DNA Damage Checkpoints Govern Global Gene Transcription and Exhibit Species-Specific Regulation on HOF1 in Candida albicans

DNA damage checkpoints are essential for coordinating cell cycle arrest and gene transcription during DNA damage response. Exploring the targets of checkpoint kinases in Saccharomyces cerevisiae and other fungi has expanded our comprehension of the downstream pathways involved in DNA damage response. While the function of checkpoint kinases, specifically Rad53, is well documented in the fungal pathogen Candida albicans, their targets remain poorly understood. In this study, we explored the impact of deleting RAD53 on the global transcription profiles and observed alterations in genes associated with ribosome biogenesis, DNA replication, and cell cycle. However, the deletion of RAD53 only affected a limited number of known DNA damage-responsive genes, including MRV6 and HMX1. Unlike S. cerevisiae, the downregulation of HOF1 transcription in C. albicans under the influence of Methyl Methanesulfonate (MMS) did not depend on Dun1 but still relied on Rad53 and Rad9. In addition, the transcription factor Mcm1 was identified as a regulator of HOF1 transcription, with evidence of dynamic binding to its promoter region; however, this dynamic binding was interrupted following the deletion of RAD53. Furthermore, Rad53 was observed to directly interact with the promoter region of HOF1, thus suggesting a potential role in governing its transcription. Overall, checkpoints regulate global gene transcription in C. albicans and show species-specific regulation on HOF1; these discoveries improve our understanding of the signaling pathway related to checkpoints in this pathogen.


Introduction
DNA damage, caused by intrinsic and extrinsic factors, is unavoidable in cells but could be lethal if not adequately repaired.To address this potentially fatal event, cells detect and repair damaged DNA through an intricate and precise mechanism referred to as the DNA damage response (DDR) [1,2].Among the components of the DDR, DNA damage checkpoints are considered pivotal hubs for coordinating cell cycle arrest and gene transcription to facilitate the repair process [3].Upon the detection of damaged DNA signals, checkpoint kinases are activated through a cascade of phosphorylation events to halt the cell cycle progression and prevent the transmission of damaged DNA to daughter cells [2].In Saccharomyces cerevisiae, Mec1 and Tel1 have been identified as two primary sensor kinases that function with partial redundancy in sensing DNA damage and initiating its repair [4,5].Once activated, these sensors transmit the signal of DNA damage to downstream effector kinases Rad53 or Chk1, with the involvement of adaptors Rad9 or Mrc1 [5][6][7].Activated Rad53 further regulates the transcription of DNA damage response genes and dNTP pools directly or in a Dun1-dependent manner [2,8,9].Understanding the cellular response to diverse types of DNA damage stresses has dramatically enriched our understanding of the DNA damage repair process across various model organisms.Particularly in S. cerevisiae, numerous transcription factors function downstream from checkpoint kinases Rad53 and Dun1 [10].For instance, multiple nucleic acid metabolism genes involved in DNA replication and repair are regulated by the transcription factor complexes MBF (Swi6-Mbp1) and SBF (Swi6-Swi4); this regulation depends on Rad53 but not its downstream kinase Dun1 [10].In the pathogenic fungus Cryptococcus neoformans, the transcription of DNA damage repair genes RAD51, RAD54, and RDH54 is induced by gamma radiation; however, the induction is significantly inhibited by RAD53 deletion rather than CHK2, thus suggesting checkpoint-specific regulation [11].
Emerging evidence suggests that most genes responding to DNA damage are not directly involved in DNA repair but instead participate in various indirect processes such as cell cycle progression, stress response, protein homeostasis, and energy metabolism [12].In addition, checkpoint kinases play a role in regulating the global gene transcription to facilitate DNA repair.In S. cerevisiae, the primary checkpoint effector kinase Rad53 governs the transcription of genes related to the vacuolar protein catabolic process by modulating the activity of transcription factor Msn4 in response to methyl methanesulfonate (MMS) [10].Moreover, Rad53 regulates the transcription of genes associated with cellular amino acids and the derivative metabolic process through transcription factor Gcn4, as well as genes involved in cell division via the Ndd1/Mcm1/Fkh2 complex; these actions are dependent on downstream kinase Dun1 [10].Specifically, Hof1, an F-BAR protein, is essential for regulating actin cytoskeleton organization and cytokinesis, and it exhibits decreased expression after MMS exposure in S. cerevisiae.Notably, this reduction can be blocked by deleting the DUN1 gene, thus highlighting its role in checkpoint-mediated transcriptional control [10].Furthermore, the regulation of HOF1 and other cell cycle elements depends on the Ndd1/Mcm1/Fkh2 complex, with Ndd1 serving as a coactivator for transcription [13].Similarly, MMS stress suppresses HOF1 expression in the pathogenic yeast Candida albicans, which is an effect that can be reversed by RAD53 deletion [14].However, the regulatory mechanism by which Rad53 governs HOF1 and other cell cycle genes remains unclear in C. albicans, primarily due to the lack of a known orthologous counterpart for Ndd1.
Checkpoint kinases regulate global cellular processes, including the virulence of various pathogens.In Cryptococcus neoformans, Rad53 is phosphorylated by the two phosphatidylinositol 3-kinase (PI3K)-like kinases Tel1 and Mec1, and it governs the expression of DNA damage repair genes in response to gamma radiation [11].The perturbation of RAD53 attenuates the virulence of C. neoformans and increases susceptibility to specific antifungal drugs such as amphotericin B [11].In Candida glabrata, the overexpression of RAD53 reduces biofilm formation and enhances biofilm susceptibility to fluconazole under hypoxia [15], while the checkpoint kinase Chk1 is crucial for the virulence of the plant pathogen Ustilago maydis [16].In C. albicans, DNA damage repair genes like RTT109 and RAD23 are implicated in virulence; however, the role of checkpoints in virulence remains uncertain [17,18].
In our previous study, we revealed the transcriptional response to MMS-induced DNA damage stress in C. albicans and identified a set of MMS-responsive genes [12].Nevertheless, the regulatory mechanisms governing these potential DNA damage-responsive genes during DNA damage responses remain unclear.Profiling checkpoint-related transcription is crucial for defining the essential DNA damage response genes.To address this, we employed RNA sequencing assays to elucidate the transcriptional consequences of RAD53 deletion in C. albicans.We discovered that Rad53 potentially regulates ribosome biogenesis, the cell cycle, and DNA replication circuits in C. albicans.Additionally, we noted the specific mechanism of the Rad53-and Rad9-mediated transcriptional regulation of HOF1 in C. albicans; this pattern differed from that seen in S. cerevisiae, as the expression of HOF1 in C. albicans was independent of Dun1.Furthermore, our findings demonstrate that checkpoint kinase Rad53 directly interacts with the promoter region of HOF1 and regulates the dynamic binding of Mcm1 to its promoter region.Overall, our study provides valuable insights into understanding checkpoint-dependent transcriptional regulation during DNA damage response in C. albicans, thus highlighting a species-specific regulation on HOF1.

Strains, Media, and Reagents
C. albicans strains were cultured in YPD media supplemented with 50 mg/L uridine, as previously described [19].Deletion strains were selected on synthetic complete (SC) media lacking uridine, histidine, or arginine.To inhibit the MET3 promoter, strains carrying this promoter were cultured in liquid SC media supplemented with 5 mM methionine and cysteine.Strains and primers used in this study are listed in Tables S1 and S2, respectively.Methyl methanesulfonate (MMS) was purchased from Sigma (St. Louis, MO, USA).Digitonin was purchased from Meryer (Shanghai, China).Additional reagents and amino acids for making media were acquired from Sangon (Shanghai, China).Solid media consisted of 2% agar.

DNA Manipulation
To construct the DUN1 deletion strain (Table S1, JC25), two copies of the DUN1 gene in wild-type strain (SN148 background) were substituted with HIS1 and ARG4 markers following previous protocols [12].Similarly, the RAD9 deletion strain was produced by replacing the two RAD9 gene copies with HIS1 markers in the SN148 background.Furthermore, based on the HOF1 deletion strain, the DUN1 or RAD9 genes were deleted using a transient CRISPR/Cas9 system to generate the HOF1 DUN1 or HOF1 RAD9 doublegene deletion strains (Table S1, JC27 and JC28).The successful knockout strains were confirmed by PCR analysis.
To investigate the influence of Fkh2 on HOF1 transcription, the FKH2 gene in the wildtype SN148 strain was knocked out using a transient CRISPR/Cas9 system as before (Table S1, JC29).To overexpress FKH2, the ORF of the FKH2 gene with its terminator was amplified by PCR and inserted into the Xhol I and Kpn I sites of the CIP10-ADH1 plasmid [20], thus generating CIP10-ADH1-FKH2.Subsequently, the linearized CIP10-ADH1-FKH2 plasmid was transformed into an SN148 strain after digestion with the Stu I enzyme (Table S1, JC30).The successful overexpression of the FKH2 gene was confirmed through qRT-PCR analysis.Similarly, the ORF of DUN1 genes was amplified and integrated into the CIP10-ADH1 plasmid to construct a DUN1 overexpression strain (Table S1, JC37).
To suppress the expression of MCM1, a DNA fragment containing the MET3 promoter was amplified from the pFA-URA3-MET3 plasmid [21] and introduced into the wild-type SN148 strain to replace its original promoter using a transient CRISPR/Cas9 system (Table S1, JC31).The successful substitution was confirmed by PCR analysis.The suppression and overexpression levels of the MCM1 gene were validated by qRT-PCR.The diagram for making the mutant strains is presented in Figure S1.

