuclear segregation patterns in the strains ASR02, ASR03, and ASR04 expressing CaDad2-FL, CaDad2-R92A, and CaDad2-ΔDSS, respectively. We observed ∼twofold increase in the frequency of unsegregated nuclei in strains expressing CaDad2-R92A and CaDad2-ΔDSS as compared with cells expressing CaDad2-FL when grown at 30°C (Fig 4C). This frequency further increased to >threefold when these strains were grown at 18°C (Fig 4C). Each of these strains was engineered to express an SPB marker Tub4-mCherry to study the spindle dynamics and correlate them with the frequency of unsegregated nuclei. In the strains ASR13 and ASR14 expressing CaDad2-R92A and CaDad2-ΔDSS, respectively, we observed a significant increase in the frequency of large-budded cells showing short spindles (<2 μm) as compared with ASR12 that expressed CaDad2-FL at 30°C (Fig 4D). This was further reflected in the reduction in the average spindle length in these mutant strains (Fig 4E). We were unable to perform this assay at 18°C as the DSS mutants showed an aberrant elongated large-budded phenotype at this temperature, suggesting that epitope-tagged Tub4 could be nonfunctional in this condition. The increased proportion of cells at metaphase with the spindle length (SPB–SPB distance) of < 2 μm and an unsegregated nuclear mass is suggestive of a delayed anaphase onset in these mutants.

The observed delay in mitotic progression could be because of the inability of these mutants to achieve a bioriented state, as observed in S. cerevisiae (Fig 2D). Such defective kinetochore–MT attachments which do not generate tension are detected by sensors like the Aurora B kinase (Ipl1 in yeast) that eventually activate the SAC until proper biorientation is achieved (Biggins & Murray, 2001; Tanaka et al, 2002; Franck et al, 2007; Akiyoshi et al, 2010). We suspected that the accumulation of cells with metaphase-like spindle in the DSS mutants could also be because of a similar defect. As mentioned above, ASR03 (CaDad2-R92A-GFP) and ASR04 (CaDad2-ΔDSS-GFP) cells were arrested completely at the G2/M stage when grown at 18°C. We selected this condition to score for bypass of the G2/M arrest in the corresponding checkpoint-deficient mutants ASR17 (Δmad2 CaDad2-R92A-GFP) and ASR18 (Δmad2 CaDad2-ΔDSS-GFP) by flow cytometry (Fig S5B). The presence of unbudded cells (2N population) in ASR17 and ASR18 mutants as compared with the G2/M-arrested phenotype in ASR03 and ASR04 cells suggested that the DSS mutants were indeed arrested at metaphase. We also analyzed these strains for segregation of the sister kinetochores marked by CaDad2-GFP when grown at 30°C and 18°C (Fig S5C). At both the temperatures tested, the percentage of large-budded cells with unsegregated sister kinetochores in the mother bud (<2 μm between CaDad2 puncta) was reduced in the checkpoint-deficient Dad2 mutants ASR17 and ASR18 as compared with their checkpoint-proficient parent strains, further validating a delay in anaphase onset in these cells.

By comparing the results obtained thus far in S. cerevisiae and C. albicans, it is evident that the function of the DSS is conserved between these species, with an obvious reduction in the severity of the DSS mutant phenotype in C. albicans. The Dam1 complex is known to play a critical role in chromosome segregation by strengthening the kinetochore–MT associations, especially in instances where a single MT binds to a chromosome like in S. cerevisiae and C. albicans. Although the number of MTs binding a chromosome is the same between these species, a single CENPA nucleosome forms the basis of MT attachment in S. cerevisiae (Winey et al, 1995). On the other hand, one among four CENPA nucleosomes anchors the MTs in C. albicans (Joglekar et al, 2008). We speculated that the presence of additional CENPA nucleosomes confers a degree of tolerance towards mutations in the DSS. To test if this correlation indeed exists, we studied the functional significance of the DSS in the ascomycete S. pombe, which is a classic example for the large regional centromere structure.

DSS is dispensable for Dad2 function in S. pombe

Unlike any of the species we tested earlier, dad2 mutants are viable in S. pombe (Liu et al, 2005; Kobayashi et al, 2007). However, these mutants undergo prolonged mitosis because of biorientation defects that result in lagging chromosomes. At lower temperatures, these mutants missegregated chromosomes causing a cold-sensitive phenotype (Kobayashi et al, 2007). We exploited this property to study functional significance of DSS in SpDad2 as follows. Null mutants of dad2 in a strain where the chromosomes are tagged by lacO-LacI-GFP system have been reported previously (Strain 1619, [Blyth et al, 2018]). We independently reintegrated SpDad2-FL (PSR01) and SpDad2-ΔDSS (PSR02) into the native genomic locus with their expression driven by the native Dad2 promoter (Fig 5A).