Microscopy
For visualizing the distribution of nuclei, C. albicans cells were stained with DAPI [14].Generally, overnight cultures of C. albicans cells were diluted 1/10 in fresh YPD media and incubated at 30 • C with shaking for 3 h before being treated with 0.02% MMS for 2 h.Subsequently, the MMS-treated cells were collected and fixed in 70% ethanol for 5 min, washed twice with 1×PBS, and then incubated in a solution containing DAPI at a concentration of 1.0 µg/mL for 10 min.Following two additional washes with PBS, the cells were mounted for analysis using a Leica DM5000B microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 40 × objective.The data regarding nuclei separation represent an average from two independent experiments.
To assess filamentation triggered by genotoxic stress in C. albicans cells, overnight cultures were diluted in fresh YPD media and incubated at 30 • C with shaking for 3 h.Subsequently, the cell culture was treated with either 0.02% MMS or 40 mM hydroxyurea (HU) and incubated for the specified duration.Imaging was performed using a Leica DM5000B microscope (Leica Microsystems) equipped with a 40 × objective lens.

RNA Preparation and RNA Sequencing (RNA-Seq) Assay
The C. albicans wild-type SN148 strain and the RAD53 deletion strain were inoculated into 3 mL liquid YPD and incubated overnight at 30 • C on a shaker rotating at 200 rpm.The overnight cultures were diluted to an optical density at 600 nm (OD600) of 0.1 in 10 mL YPD media and grown with shaking at 30 • C until reaching an OD600 of 0.6-0.8.Subsequently, the cells were treated with 0.015% MMS for 90 min before being harvested for RNA extraction.Total RNA was extracted using a trizol reagent kit from Invitrogen (Waltham, MA, USA) according to the manufacturer's protocol.RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and checked using RNasefree agarose gel electrophoresis.Following total RNA extraction, mRNA was enriched using Oligo (dT) beads (Illumina, San Diego, CA, USA) and fragmented into short pieces with a fragmentation buffer.The fragments were then reverse transcribed into cDNA using the NEB Next Ultra RNA Library Prep Kit (NEB #7530, Ipswich, MA, USA).The resulting double-stranded cDNA fragments were purified and subjected to end repair, followed by the addition of an A base and ligation to Illumina sequencing adapters.Subsequently, the ligated fragments were subjected to size selection by agarose gel electrophoresis and polymerase chain reaction (PCR)-amplified.RNA library sequencing was performed on the Illumina Novaseq6000 by Gene Denovo Biotechnology Co., Ltd.(Guangzhou, China).The reads from each sample were aligned and assembled using StringTie v1.3.1 in a referencebased manner.The mapped reads of each sample were assembled by using StringTie v1.3.1 in a reference-based approach.The C. albicans SC5314 genome data downloaded from NCBI (https://www.ncbi.nlm.nih.gov/genome/21?genome_assembly_id=294796(accessed on 28 December 2021) were used as a reference genome.Raw data have been deposited in the NCBI SRA database (PRJNA811694 and PRJNA985884).
Transcript analysis was conducted using the DESeq2 software 1.24.0,thus focusing on transcripts with adjusted p values ≤ 0.05 between the two groups.Pathway enrichment (KEGG) analysis was conducted to identify significantly enriched metabolic pathways or signal transduction pathways in differentially expressed genes (DEGs) compared to the whole genome background.The calculated p value went through FDR correction, with a threshold set at FDR ≤ 0.05.Pathways meeting this criterion were defined as significantly enriched pathways in DEGs.Gene set enrichment analysis (GSEA) was performed using the GseaPreranked tool (v.2.2.0) and the weighted enrichment statistics on 6387 (for C. albicans) gene sets, with each containing 5 to 500 genes [22].

Real-Time PCR (qRT-PCR)
To validate the transcription of specific genes, qRT-PCRs were conducted.The samples of the wild-type SN148 strain and the RAD53 deletion strain, treated or untreated with 0.015% MMS for 90 min, were collected as previously described.Total RNA was extracted using an RNA-easy isolation reagent (R701, Vazyme, Nanjing, China), and cDNA was synthesized using the cDNA synthesis kit (R212, Vazyme, Nanjing, China) incorporating DNase to eliminate residual DNA in the template.qRT-PCRs were carried out utilizing ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) according to the protocol from the manufacturer.Specific primers for the target genes, along with the GAPDH primers serving as controls, are listed in Table S2.The data for each gene represent an average derived from at least three independent experiments.

ChIP Assay
The overnight culture of the strains harboring the Fkh2-HA, Mcm1-HA, or Rad53-HA fusion was transferred to 40 mL liquid YPD and grown until reaching OD600 around 0.6.The cells were then exposed to 0.02% MMS for 90 min, with an untreated wild-type cell culture of comparable growth time used as a control.Following this treatment, the samples were fixed with 1% formaldehyde for 30 min before being harvested for ChIP analysis [23].Cell disruption was achieved by agitating the cells with glass beads in a disruptor (SI-DD48, Scientific Industries, Bohemia, NY, USA) for five rounds of 6 min each, thus generating whole-cell extract (WCE).The WCE was subsequently sonicated 9 times for 10 s each using an ultrasonic cell disruptor (0.2 kW, JY92-2D, Ningbo, China), thus resulting in DNA fragments with an average length of 200 bp.Immunoprecipitation was performed using anti-HA beads from ROCHE.The bound DNA fragments were eluted and purified utilizing a MicroElute Cycle Pure Kit (Omega Bio-tek, Norcross, GA, USA).Finally, potential binding events at the promoter of HOF1 were detected by PCR.

ChEC Assay
To quantify the binding of Mcm1 and Fkh2 to the HOF1 promoter, a chromatin endogenous cleavage (ChEC) coupled with qPCR assay was used [24].Initially, a pFA-HA-Mnase-Ura3 plasmid was synthesized following the sequence derived from pFA-MNase-CaURA3.Mcm1 or Fkh2 were then tagged with an HA-Mnase fusion using the PCR products amplified from pFA-HA-Mnase-Ura3 plasmid.The successful integration of the HA-Mnase fusion constructs was confirmed by PCR and western blot analyses.In the ChEC experiment, overnight cultures were diluted to an initial OD600 of 0.1 in 20 mL liquid YPD medium and cultured at 30 • C until reaching an OD600 of 0.6-0.8.Subsequently, cells were exposed to 0.02% MMS for 90 min, while untreated cells under the same culture conditions served as controls.Harvested cells were washed and treated with 0.1% digitonin, as previously described.Mnase digestion was performed with 5 mM CaCl 2 for 5 min, followed by quenching with 60 µL of 250 mM EGTA solution.Total DNA was extracted using a kit from Yuanye Bio-Technology (Shanghai, China), and DNA fragments ranging from 100 bp to 400 bp were selectively isolated using a Gel DNA Extraction Mini Kit (Vazyme, China).The enriched DNA fragments were then employed for qPCR analysis, with GAPDH primers used as internal controls for quantification.The data presented here are an average derived from a minimum of three independent experiments.

Yeast One-Hybrid Assay
The pGADT7 and pHIS2 plasmids were employed for yeast one-hybrid assay [25].The HOF1 promoter, in different lengths, was amplified by PCR and inserted into the EcoR I site of the pHIS2 plasmid as bait using the Universal One Step Cloning Kit (Yeasen, Shanghai, China).The pGADT7 plasmids containing N-terminus, C-terminus, or full-length Rad53 were obtained from our previous study [26].Yeast Y187 strain was transformed with various combinations of pGADT7 and pHIS2 plasmids using the lithium acetate method and selected on solid SC medium lacking tryptophan and leucine (SC−Trp−Leu).The transformants were picked, resuspended in distilled water, and standardized to a consistent cell density.Subsequently, 3 µL cell suspension was dropped onto SC medium lacking tryptophan, leucine, and histidine (SC−Trp−Leu−His) containing 50 or 75 mM 3-amino-1,2,4-triazole (3-AT).The plates containing cotransformed yeast cells were cultured at 30 • C for 2-3 days.Yeast cells containing pGADT7 and pHIS2-HOF1p-1 plasmids served as negative controls.

Checkpoint Kinases Play Distinct Roles in Response to Genotoxic Stresses
Checkpoint kinases are essential for cells to coordinate the cell cycle progression and gene transcription in response to DNA damage.However, the specific functions of these checkpoint kinases in C. albicans remain unclear.In this study, we generated deletion strains lacking Dun1 or Rad9 and evaluated their importance in DNA damage stress response in comparison to Rad53.MMS is a commonly employed DNA-damaging agent that induces alkylating damage to DNA, thus resulting in single-strand breaks (SSBs) that can progress to double-strand breaks (DSBs) [12].Meanwhile, hydroxyurea (HU) serves as a widely utilized genotoxic agent by inhibiting ribonucleotide reductase (RNR), thus leading to cell cycle arrest in the S-phase through the induction of replication stress [27].Under normal conditions without MMS stress, the deletion of RAD53 caused a significant growth defect, while the deletion of RAD9 or DUN1 allowed for similar growth rates as the wild-type strain on solid YPD media.However, under stress conditions, the deletion of RAD53 led to strong sensitivity to MMS, HU, or UV (Figure 1A).In contrast, the deletion of RAD9 exhibited pronounced sensitivity to MMS-or UV-induced DNA damage stress while displaying only moderate sensitivity to HU-induced DNA replication stress (Figure 1A).In contrast, the deletion of DUN1 showed strong sensitivity to HU but mild sensitivity to MMS or UV (Figure 1A).

Profiling the Rad53-Related Transcriptome in C. albicans
Rad53 has been recognized as a key player in the cellular stress response [28,29].However, in growth conditions devoid of stress, the absence of RAD53 led to a reduced growth rate.A thorough transcriptional analysis was carried out to explore the effects of Rad53 depletion under nutrient-rich conditions, thus revealing notable expression variations; a total of 6104 transcripts were identified, with 2026 showing significant changes based on a log2-fold cut-off of 1.0 (Table S3).Notably, among these differentially expressed genes, 1211 exhibited upregulation, with significant alterations observed in 21 genes displaying log2-fold cut-offs greater than six (Figure S2A and Table S3).Noteworthy among them was Orf19.4653,thus encoding a putative GPI-linked cell wall protein exhibiting the highest upregulation (log2-fold cut-off = 14.91).In addition, genes encoding cell The primary role of checkpoint kinases in response to DNA damage stress is in ensuring the proper arrest of the cell cycle.Previous studies have highlighted the essential functions of Rad53 and Rad9 in arresting the cell cycle under various genotoxic stresses in C. albicans [28].To investigate whether Dun1 plays a similar role in regulating the cell cycle progression, we treated the mutant cells lacking DUN1, RAD9, or RAD53 with MMS and examined nuclear separation as an indicator of expected arrest.Upon exposure to MMS-induced damaged DNA that triggers checkpoint activation, wild-type cells displayed restrained nuclei separation; a total of 69.6% of treated WT cells showed arrested nuclei separation, and only 17.1% escaped this blockage (Figure 1B).In contrast, 40.6% of the RAD53 deletion cells escaped the blockage, and only 41.9% exhibited checkpoint-mediated arrest; likewise, for RAD9-deleted cells, 29.3% escaped the blockage, while 61.5% exhibited checkpoint-mediated arrest, thus demonstrating their consistent roles in cell cycle arrest as previously reported [28].In the case of DUN1-deleted cells, 25.5% of cells bypassed the block, while 59.1% arrested the cell cycle, thus indicating a moderate role of Dun1 in cell cycle regulation.
The checkpoints play a critical role in controlling the filamentous growth induced by genotoxic stresses in C. albicans.The deletion of RAD53 significantly triggered MMS-or HU-induced filamentous growth, while the deletion of RAD9 only hindered filamentous growth in response to MMS, but not HU [28].Since Dun1 is a potential downstream kinase of Rad53, we investigated whether it shares a similar function in regulating genotoxic stress-induced filamentous growth.After a 7 h MMS treatment, both wild-type cells and DUN1-deleted cells exhibited elongation without significant difference (Figure 1C).Following a 6 h HU treatment, wild-type cells showed significant elongation, while DUN1deleted cells also elongated but with impaired bud development; the mutant cells showed much shorter buds compared to the wild-type cells (Figure 1C,D), thus suggesting a role of Dun1 in regulating HU-induced filamentous growth in C. albicans.
In summary, the checkpoint kinase Rad53 plays a central role in response to genotoxic stresses, whereas Dun1 primarily functions during replication stress, and Rad9 mainly responds to DNA damage stress.

Profiling the Rad53-Related Transcriptome in C. albicans
Rad53 has been recognized as a key player in the cellular stress response [28,29].However, in growth conditions devoid of stress, the absence of RAD53 led to a reduced growth rate.A thorough transcriptional analysis was carried out to explore the effects of Rad53 depletion under nutrient-rich conditions, thus revealing notable expression variations; a total of 6104 transcripts were identified, with 2026 showing significant changes based on a log2-fold cut-off of 1.0 (Table S3).Notably, among these differentially expressed genes, 1211 exhibited upregulation, with significant alterations observed in 21 genes displaying log2-fold cut-offs greater than six (Figure S2A and Table S3).Noteworthy among them was Orf19.4653,thus encoding a putative GPI-linked cell wall protein exhibiting the highest upregulation (log2-fold cut-off = 14.91).In addition, genes encoding cell wall proteins such as CDA2, PGA31, and PGA23, along with the chromosome stability-related gene REC8, showed upregulation.Conversely, we observed downregulation in 815 genes, including notable repression in 30 genes displaying log2-fold cut-offs over 10 (Figure S2B and Table S3).For instance, Orf19.446.1, encoding a protein with a NADH-ubiquinone oxidoreductase B18 subunit domain, and IMG2, encoding a mitochondrial ribosomal protein of the large subunit, demonstrated significant downregulation, thus indicating compromised respiration capacity.Notably, the downregulation of Orf19.5042implicated its potential role in rDNA maintenance and mitotic exit, thereby providing insights into how Rad53 influences the cell cycle progression.
To elucidate the comprehensive transcriptome associated with Rad53, we performed KEGG term analysis on genes with altered transcription.Notably, ribosome biogenesis exhibited significant enrichment, thus comprising 30 upregulated genes and five downregulated genes.Additionally, the pathways associated with steroid biosynthesis, aminoacyl-tRNA biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, as well as terpenoid backbone biosynthesis, were enriched.These findings underscore the crucial role of Rad53 in maintaining the normal growth of C. albicans cells.Consistent with its involvement in cell cycle regulation, a set of 30 upregulated genes and 10 downregulated genes were sig-nificantly enriched in the cell cycle process, including CAK1 (a monomeric CDK-activating kinase), and several CDC genes, such as CDC28 and CDC53 (Figure S2C and Table S3).Collectively, our results highlight the extensive transcriptional regulation mediated by Rad53 under nutrient-rich conditions.In the presence of MMS stress, a total of 6103 transcripts were detected, with 2404 transcripts exhibiting significant changes upon RAD53 deletion using a log2-fold cut-off of 1.0 (Figure 2A, Table S4).Among them, 1234 genes were upregulated, while 1170 genes were downregulated.Notable upregulated genes included BUD20, PGA31, and Orf19.4653,which are associated with cell wall synthesis (Table S4).As well, CSA2 encoding an extracellular heme-binding protein was also significantly upregulated.Conversely, NUE1, encoding a mitochondrial protein required for the expression of mitochondrial respiratory chain complex I, and PEX17, encoding a putative peroxin, were among the prominently downregulated genes.Despite Rad53's critical role in DNA damage response, only a limited number of genes, like RTT101 and DAP1, exhibited significant alterations upon its deletion; most differentially expressed genes were associated with antioxidation response, cell wall integrity, or other DNA damage-unrelated pathways.