Spot dilution assays did not reveal any apparent growth difference between dad2 null mutants and the reintegrant strains PSR01 and PSR02 expressing SpDad2-FL and SpDad2-ΔDSS, respectively, at ambient growth temperatures (Fig 5B). Reintegrating Sp-Dad2FL rescued the cold-sensitive phenotype observed in dad2 null mutant strain SP1619 as expected. Furthermore, we also observed a rescue in PSR02 cells expressing SpDad2-ΔDSS, suggesting that DSS is not essential for Dad2 function in S. pombe. We strengthened our correlation further by testing for the essentiality of DSS in Dad2 function in another large regional centromere containing species C. neoformans. Unlike the species studied above, C. neoformans belongs to Basidiomycota and diverged from a common ancestor it shared with S. pombe ∼642 MYA (Kumar et al, 2017).

The conserved arginine R102 in CnDad2 is dispensable for mitotic progression in C. neoformans

We generated constructs to introduce an alanine substitution mutation in the conserved arginine residue (R102 in CnDad2) and for the deletion of the entire DSS region in CnDad2 and express them along with mCherry tagged at the C-terminus in C. neoformans (Fig 5C). As a control, another construct to express an unaltered full-length CnDad2 sequence was also generated. Each of these constructs was used to transform the strain CNV108 (expressing GFP-H4) (Kozubowski et al, 2013) to generate CNSD169 (CnDad2-FL-mCherry, GFP-H4), CNSD170 (CnDad2-R102A-mCherry, GFP-H4), and CNSD171 (CnDad2-ΔDSS-mCherry, GFP-H4).

Spot dilution assays performed at routine growth conditions in YPD at 30°C revealed that neither the point mutation R102A nor the deletion of the entire DSS motif of CnDad2 resulted in an obvious growth defect, suggesting that the DSS mutants are viable in C. neoformans as well (Fig 5D). We also examined growth inhibitions of these mutants at a lower temperature (14°C) or in the presence of thiabendazole (6 μg/ml), conditions used to test mutants for defective kinetochore–MT associations. We failed to observe any growth defects in the mutant strains CNSD170 and CNSD171 expressing CnDad2-R102A and CnDad2-ΔDSS, respectively, as compared with CNSD169 cells expressing CnDad2-FL (Fig 5D). To corroborate these observations, we grew each of these strains to log phase at 30°C. We did not observe a significant increase in the number of large-budded cells in strains expressing WT or mutant versions of Dad2-bearing alanine substituted conserved arginine residue in the DSS or deletion of the entire DSS itself (Fig 5E). However, further analysis of nuclear segregation by localization of GFP-tagged histone H4 revealed that the CNSD171 mutant strain expressing the DSS deletion, but not CNSD170 with the point mutation CnDad2-R102A, had a significant increase in the proportion of large-budded cells with an unsegregated nuclear mass (Fig 5F and G). We do not find any significant difference in the fluorescence intensities between CnDad2-FL, CnDad2-R102A, and CnDad2-ΔDSS, suggesting that the kinetochore structure was not compromised by the mutations introduced (Fig 5H). We validated this further by testing the levels of another outer kinetochore protein Dad1in the strains CNSD172, CNSD173, and CNSD174 expressing CnDad2-FL, CnDad2-R102A, and CnDad2-ΔDSS, respectively (Fig 5I).

These results suggest that the conserved arginine residue in Dad2 is dispensable for viability and timely mitotic progression in C. neoformans unlike in S. cerevisiae or C. albicans. However, a small but significant increase in the large bud arrested cells when the entire DSS was deleted, suggesting the importance of the motif in chromosome segregation in C. neoformans.

The tolerance of S. pombe and C. neoformans to mutations in the DSS is suggestive of a nonessential role for the conserved arginine residue/DSS in Dam1 complex function in these species as compared with S. cerevisiae or C. albicans. Despite a high level of conservation at the amino acid sequence in the DSS, the striking differences in the dependence on this conserved arginine residue for mitotic progression across the evolutionarily diverged species represent a strong inverse correlations with the centromere length.