Pooling RAD53-Dependent DNA Damage-Responsive Genes
The transcripts that differ by deleting RAD53 may encompass genes that did not exhibit significant changes in response to MMS-induced DNA damage.In a previous study, we identified 306 defined (genes with consistent transcription in two independent RNAseq data sets) and 705 putative (the remaining genes from two independent RNA-seq data sets selected by a p value less than 0.05) MMS-responsive genes [12].To identify Rad53dependent DNA damage-responsive genes, we examined the transcription of these MMSresponsive genes in the present study.Most of the defined MMS-responsive gene clusters The transcriptome affected by deleting RAD53 in both normal and MMS stress conditions demonstrated high consistency, with 797 genes being commonly upregulated, 414 genes being specifically induced in nutrient-rich conditions, and 437 genes being specifically induced under MMS stress (Figure S3 and Table S5).Among these commonly upregulated genes, Orf19.4653,Orf19.6487, and LDG3 consistently showed over 10-fold upregulation under both conditions.Similarly, there were 703 commonly suppressed genes; 112 genes were specifically suppressed in rich media, and 467 genes were specifically suppressed under the MMS stress condition (Figure S3 and Table S6).Noteworthy downregulated genes like LEU2, ARC18, and Orf19.6308consistently exhibited an over 10-fold decrease in expression under both conditions.
The enrichment of KEGG terms was compared between normal and MMS stress conditions after deleting RAD53.In the MMS stress condition, several KEGG terms, including ribosome biogenesis, cell cycle, starch, and sucrose terms, were enriched (Figure 2B).Notably, there was high consistency in the top-20 KEGG terms identified under both normal or MMS stress conditions; specifically, cell cycle, DNA replication, ribosome biosynthesis, and other metabolism terms were consistently enriched in these two conditions.This observation suggests a well-defined regulation mediated by Rad53.Collectively, our findings highlight the critical role of Rad53 in governing global gene transcription.
Cell cycle-related genes were enriched in both normal and MMS stress conditions upon the deletion of RAD53, which is known to function in checkpoints for cell cycle arrest following DNA damage.In normal conditions, 37 cell cycle-related genes were enriched, while 55 cell cycle-related genes were enriched in the MMS stress condition (Figure 2C).Moreover, high consistency was observed in the enriched genes across both conditions.Among the 37 genes affected by RAD53 deletion in normal conditions, 33 were also found in the MMS stress group.Only four genes (MEC1, CDC7, Orf19.5692, and CDC20) were specifically affected by Rad53 under nonstress conditions.The top-3 commonly upregulated genes in both conditions were MCD1, GRF10, and MCM3; where MCD1 encodes an Alphakleisin cohesin subunit involved in sister chromatid cohesion in mitosis and meiosis, thus supporting its role in cell cycle arrest.Additionally, CDC28, encoding a cyclin-dependent protein kinase [30,31], was downregulated upon RAD53 deletion.
The genes associated with DNA replication were significantly enriched upon the deletion of RAD53 under both normal and MMS stress conditions (Figure 2D).Moreover, there was a remarkable consistency in the genes affected by Rad53 between these conditions.Particularly, the upregulation of POL1, a putative DNA-directed DNA polymerase alpha, and POL3, the large subunit of DNA polymerase III, indicates their close functional association with Rad53.However, deleting RAD53 resulted in the downregulation of POL30 (PCNA) in both normal and MMS stress conditions (log2-fold change = −0.75,p < 0.05).
Recent studies have implicated ribosomes in cell cycle regulation [32], and our findings reveal that ribosome biogenesis ranks highest in KEGG terms under both conditions (Figure 2E).Specifically, we noted the upregulation of 28 genes, including HBR3, participating proteasomal and 40S ribosomal subunit biogenesis; NOP4, encoding a putative nucleolar protein; and UTP18, encoding a U3 snoRNA-associated protein.Additionally, five genes, such as CKA2, REX2, Orf19.2708,RPP1, and Orf19.68.2, displayed consistent downregulation under normal and MMS stress conditions according to RNA-seq data.
Unlike the top-20 KEGG pathways affected by RAD53 in the absence of stress, DNA damage repair pathways were specifically enriched under MMS stress conditions.In this context, we observed a significant enrichment of several genes involved in homologous recombination (HR, Figure 2F) and nonhomologous end joining (NHEJ, Figure 2F).Notably, RAD52, a well-known HR-related gene, exhibited moderate upregulation, along with increased transcription levels of other HR-related genes like RAD50 and RAD57.However, RDH54 and SEM1 displayed downregulation.Furthermore, classical NHEJ-associated genes, including LIG4, RAD27, RAD50, and MRE11, were enriched [33].

Pooling RAD53-Dependent DNA Damage-Responsive Genes
The transcripts that differ by deleting RAD53 may encompass genes that did not exhibit significant changes in response to MMS-induced DNA damage.In a previous study, we identified 306 defined (genes with consistent transcription in two independent RNA-seq data sets) and 705 putative (the remaining genes from two independent RNA-seq data sets selected by a p value less than 0.05) MMS-responsive genes [12].To identify Rad53dependent DNA damage-responsive genes, we examined the transcription of these MMSresponsive genes in the present study.Most of the defined MMS-responsive gene clusters were not found among the gene list affected by deleting RAD53, thus suggesting their transcription is likely independent of RAD53.However, among these genes, 12 defined and 25 putative upregulated genes, including MRV6 and GST1, exhibited higher transcription levels in the RAD53 deletion strain compared to the level in wild-type strain when treated with MMS (Figure 3A,B), thus indicating a suppressive role for Rad53 on these specific MMS-responsive genes.In addition, under MMS stress, 72 defined and 145 putative upregulated genes displayed reduced transcription levels in the RAD53 deletion strain compared to the wild-type strain (Figure 3A,B).These findings indicate that Rad53 is responsible for, to some extent, the transcription of these 217 upregulated genes induced by MMS.(left column) or by deleting RAD53 upon exposure to MMS (right column) is listed after the gene name.Genes highlighted in red represent the defined MMS-responsive genes, and those genes in black represent the putative MMS-responsive genes.(C) Relative transcription of MMS-responsive genes affected by deleting RAD53 in MMS stress conditions.The transcription of indicated genes in wild-type strain under MMS stress was compared to the level in wild-type strain without MMS treatment, and the transcription of indicated genes in the RAD53 deletion strain was compared to the level in wild-type strain with or without MMS treatment.(D) Relative transcription levels of Rad53regulated genes affected by deleting RAD9 or DUN1 in the MMS stress condition.The transcriptional levels of indicated genes in the RAD9 or DUN1 deletion strains were compared to those in wild-type strain under MMS treatment.The qRT-PCR assays for each strain were repeated at least 3 times.The difference between each group was compared using paired t test with GraphPad Prism 8.0.1 software.* represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001.NS represents no significant difference.
In the gene cluster suppressed by MMS, 50 defined and 134 putative MMS-responsive genes exhibited higher transcription levels in the RAD53 deletion strain compared to the wild type under MMS treatment (Figure 3A,B), thus indicating a repressive role of Rad53 on these MMS-responsive genes.Moreover, among the downregulated genes, three defined and 36 putative ones showed even lower transcription levels in the RAD53 deletion under the MMS stress condition compared to the wild-type strain (Figure 3A,B).Collectively, these findings suggest that Rad53 may influence a total of 137 defined and 340 putative MMS-responsive genes in response to DNA damage induced by MMS.
We further validated the regulatory role of Rad53 in controlling these potential targets under MMS stress.In the wild-type strain, MRV6 was upregulated as expected after MMS treatment; however, this upregulation was amplified upon RAD53 deletion (Figure 3C).In addition, IQG1 and CSE4 were downregulated in response to MMS; nevertheless, the deletion of RAD53 increased their transcriptional level (Figure 3C).Furthermore, HMX1 was downregulated upon exposure to MMS stress, but its transcription was exacerbated by the deletion of RAD53.Under normal conditions, RAD53 deletion consistently influenced the transcription of MRV6, CSE4, and HMX1.In general, our findings suggest that Rad53 plays a regulatory role on potential targets in response to MMS-induced DNA damage stress.
In light of the functional correlation between Rad53 and Rad9 or Dun1, we explored the transcription of potential targets of Rad53 by deleting RAD9 or DUN1.The deletion of DUN1, but not RAD9, led to decreased transcription levels of MRV6, IQG1, and CSE4 under MMS-induced stress conditions (Figure 3D).In contrast, both RAD9 and DUN1 deletions caused decreased transcription levels of HMX1 upon MMS treatment (Figure 3D).Taken together, these findings highlight the diverse regulatory roles played by Rad53, Rad9, and Dun1 on downstream genes.

Transcription of HOF1 Depends on Checkpoint Kinases Rad9 and Rad53
Within the potential targets of Rad53 (Figure 3B), we observed the downregulation of HOF1, a previously reported checkpoint-related gene, following MMS treatment; however, its expression increased post RAD53 deletion.This discovery is consistent with our previous result demonstrating a decline in the protein level of Hof1 with MMS treatment, which then returned to normal after RAD53 removal [14].To validate this observation, qRT-PCRs were conducted.In the wild-type strain, the transcriptional suppression of HOF1 was evident under MMS stress, as its expression declined to approximately half compared to basal levels.Conversely, in the RAD53 deletion strain, HOF1 transcription remained unchanged, thus resembling wild-type levels without MMS stress (Figure 4A) and indicating that the Rad53-mediated repression of HOF1 originates from transcriptional mechanisms.mild sensitivity to the low concentration of MMS; however, its double mutant with HOF1 demonstrated pronounced hypersensitivity towards MMS (Figure 4D), thus suggesting an independent role for HOF1 in DNA damage response that is unrelated to Dun1.Taken together, our findings indicate that both the transcriptional regulation and functional involvement of HOF1 in MMS-induced DNA damage response rely on checkpoint kinases Rad53 and Rad9, but probably not on Dun1.Rad53 acts as a critical effector in the signaling pathways of checkpoints, thus relying on the participation of additional kinases like adaptor Rad9 and potential downstream kinase Dun1 [29].To determine the direct association of HOF1 regulation with Rad53 or its broader connection to the checkpoint pathway, we examined the transcriptional levels of HOF1 in the RAD9 or DUN1 mutants.Consistent with findings from RAD53 mutant analysis, the transcriptional level of HOF1 in the RAD9 mutant resembled that observed in the wild-type strain; however, under the MMS stress condition, instead of decreasing, HOF1 transcription increased beyond wild-type levels (Figure 4A).Conversely, in the DUN1 mutant strain, HOF1 transcription exhibited a pattern similar to that seen in the wild-type strain; following exposure to MMS, a decrease in HOF1 transcription was observed (Figure 4A).In general, our results indicate that the proper regulation on HOF1 transcription upon MMS-induced DNA damage relies on checkpoint kinases Rad53 and Rad9 rather than Dun1.
Given that the deletion of DUN1 only resulted in moderate sensitivity to MMS in C. albicans, it may elucidate the Dun1-independent regulation on HOF1 under the MMS stress condition.In contrast, the deletion of DUN1 conferred strong sensitivity to HU in C. albicans, as evidenced by our experimental findings.Hence, we investigated whether Dun1 governs the transcription of HOF1 under the HU stress condition.Consistent with the observations during MMS stress, HU treatment significantly suppressed the transcription of HOF1 to 52% of the level in the wild-type strain; likewise, in the DUN1 deletion strain, HU exposure led to a significant decrease in HOF1 transcription compared to the stress-free state in wild-type or DUN1-deficient cells (Figure 4B).Consequently, our results indicate that the transcriptional regulation of HOF1 is independent of Dun1 in C. albicans.
In S. cerevisiae, checkpoint kinases govern global transcriptional shifts, with HOF1 identified as a target downstream of Dun1, in conjunction with the Ndd1/Fkh2/Mcm1 transcription factor complex [10].Since the transcription of HOF1 was not Dun1-related in C. albicans, we examined the transcription pattern of the orthologs for other targets of Dun1 in S. cerevisiae.ADE4 is downregulated upon MMS treatment in S. cerevisiae, and its downregulation can be blocked by deleting DUN1 [10].However, in C. albicans, the deletion of DUN1 did not induce notable alterations in the ADE4 transcription under both treated and untreated conditions with MMS (Figure S4).Furthermore, RNR3 shows increased transcription following MMS treatment in S. cerevisiae, and its upregulation can be repressed by deleting DUN1 [10].In contrast to this finding, we observed a slight decrease in RNR3 transcription in C. albicans post MMS treatment, with this reduction prevented by DUN1 deletion (Figure S4).In general, our results indicate nonconserved regulation between C. albicans and S. cerevisiae for both HOF1 and other cell cycle-related targets of Dun1.
Dun1 is recognized as a downstream kinase of Rad53 [9].To investigate whether elevating DUN1 levels can restore the compromised downregulation of HOF1 in the absence of RAD53, we introduced an overexpression cassette for DUN1 into the RAD53 deletion strain.Interestingly, despite DUN1 overexpression in the wild-type strain, HOF1 transcription decreased following MMS treatment (Figure 4C).Furthermore, when the RAD53 deletion strain harbored the DUN1 overexpression cassette, the HOF1 transcription remained unaffected by MMS treatment (Figure 4C).Thus, our findings indicate that overexpressing DUN1 cannot compensate for the Rad53-dependent decrease in HOF1 transcription.
Previously, we investigated the genetic interaction between RAD53 and HOF1, and we observed that RAD53 is epistatic to HOF1 in response to MMS-induced DNA damage.To further elucidate the relationship between Hof1 and checkpoint kinases, we constructed the double deletion strains of HOF1 with either RAD9 or DUN1 and assessed their sensitivity to MMS.Spot assays revealed that single-gene deletions of RAD9 or HOF1 both showed similar MMS sensitivity levels, whereas the double deletion strain of RAD9 with HOF1 exhibited comparable MMS sensitivity to their single mutants, with no significant increase in susceptibility to MMS (Figure 4D).In contrast, the DUN1 mutant showed mild sensitivity to the low concentration of MMS; however, its double mutant with HOF1 demonstrated pronounced hypersensitivity towards MMS (Figure 4D), thus suggesting an independent role for HOF1 in DNA damage response that is unrelated to Dun1.Taken together, our findings indicate that both the transcriptional regulation and functional involvement of HOF1 in MMS-induced DNA damage response rely on checkpoint kinases Rad53 and Rad9, but probably not on Dun1.

Mcm1 and Fkh2 Target the Promoter of HOF1
Given that transcription factors Fkh2 and Mcm1 are essential for the transcription of HOF1, we conducted a ChIP analysis to explore their binding to the HOF1 promoter under normal conditions.A band representing the promoter region (−532 bp to −261 bp, Furthermore, we examined the impact of FKH2 and MCM1 overexpression on HOF1 transcription levels to further understand their regulatory roles.Notably, the ADH1 promoter significantly enhanced the transcription of FKH2 (Figure 5A, right panel); nevertheless, overexpressing FKH2 did not elevate HOF1 transcription but instead caused a slight decrease.In contrast, employing the MET3 promoter increased the MCM1 transcript levels by a nearly 3-fold amount compared to the wild-type strain; moreover, elevated MCM1 further augmented the transcription of HOF1 (Figure 5B, right panel).These findings suggest that Mcm1 may serve as a crucial regulator governing the transcription of HOF1.

Mcm1 and Fkh2 Target the Promoter of HOF1
Given that transcription factors Fkh2 and Mcm1 are essential for the transcription of HOF1, we conducted a ChIP analysis to explore their binding to the HOF1 promoter under normal conditions.A band representing the promoter region (−532 bp to −261 bp, including a potential binding motif of ScMcm1) of HOF1 was observed from the Mcm1-HA bound samples (Figure 6A), yet MMS treatment notably reduced this binding.Likewise, Fkh2 exhibited binding to the HOF1 promoter under normal conditions, but it showed reduced binding upon MMS treatment (Figure 6B). on the HOF1 promotor using a ChEC-qPCR assay in RAD53 deletion cells (Figure 6D).The activation of Mcm1-Mnase in the RAD53 deletion cells by Ca 2+ for 5 min or 20 min did not lead to noticeable changes in the enrichment of the HOF1 promoter under normal or MMS stress conditions.Therefore, RAD53 deletion hinders the liberation of Mcm1 from the HOF1 promoter.To quantify the altered binding of Mcm1 and Fkh2 to the promoter of HOF1 post MMS treatment, we employed a ChEC assay coupled with qPCR.Generally, the fusion proteins Mcm1-Mnase or Fkh2-Mnase triggered specific cleavage at their respective DNA surroundings, thus resulting in the liberation of enriched DNA fragments (Figure S5) and demonstrating their binding capability to the HOF1 promoter.The enrichment of the HOF1 promoter with Mcm1 decreased to 43% compared to the untreated group following MMS treatment (Figure 6C), as indicated by our findings.Similarly, a notable reduction in the enrichment of the HOF1 promoter with Fkh2 was observed under MMS treatment conditions (Figure 6D).Hence, both Mcm1 and Fkh2 exhibit dynamic binding patterns towards the HOF1 promoter depending on the cellular status.
The deletion of RAD53 in C. albicans has been shown to impede the downregulation of HOF1 expression during MMS-induced stress [14].Considering that Mcm1 plays a critical role in regulating the transcription of HOF1, we investigated whether this process involves altering the dynamic binding between the Mcm1-Mnase fusion and its target site on the HOF1 promotor using a ChEC-qPCR assay in RAD53 deletion cells (Figure 6D).The activation of Mcm1-Mnase in the RAD53 deletion cells by Ca 2+ for 5 min or 20 min did not lead to noticeable changes in the enrichment of the HOF1 promoter under normal or MMS stress conditions.Therefore, RAD53 deletion hinders the liberation of Mcm1 from the HOF1 promoter.

Rad53 Targets the Promoter of HOF1
A recent study has revealed that checkpoint kinase Rad53 interacts with the promoters of approximately 20% of genes and coordinates genome-wide replication and transcription under replication stress in S. cerevisiae [34].Moreover, studies on localization have shown that the majority of GFP-tagged Rad53 proteins in S. cerevisiae are localized within the nucleus [35].Considering that Rad53 functions as a suppressor of HOF1 transcription without being a transcription factor itself, we employed a yeast one-hybrid system to explore the potential interaction between Rad53 and the promoter region of HOF1.A series of pGADT7 plasmids containing either the FHA1 domain, the kinase catalytic domain (KD) (Rad53-N), or the FHA2 domain (Rad53-C) from our previous study were used as the prey [26].Additionally, distinct segments of the HOF1 promoter were integrated into the pHIS2 plasmid using it as the bait (Figure 7A).Our findings revealed no substantial interaction between the complete Rad53 protein and the HOF1 promoter (Figure 7B).The FHA1/KD domains of Rad53 exhibited no discernible interaction with various regions of the HOF1 promoter, except for a weak interaction detected with the complete sequence of the HOF1 promoter (Figure 7B).However, a distinct interaction was detected between the FHA2 domain and the complete HOF1 promoter but not its truncated versions, thus indicating a possible binding motif within the DNA segment ranging from −991 bp to −710 bp.To further validate the positive interaction between the FHA2 domain of Rad53 and the HOF1 promoter, we integrated the DNA fragment encompassing the −991 bp to −693 bp region of the HOF1 promoter into the pHIS2 plasmid and assessed its interaction with the FHA2 domain of Rad53; our results validated a robust interaction between the upstream region of HOF1 promoter and the FHA2 domain of Rad53 (Figure 7C).These findings align well with previous observations showing increased signal intensity upstream rather than downstream from the transcription start site in the HOF1 promoter of S. cerevisiae when probed by the Rad53 protein [34].Therefore, our yeast one-hybrid assays provide evidence for a direct association between the checkpoint kinase Rad53 and the HOF1 promoter.
To further investigate the interaction between the Rad53 and the HOF1 promoter in C. albicans, a ChIP assay was conducted.Under normal conditions, a faint band representing HOF1 was detected in the Rad53-HA-coated sample; upon treatment with MMS, a prominent signal of the promoter of HOF1 was observed (Figure 7D).However, the Rad53-Mnase fusion exhibited no apparent DNA digestion activity, and the ChEC assay was not performed for Rad53.But collectively, these findings suggest that Rad53 directly contributes to the modulation of HOF1 transcription in C. albicans, thus involving dynamic enrichment at its promoter region.The log phase wild-type cells carrying the Rad53-HA fusion, with or without 0.02% MMS treatment, were fixed with 1% formaldehyde.A wild-type strain without an HA tag was used as a control.Immunoprecipitated pellets were used as templates for PCR with the primer pairs HOF1-pro-F/R.The band intensity was quantified using ImageJ software.Under no stress conditions, the ratio of the band in the ChIP group to the input group was normalized as 1.The result was averaged from two independent experiments.