Media and growth conditions

S. cerevisiae, C. albicans, and C. neoformans strains used in this study were grown in YPD (2% dextrose, 2% peptone, 1% yeast extract supplemented with 0.01% adenine) and incubated at 30°C at 180 rpm. Conditional expression strains in S. cerevisiae were propagated in galactose media (YPG, 2% galactose, 0.3% raffinose, 2% peptone, 1% yeast extract) unless repression in YPD is mentioned. Transformation of S. cerevisiae and C. albicans were performed by standard lithium acetate–PEG method (Sanyal & Carbon, 2002; Geitz & Schiestl, 2007). Biolistic transformation method was used for C. neoformans strains (Davidson et al, 2000). Selection of transformants was based on prototrophy for the metabolic markers used. In case of antibiotic marker NAT, selection was done in media supplemented with 100 μg/ml nourseothricin (ClonNAT; CAS 96736-11-7; Werner Bioagents). For cold sensitivity assays, the growth temperature was reduced to 18°C for C. albicans and 14°C for C. neoformans. For thiabendazole sensitivity assay, the growth media were supplemented with indicated concentration of thiabendazole (10 mg/ml stock in dimethyl formamide; Sigma-Aldrich).

Construction of S. cerevisiae strains

Construction of C. albicans strains

Construction of C. neoformans strains

Construction of S. pombe strains

Identification of DSS

The HMM file for the Dad2 domain (PF08654) was downloaded from the Pfam database page for this domain. This model was used as the query to perform a HMMsearch (hmmsearch search | HMMER (ebi.ac.uk)) routinely used to search for a HMM profile against a protein sequence database. The search was targeted against fungal genomes using the option “Ensembl Genomes Fungi.” The E-value cutoff for the search was 0.01 by default. The first round of HMM search resulted in 912 hits from all the major fungal subphyla (Table S5, Sheet- Hits_Ensembl Fungi). The hits were manually curated to remove duplicate entries of any given species for which multiple genome sequences were available. In addition to this, hits from nine species that were unusually long were also removed for subsequent analysis to avoid alignment artifacts (Table S5, sheet-long hits). This resulted in a final pool of 466 hits from which a multiple sequence alignment was generated using MAFFT. Apart from the known Dad2 domain at the N-terminus, the alignment revealed a second conserved domain Dad2 centered around a highly conserved arginine. This 10aa-long domain was named DSS. The amino acid logo indicating the consensus sequence of DSS (in Fig 1A) was generated with this multiple sequence alignment using Weblogo tool (WebLogo - Create Sequence Logos (berkeley.edu)).

Flow cytometry

Overnight grown cultures of S. cerevisiae or C. albicans were subcultured to 0.2 OD₆₀₀ in desired media/growth condition as described for the experiment. Cells were harvested at various time intervals post-inoculation. Harvested samples were fixed by dropwise addition of ice-cold 70% ethanol, followed by RNAse treatment and propidium iodide (PI) staining as described before (Sanyal & Carbon, 2002; PNAS). Stained cells were diluted to the desired cell density in 1x PBS and analyzed (30,000 cells) by flow cytometry (FACSAria III; BD Biosciences) at a rate of 500–2,000 events/s. The output was analyzed using the FLOWJO software. The 561-nm laser was used to excite PI and 610/20 filter to detect its emission signals.

Fluorescence microscopy and analysis

Cells were grown in media and growth conditions as described for each experiment. Before imaging, the cells were washed thoroughly with sterile water twice and resuspended in sterile water to achieve optimum cell density for imaging. The cells were imaged in Zeiss Axio observer equipped with Colibri 7 as the LED light source, 100x Plan Apochromat 1.4 NA objective, and PCO edge 4.2 sCMOS camera. Z sections were obtained at an interval of 300 nm. All the images were displayed after the maximum intensity projection using ImageJ. For visualizing nuclear mass, S. cerevisiae and C. albicans cells were stained with Hoechst (50 ng/ml) before imaging. GFP and mCherry fluorescence were acquired using the filter set 92 HE (excitation 455–483 nm and emission 501–547 nm for GFP, and excitation 583–600 nm, and emission 617–758 nm for mCherry).

Western blotting

Protein lysates for Western blot were prepared by the TCA method. From overnight grown cultures, 3OD₆₀₀ equivalent cells were harvested, washed, and resuspended in 400 μl of 12.5% ice-cold TCA solution. The suspension was vortexed briefly and stored at −20°C for 12 h. The suspension was thawed on ice, pelleted at 14,000 rpm for 10 min, and washed twice with 350 μl of 80% acetone (ice cold). The washed pellets were air dried completely and resuspended in desired volume of lysis buffer (0.1N NaOH+1% SDS). Rabbit anti-Protein A antibody (P3775; Sigma-Aldrich) and the HRP-conjugated goat anti-rabbit secondary antibody, both were used at 1:5,000 dilution in 2.5% skim milk powder in 1XPBS. The blots were developed using chemiluminescent substrate (Bio-Rad) and imaged using Chemidoc system (Bio-Rad).

Expression and purification Dam1 complex

Statistical analysis

All graphs were generated and analyzed using GraphPad Prism (v8). One-way ANOVA or paired t test with Welsch’s correction was used based on the sample size to test for the statistical significance of acquired results using a P-value cut off of 0.05.