Discussion
In this study, we investigated the transcriptional response related to the cell cycle checkpoint kinases in C. albicans cells lacking Rad53 and observed a significant alteration in the gene transcription upon the deletion of RAD53, both in nutrient-rich and MMS stress conditions.In addition, our findings demonstrated that Rad53 and Rad9, rather than Dun1, play a role in restricting the MMS-mediated repression of HOF1 in C. albicans.Moreover, we uncovered that Rad53 is crucial for regulating the dynamic binding of Mcm1 to the promoter region of HOF1 and can directly bind to its promoter as well.
The DNA damage response is pivotal for cells to maintain genome integrity and fidelity during growth and development.Checkpoints serve as a critical hub for halting the cell cycle and repairing DNA damage, thus safeguarding daughter cells from inheriting such damage.In this study, we investigated the essentiality of checkpoint kinases Rad53, Rad9, and Dun1 in C. albicans under various genotoxic stress conditions.Our findings revealed diverse levels of essentiality among these checkpoint kinases, with Rad53 being identified as the most critical and Rad9 and Dun1 showing partial essentiality in addressing DNA damage or replication stress.The deletion of RAD53 caused dramatic sensitivity to various genotoxic agents; Rad9 played a critical role in responding to DNA damage caused by MMS and UV radiation, while its significance was limited in replication stress induced by HU; Dun1, a potential downstream kinase of Rad53, appeared dispensable for The log phase wild-type cells carrying the Rad53-HA fusion, with or without 0.02% MMS treatment, were fixed with 1% formaldehyde.A wild-type strain without an HA tag was used as a control.Immunoprecipitated pellets were used as templates for PCR with the primer pairs HOF1-pro-F/R.The band intensity was quantified using ImageJ software.Under no stress conditions, the ratio of the band in the ChIP group to the input group was normalized as 1.The result was averaged from two independent experiments.

Discussion
In this study, we investigated the transcriptional response related to the cell cycle checkpoint kinases in C. albicans cells lacking Rad53 and observed a significant alteration in the gene transcription upon the deletion of RAD53, both in nutrient-rich and MMS stress conditions.In addition, our findings demonstrated that Rad53 and Rad9, rather than Dun1, play a role in restricting the MMS-mediated repression of HOF1 in C. albicans.Moreover, we uncovered that Rad53 is crucial for regulating the dynamic binding of Mcm1 to the promoter region of HOF1 and can directly bind to its promoter as well.
The DNA damage response is pivotal for cells to maintain genome integrity and fidelity during growth and development.Checkpoints serve as a critical hub for halting the cell cycle and repairing DNA damage, thus safeguarding daughter cells from inheriting such damage.In this study, we investigated the essentiality of checkpoint kinases Rad53, Rad9, and Dun1 in C. albicans under various genotoxic stress conditions.Our findings revealed diverse levels of essentiality among these checkpoint kinases, with Rad53 being identified as the most critical and Rad9 and Dun1 showing partial essentiality in addressing DNA damage or replication stress.The deletion of RAD53 caused dramatic sensitivity to various genotoxic agents; Rad9 played a critical role in responding to DNA damage caused by MMS and UV radiation, while its significance was limited in replication stress induced by HU; Dun1, a potential downstream kinase of Rad53, appeared dispensable for MMS-or UVinduced DNA damage stress but played an indispensable role in HU-induced replication stress.Interestingly, our results indicate that the significance of these checkpoint kinases varies across different organisms, thus encompassing C. albicans and other eukaryotes.Although RAD53 is essential in S. cerevisiae, it is nonessential in either C. albicans or C. glabrata [36,37].Particularly, Rad53 exhibits a noncanonical role in DNA damage response within C. glabrata, where it remains unphosphorylated upon MMS treatment [37].Moreover, the loss of DUN1 leads to pronounced sensitivity to both MMS and HU stresses in S. cerevisiae [38]; however, its role seems less significant in responding to DNA damage but more evident in dealing with replication stress in C. albicans.Thus, the orthologs of checkpoint kinases in different organisms may have nonconserved roles in the DNA damage response.
Checkpoint kinases are activated to arrest the cell cycle progression and regulate transcriptional changes to facilitate DNA damage repair.Our research uncovered that the deletion of RAD53 influenced the transcription of genes related to DNA replication and the cell cycle.MCD1 encodes an Alpha-Kleisin cohesin complex subunit for sister chromatid cohesion in S. cerevisiae [39].Additionally, Smc4 is a subunit of the nuclear condensin complex responsible for chromatin binding, chromosome condensation, and meiotic chromosome separation [40].The increased transcription of cell cycle genes may indicate disrupted cell cycle control due to checkpoint malfunction, thus resulting in the sustained expression of these genes.Consistently, most identified DNA replication genes showed increased transcription upon RAD53 deletion, thus indicating unrestricted DNA replication under MMS stress without checkpoint control.Furthermore, we conducted a comparison between our RNA-seq data and the documented cell cycle-modulated genes through GSEA analysis.The altered genes resulting from RAD53 deletion demonstrated significant similarity to cell cycle-related genes influenced by Swi4/Swi6 and Ndt50, thus validating its involvement in cell cycle regulation (Table S7).
Interestingly, RNA-seq data revealed enrichment in pathways unrelated to DNA damage repair upon RAD53 deletion, particularly those associated with ribosome biogenesis.The KEGG term for ribosome biogenesis was enriched by deleing RAD53 under both richnutrient and MMS stress conditions.Ribosome biogenesis has been linked to disease and DNA repair processes [32].Previous studies have shown that nonfunctional rRNA decay in yeast requires the involvement of DNA repair factors Rtt101 and Mms1, thus suggesting a cellular response to genotoxic stress affecting both rRNA and DNA integrity [41,42].Ribosome biogenesis is also related to cell size; Dot6 acts as an activator in mediating the transcription of ribosome biogenesis genes, and the deletion of DOT6 results in decreased cell size.Here, the deletion of RAD53 resulted in disrupted cell cycle and caused a bulged cell form, thus indicating a potential link between checkpoint kinases and cell size through regulators like Dot6.Furthermore, deleting RAD53 resulted in slow growth under both nutrient-rich conditions and MMS stress conditions, with several amino acid biosynthesis genes being downregulated consistently.These observations suggest that checkpoint kinases may downregulate biosynthesis gene expression as part of their role in slowing down cell growth.In summary, our results highlight the significant regulatory roles played by checkpoint kinases in coordinating gene transcription under different stress conditions.
Rad53 plays multiple roles in regulating the transcription of various DNA damageresponsive genes in C. albicans.Our previous research revealed transcriptional alterations in response to MMS, thus showing that Rad53 positively regulates the transcription of potential target genes like RAD7 and HTA2 [12].However, the deletion of RAD53 affected only a limited number of MMS-responsive genes, thus leaving a significant portion unaffected by Rad53.This discrepancy suggests the involvement of alternative mechanisms independent of Rad53 control.Notably, among the Rad53-related genes, several MMS-induced genes like MRV6 were further upregulated by losing RAD53; similarly, MMS-repressed genes, including IQG1 and CSE4, exhibited increased transcription by losing RAD53.The upregulation of these MMS-responsive genes indicates that Rad53 exerts a negative influence on their expression, thus affecting DNA damage response either directly or indirectly.Strikingly, selected MMS-responsive genes displayed significant differences in mutants lacking specific checkpoints; particularly noteworthy was the downregulation observed for MRV6, CSE4, and IQG1 upon the deletion of DUN1 compared to their upregulation upon RAD53 deletion-thus providing further evidence of the distinct roles for Rad53 and Dun1 in the DNA damage response.
The transcriptional regulation of HOF1 in C. albicans appears to differ from that in S. cerevisiae in terms of both the involved transcription factors and the underlying mechanism.In S. cerevisiae, the Fkh2/Mcm1/Ndd1 complex serves as a transcription factor complex for HOF1, with Mcm1 binding to its promoter throughout the cell cycle, Fkh2 acting as an inhibitor, and Ndd1 functioning as an activator [10].However, it is possible that Ndd1 may have been lost during evolution in C.   Overall, our study presents a comprehensive overview of checkpoint-dependent transcriptional regulation during the MMS-induced DNA damage response in C. albicans, thus shedding light on species-specific control over cell cycle-specific genes such as HOF1.Furthermore, our study demonstrates how the checkpoint kinase Rad53 can specifically interact with the HOF1 promoter, thereby potentially influencing gene transcription.These findings offer valuable insights into the checkpoint-dependent transcriptional regulation, morphogenesis, and virulence mechanisms employed by C. albicans.

Supplementary Materials:
The following supporting information can be downloaded at https:// www.mdpi.com/article/10.3390/jof10060387/s1, Figure S1.The diagram of constructing the mutant strains for RAD9, DUN1, FKH2, and MCM1.S1.Strains used in this study.Table S2.Primers used in this study.Table S3.RNA-seq data of the RAD53 deletion strain under normal condition.Table S4.RNA-seq data of the RAD53 deletion strain under MMS stress condition.Table S5.Common upregulated genes by deleting RAD53 under normal and MMS stress conditions.Table S6.Common downregulated genes by deleting RAD53 under normal and MMS stress conditions.Table S7.GSEA analysis of genes affected by Rad53.

Figure 1 .
Figure 1.Functional characterization of checkpoint kinases responding to genotoxic stresses in C. albicans.(A) Phenotypic assay of the RAD53 deletion, the RAD9 deletion, and the DUN1 deletion strains under genotoxic stresses.Two independent mutants for each strain were used for phenotypic assays, thus showing consistent results.(B) Nuclei separation of the wild type (SN148), the RAD53 deletion, the RAD9 deletion, and the DUN1 deletion strains.The log phase cells were treated with 0.02% MMS for 120 min and then stained with DAPI.Cells with buds containing different types of nuclei were divided into three groups as indicated.The result was averaged from two independent experiments.(C) Filamentous growth of the DUN1 strain induced by genotoxic stress.The wildtype and the DUN1 deletion cells were treated with 0.02% MMS or 40 mM HU for the indicated time.The cell morphology was checked and imaged (400×).(D) The long bud of the DUN1 deletion cells induced by 40 mM HU for 6 h was measured using Image J software 1.42.Over 30 cells were checked for each strain.The difference was compared using a paired t test with GraphPad Prism 8.0.1 software.** represents p < 0.01.

Figure 1 .
Figure 1.Functional characterization of checkpoint kinases responding to genotoxic stresses in C. albicans.(A) Phenotypic assay of the RAD53 deletion, the RAD9 deletion, and the DUN1 deletion strains under genotoxic stresses.Two independent mutants for each strain were used for phenotypic assays, thus showing consistent results.(B) Nuclei separation of the wild type (SN148), the RAD53 deletion, the RAD9 deletion, and the DUN1 deletion strains.The log phase cells were treated with 0.02% MMS for 120 min and then stained with DAPI.Cells with buds containing different types of nuclei were divided into three groups as indicated.The result was averaged from two independent experiments.(C) Filamentous growth of the DUN1 strain induced by genotoxic stress.The wild-type and the DUN1 deletion cells were treated with 0.02% MMS or 40 mM HU for the indicated time.The cell morphology was checked and imaged (400×).(D) The long bud of the DUN1 deletion cells induced by 40 mM HU for 6 h was measured using Image J software 1.42.Over 30 cells were checked for each strain.The difference was compared using a paired t test with GraphPad Prism 8.0.1 software.** represents p < 0.01.

3. 3 .
Profiling the Rad53-Related Transcriptome under DNA Damage Stress in C. albicans

Figure 2 .
Figure 2. Overview of RAD53-related transcriptome under MMS stress in C. albicans.(A) Volcano plot showing the global transcriptional changes affected by Rad53 under the stress of MMS.(B) Top-20 KEGG terms of differed genes in response to MMS by deleting RAD53.Cell cycle-involved genes (C), DNA replication-involved genes (D), and ribosome biogenesis-involved genes (E) affected by Rad53 in C. albicans.The fold change for each gene under the normal condition (first column) or the MMS stress condition (second column) is shown after the gene name, with blanks indicating no significant change based on transcriptome data.Genes highlighted in red indicate consistent changes between normal and MMS stress conditions.(F) DNA damage repair genes were affected by deleting RAD53 under MMS stress conditions.The fold change for each gene is shown after the gene name.

Figure 2 .
Figure 2. Overview of RAD53-related transcriptome under MMS stress in C. albicans.(A) Volcano plot showing the global transcriptional changes affected by Rad53 under the stress of MMS.(B) Top-20 KEGG terms of differed genes in response to MMS by deleting RAD53.Cell cycle-involved genes (C), DNA replication-involved genes (D), and ribosome biogenesis-involved genes (E) affected by Rad53 in C. albicans.The fold change for each gene under the normal condition (first column) or the MMS stress condition (second column) is shown after the gene name, with blanks indicating no significant change based on transcriptome data.Genes highlighted in red indicate consistent changes between normal and MMS stress conditions.(F) DNA damage repair genes were affected by deleting RAD53 under MMS stress conditions.The fold change for each gene is shown after the gene name.

Figure 3 .Figure 3 .
Figure 3. Uncovering RAD53-dependent DNA damage responsive genes in C. albicans.(A) Overview of MMS-responsive genes affected by deleting RAD53.The number without brackets represents the defined MMS-responsive genes, while the number with brackets represents the putative MMS-responsive genes.(B) List of MMS-induced (left panel) or -repressed genes (right panel) affected by Rad53 in C. albicans.The fold change for each gene affected by MMS stress in wild-type strain (left column) or by deleting RAD53 upon exposure to MMS (right column) is listed after the gene name.Genes highlighted in red represent the defined MMS-responsive genes, and those genes in black represent the putative MMS-responsive genes.(C) Relative transcription of MMS-responsive genes affected by deleting RAD53 in MMS stress conditions.The transcription of indicated genes in wild-type strain under MMS stress was compared to the level in wild-type strain without MMS treatment, and the transcription of indicated genes in the RAD53 deletion strain was compared to the level in wild-type strain with or without MMS treatment.(D) Relative transcription levels of Rad53-regulated genes affected by deleting RAD9 or DUN1 in the MMS stress condition.The tran-Figure 3. Uncovering RAD53-dependent DNA damage responsive genes in C. albicans.(A) Overview of MMS-responsive genes affected by deleting RAD53.The number without brackets represents the defined MMS-responsive genes, while the number with brackets represents the putative MMSresponsive genes.(B) List of MMS-induced (left panel) or -repressed genes (right panel) affected by Rad53 in C. albicans.The fold change for each gene affected by MMS stress in wild-type strain

Figure 4 .
Figure 4. Checkpoint kinases Rad53 and Rad9 regulate the transcription of HOF1 in C. albicans.(A) The transcription of HOF1 after deleting RAD9, RAD53, and DUN1 was checked by qRT-PCR.The wild-type strain (SN148), the RAD9 deletion strain, the RAD53 deletion strain, and the DUN1 deletion strain were treated with 0.015% MMS for 90 min before being harvested for RNA extraction.The transcription of HOF1 in each strain was compared to the level in wild type with no MMS stress.(B) The transcription of HOF1 after deleting DUN1 was checked by qRT-PCR.The wild-type strain (SN148) and the DUN1 deletion strain were treated with 40 mM HU for 90 min before being harvested for RNA extraction.The transcription of HOF1 in each strain was compared to the level in wild type without MMS stress.(C) The transcription of HOF1 after overexpressing DUN1.The wildtype strain or the RAD53 deletion strain with or without the DUN1 overexpression cassette was treated with MMS, as mentioned in panel A. The qRT-PCR assay for each strain was repeated at least 3 times.The transcription of HOF1 in each group was compared to the level in wild type without MMS stress.The difference between each group was compared using paired t test with GraphPad Prism 8.0.1 software.* represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.NS represents no significant difference.(D) Phenotypic assay of the HOF1 RAD9 double deletion and the HOF1 DUN1 double deletion strains to MMS stress.

Figure 4 .
Figure 4. Checkpoint kinases Rad53 and Rad9 regulate the transcription of HOF1 in C. albicans.(A) The transcription of HOF1 after deleting RAD9, RAD53, and DUN1 was checked by qRT-PCR.The wild-type strain (SN148), the RAD9 deletion strain, the RAD53 deletion strain, and the DUN1 deletion strain were treated with 0.015% MMS for 90 min before being harvested for RNA extraction.The transcription of HOF1 in each strain was compared to the level in wild type with no MMS stress.(B) The transcription of HOF1 after deleting DUN1 was checked by qRT-PCR.The wild-type strain (SN148) and the DUN1 deletion strain were treated with 40 mM HU for 90 min before being harvested for RNA extraction.The transcription of HOF1 in each strain was compared to the level in wild type without MMS stress.(C) The transcription of HOF1 after overexpressing DUN1.The wild-type strain or the RAD53 deletion strain with or without the DUN1 overexpression cassette was treated with MMS, as mentioned in panel A. The qRT-PCR assay for each strain was repeated at least 3 times.The transcription of HOF1 in each group was compared to the level in wild type without MMS stress.The difference between each group was compared using paired t test with GraphPad Prism 8.0.1 software.* represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.NS represents no significant difference.(D) Phenotypic assay of the HOF1 RAD9 double deletion and the HOF1 DUN1 double deletion strains to MMS stress.

3. 6 .
Transcription Factors Mcm1 and Fkh2 Regulate the Transcription of HOF1 In S. cerevisiae, the regulation of HOF1 involves an Ndd1/Fkh2/Mcm1 transcription factor complex [10].However, C. albicans lacks an ortholog of Ndd1, with only Fkh2 and Mcm1 having been identified.To investigate the function of Fkh2 and Mcm1 in regulating HOF1 in C. albicans, we deleted FKH2 and also suppressed MCM1 to assess the transcription of HOF1, respectively.The loss of Fkh2 notably reduced HOF1 transcription levels to around 30% compared to wild-type strains (Figure 5A, left panel).Likewise, the suppression of MCM1 led to a significant decrease in HOF1 transcription (Figure 5B, left panel).Therefore, both Fkh2 and Mcm1 are essential for the proper transcriptional regulation of HOF1.moter significantly enhanced the transcription of FKH2 (Figure5A, right panel); nevertheless, overexpressing FKH2 did not elevate HOF1 transcription but instead caused a slight decrease.In contrast, employing the MET3 promoter increased the MCM1 transcript levels by a nearly 3-fold amount compared to the wild-type strain; moreover, elevated MCM1 further augmented the transcription of HOF1 (Figure5B, right panel).These findings suggest that Mcm1 may serve as a crucial regulator governing the transcription of HOF1.

Figure 5 .
Figure 5. Transcription of HOF1 affected by Mcm1 and Fkh2.(A) The transcription of HOF1 was affected by deleting (left panel) or overexpressing FKH2 (right panel).The log phase cells of indicated strains were used for qRT-PCR assays.(B) The transcription of HOF1 was affected by repressing (left panel) or overexpressing MCM1 (right panel).The promoter of MCM1 was replaced by a MET3 promoter in SN148 background.The overnight culture of the indicated strains was inoculated into SC media plus Met/Cys (5 mM for each) or SC-Met/Cys for 4 h before being harvested for RNA extraction.The qRT-PCR assay for each strain was repeated at least 3 times.The transcription of indicated genes was compared to the level in wild type using a paired t test with GraphPad Prism 8.0.1 software.* represents p < 0.05, and ** represents p < 0.01.

Figure 5 .
Figure 5. Transcription of HOF1 affected by Mcm1 and Fkh2.(A) The transcription of HOF1 was affected by deleting (left panel) or overexpressing FKH2 (right panel).The log phase cells of indicated strains were used for qRT-PCR assays.(B) The transcription of HOF1 was affected by repressing (left panel) or overexpressing MCM1 (right panel).The promoter of MCM1 was replaced by a MET3 promoter in SN148 background.The overnight culture of the indicated strains was inoculated into SC media plus Met/Cys (5 mM for each) or SC-Met/Cys for 4 h before being harvested for RNA extraction.The qRT-PCR assay for each strain was repeated at least 3 times.The transcription of indicated genes was compared to the level in wild type using a paired t test with GraphPad Prism 8.0.1 software.* represents p < 0.05, and ** represents p < 0.01.

Figure 6 .
Figure 6.Transcription factors Mcm1 and Fkh2 bind to the promoter of HOF1.(A,B) Detection of the binding of Mcm1 or Fkh2 to the promoter of HOF1 by ChIP analysis.The log phase wildtype cells (SN148) carrying Mcm1-HA or Fkh2-HA fusion, with or without 0.02% MMS treatment, were fixed with 1% formaldehyde.A wild-type strain without an HA tag was used as a control.Immunoprecipitated pellets were used as templates for PCR with the primer pairs HOF1-Chip-F and R. The intensity of the band was quantified using ImageJ software.Under no stress conditions, the ratio of the band in the ChIP group to the input group was normalized as 1.The result was averaged from two independent experiments.(C,D) The enrichment of HOF1 to Mcm1 and Fkh2 was checked by ChEC assay coupled with qPCR.Wild-type cells carrying Mcm1-Mnase or Fkh2-Mnase fusions were used.The extracted DNA fragments around 100 bp to 400 bp from cells with or without MMS treatment were applied for qPCR assays, and the GAPDH level was used as a control.The difference was compared using a paired t test with GraphPad Prism 8.0.1 software.** represents p < 0.01.(E) Rad53 regulates the dynamic enrichment of the HOF1 promoter to Mcm1.The RAD53 deletion cells carrying Mcm1-Mnase fusions were used.The extracted DNA fragments around 100 bp to 400 bp from cells with or without MMS treatment were applied to check the signal of the HOF1 promoter with primers HOF1-pro-F/R, and the GAPDH level was used as a control.The difference was compared using a paired t test with GraphPad Prism 8.0.1 software.NS represents no significant difference.

Figure 7 .
Figure 7. Rad53 is involved in the direct regulation of HOF1.(A) Diagram of yeast one-hybrid assay.(B) The FHA2 region of Rad53 binds to the promoter of HOF1.The different domains of Rad53 were cloned into the pGADT7 plasmid, and various regions of the HOF1 promoter were cloned into the pHIS2 plasmid before being transformed into the yeast Y187 strain.The transformants were dissolved in distilled water and dropped onto SC-Trp-Leu-His plates containing different concentrations of 3-AT.The plates were kept at 30 °C for 2-3 days.(C) Checkpoint kinase Rad53 binds to the promoter of HOF1.The log phase wild-type cells carrying the Rad53-HA fusion, with or without 0.02% MMS treatment, were fixed with 1% formaldehyde.A wild-type strain without an HA tag was used as a control.Immunoprecipitated pellets were used as templates for PCR with the primer pairs HOF1-pro-F/R.The band intensity was quantified using ImageJ software.Under no stress conditions, the ratio of the band in the ChIP group to the input group was normalized as 1.The result was averaged from two independent experiments.

Figure 7 .
Figure 7. Rad53 is involved in the direct regulation of HOF1.(A) Diagram of yeast one-hybrid assay.(B) The FHA2 region of Rad53 binds to the promoter of HOF1.The different domains of Rad53 were cloned into the pGADT7 plasmid, and various regions of the HOF1 promoter were cloned into the pHIS2 plasmid before being transformed into the yeast Y187 strain.The transformants were dissolved in distilled water and dropped onto SC-Trp-Leu-His plates containing different concentrations of 3-AT.The plates were kept at 30 • C for 2-3 days.(C) Checkpoint kinase Rad53 binds to the promoter of HOF1.The log phase wild-type cells carrying the Rad53-HA fusion, with or without 0.02% MMS treatment, were fixed with 1% formaldehyde.A wild-type strain without an HA tag was used as a control.Immunoprecipitated pellets were used as templates for PCR with the primer pairs HOF1-pro-F/R.The band intensity was quantified using ImageJ software.Under no stress conditions, the ratio of the band in the ChIP group to the input group was normalized as 1.The result was averaged from two independent experiments.
albicans, thus leading to the assumption of its role by other transcription factors.In particular, Mcm1 may functionally replace Ndd1, as evidenced by the effective increase in HOF1 transcription upon MCM1 overexpression in our study.Consistent with this, we observed the consistent upregulation of the target genes of Mcm1, PCL1, MRD1, and SIM1, along with the downregulation of RAM1, under both nonstress and MMS stress conditions, thus indicating that the loss of Rad53 influenced the transcriptional activity of Mcm1.Moreover, Mcm1/Fkh2 was found to bind to the promoter of HOF1; however, this binding decreased upon MMS treatment in C. albicans.The dynamic binding of transcription factor Mcm1 to the promoter of HOF1 is consistent with the transcription of HOF1, thus suggesting that Mcm1 binding stimulates HOF1 transcription and ensures normal cytokinesis by maintaining elevated HOF1 levels (Figure8A).Nevertheless, upon MMS treatment, the binding affinity between Mcm1 and the HOF1 promoter decreased, thus leading to the downregulation of HOF1 transcription as a response to arrest cytokinesis during DNA damage repair (Figure8A).The deletion of RAD53, but not DUN1, abrogated this MMS-induced downregulation of HOF1, thus suggesting its potential dependence on a transcription factor-mediated mechanism.In the absence of Rad53 phosphorylation, Mcm1 and Fkh2 likely remain bound to the promoter region, thus leading to unaltered transcription under MMS stress conditions (Figure8A).Therefore, species-specific mechanisms govern the transcriptional regulation of HOF1 and potentially other cell cycle genes in C. albicans and S. cerevisiae.

Figure 8 .
Figure 8. Overview of checkpoint-related regulation on HOF1 in response to MMS in C. albicans.(A) The dynamic binding of Rad53 and Mcm1/Fkh2 to the promoter of HOF1.Under normal conditions, Mcm1 and Fkh2 bind to the promoter of HOF1 and regulate the transcription of HOF1 to ensure regular and timely cytokinesis.Upon DNA damage stress, Mcm1 or Fkh2 dissociates from the promoter of HOF1 either by activated Rad53 or through a competition with activated Rad53, thereby subsequently diminishing the transcription of HOF1 to impede cytokinesis and giving enough time for cells to repair damaged DNA.(B) The binding region for Rad53 and Mcm1/Fkh2 in the HOF1 promoter.The red box represents the binding region of Rad53, and the green box represents the detected binding region for Mcm1 and Fkh2.

Figure 8 .
Figure 8. Overview of checkpoint-related regulation on HOF1 in response to MMS in C. albicans.(A) The dynamic binding of Rad53 and Mcm1/Fkh2 to the promoter of HOF1.Under normal conditions, Mcm1 Figure S2.Overview of Rad53-related transcriptome in C. albicans.Figure S3.Comparison of transcriptome affected by deleting RAD53 in normal condition or MMS stress condition in C. albicans.Figure S4.The transcription of ADE4 and RNR3 after deleting DUN1. Figure S5.The digestion of Fkh2-Mnase and Mcm1-Mnase on genomic DNA.Table