Yeast 9-1-1 complex acts as a sliding clamp for DNA synthesis by DNA polymerase ε.

Eukaryotic cells harbor two DNA-binding clamps, proliferating cell nuclear antigen (PCNA), and another clamp commonly referred to as 9-1-1 clamp. In contrast to the essential role of PCNA in DNA replication as a sliding clamp for DNA polymerase (Pol) δ, no such role in DNA synthesis has been identified for the human 9-1-1 clamp or the orthologous yeast 17-3-1 clamp. The only role identified for either the 9-1-1 or 17-3-1 clamp is in the recruitment of signal transduction kinases, which affect the activation of cell cycle checkpoints in response to DNA damage. However, unlike the loading of PCNA by the replication factor C (RFC) clamp loader onto 3'-recessed DNA junctions for processive DNA synthesis by Polδ, the 17-3-1 clamp or the 9-1-1 clamp is loaded by their respective clamp loader Rad24-RFC or RAD17-RFC onto the 5'-recessed DNA junction of replication protein A-coated DNA for the recruitment of signal transduction kinases. Here, we identify a novel role of 17-3-1 clamp as a sliding clamp for DNA synthesis by Polε. We provide evidence that similar to the loading of PCNA by RFC, the 17-3-1 clamp is loaded by the Rad24-RFC clamp loader at the 3'-recessed DNA junction in an ATP-dependent manner. However, unlike PCNA, the 17-3-1 clamp does not enhance the processivity of DNA synthesis by Polε; instead, it greatly increases the catalytic efficiency of Polε for correct nucleotide incorporation. Furthermore, we show that the same PCNA-interacting peptide domain in the polymerase 2 catalytic subunit mediates Polε interaction with the 17-3-1 clamp and with PCNA.

RAD51-WSS1-dependent genetic pathways are essential for DNA-protein crosslink repair and pathogenesis in Candida albicans.

Genetic analyses in Saccharomyces cerevisiae suggest that nucleotide excision repair (NER), homologous recombination (HR), and protease-dependent repair pathways coordinately function to remove DNA-protein crosslinks (DPCs) from the genome. DPCs are genomic cytotoxic lesions generated because of the covalent linkage of proteins with DNA. Although NER and HR processes have been studied in pathogenic Candida albicans, their roles in DPC repair (DPCR) are yet to be explored. Proteases like Wss1 and Tdp1 (tyrosyl-DNA phosphodiesterase-1) are known to be involved in DPCR; however, Tdp1 that selectively removes topoisomerase-DNA complexes is intrinsically absent in C. albicans. Therefore, the mechanism of DPCR might have evolved differently in C. albicans. Herein, we investigated the interplay of three genetic pathways and found that RAD51-WSS1-dependent HR and protease-dependent repair pathways are essential for DPC removal, and their absence caused an increased rate of loss of heterozygosity in C. albicans. RAD1 but not RAD2 of NER is critical for DPCR. In addition, we observed truncation of chromosome #6 in the cells defective in both RAD51 and WSS1 genes. While the protease and DNA-binding activities are essential, a direct interaction of Wss1 with the eukaryotic DNA clamp proliferating cell nuclear antigen is not a requisite for the function of Wss1. DPCR-defective C. albicans cells exhibited filamentous morphology, reduced immune cell evasion, and attenuation in virulence. Thus, we concluded that RAD51-WSS1-dependent DPCR pathways are essential for genome stability and candidiasis development. Since no vaccine against candidiasis is available for human use yet, we propose to explore DPCR-defective attenuated strains (rad51ΔΔwss1ΔΔ and rad2ΔΔrad51ΔΔwss1ΔΔ) for whole-cell vaccine development.

The ubiquitin-binding domain of DNA polymerase η directly binds to DNA clamp PCNA and regulates translesion DNA synthesis.

DNA polymerase eta (Polη) is a unique translesion DNA synthesis (TLS) enzyme required for the error-free bypass of ultraviolet ray (UV)-induced cyclobutane pyrimidine dimers in DNA. Therefore, its deficiency confers cellular sensitivity to UV radiation and an increased rate of UV-induced mutagenesis. Polη possesses a ubiquitin-binding zinc finger (ubz) domain and a PCNA-interacting-protein (pip) motif in the carboxy-terminal region. The role of the Polη pip motif in PCNA interaction required for DNA polymerase recruitment to the stalled replication fork has been demonstrated in earlier studies; however, the function of the ubz domain remains divisive. As per the current notion, the ubz domain of Polη binds to the ubiquitin moiety of the ubiquitinated PCNA, but such interaction is found to be nonessential for Polη's function. In this study, through amino acid sequence alignments, we identify three classes of Polη among different species based on the presence or absence of pip motif or ubz domain and using comprehensive mutational analyses, we show that the ubz domain of Polη, which intrinsically lacks the pip motif directly binds to the interdomain connecting loop (IDCL) of PCNA and regulates Polη's TLS activity. We further propose two distinct modes of PCNA interaction mediated either by pip motif or ubz domain in various Polη homologs. When the pip motif or ubz domain of a given Polη binds to the IDCL of PCNA, such interaction becomes essential, whereas the binding of ubz domain to PCNA through ubiquitin is dispensable for Polη's function.

Interdomain connecting loop and J loop structures determine cross-species compatibility of PCNA.

Eukaryotic proliferating cell nuclear antigen (PCNA) plays an essential role in orchestrating the assembly of the replisome complex, stimulating processive DNA synthesis, and recruiting other regulatory proteins during the DNA damage response. PCNA and its binding partner network are relatively conserved in eukaryotes, and it exhibits extraordinary structural similarity across species. However, despite this structural similarity, the PCNA of a given species is rarely functional in heterologous systems. In this report, we determined the X-ray crystal structure of Neurospora crassa PCNA (NcPCNA) and compared its structure-function relationship with other available PCNA studies to understand this cross-species incompatibility. We found two regions, the interdomain connecting loop (IDCL) and J loop structures, vary significantly among PCNAs. In particular, the J loop deviates in NcPCNA from that in Saccharomyces cerevisiae PCNA (ScPCNA) by 7 Å. Differences in the IDCL structures result in varied binding affinities of PCNAs for the subunit Pol32 of DNA polymerase delta and for T2-amino alcohol, a small-molecule inhibitor of human PCNA. To validate that these structural differences are accountable for functional incompatibility in S. cerevisiae, we generated NcPCNA mutants mimicking IDCL and J loop structures of ScPCNA. Our genetic analyses suggested that NcPCNA mutants are fully functional in S. cerevisiae. The susceptibility of the strains harboring ScPCNA mimics of NcPCNA to various genotoxic agents was similar to that in yeast cells expressing ScPCNA. Taken together, we conclude that in addition to the overall architecture of PCNA, structures of the IDCL and J loop of PCNA are critical determinants of interspecies functional compatibility.

Quaternary structural diversity in eukaryotic DNA polymerases: monomeric to multimeric form.

Sixteen eukaryotic DNA polymerases have been identified and studied so far. Based on the sequence similarity of the catalytic subunits of DNA polymerases, these have been classified into four A, B, X and Y families except PrimPol, which belongs to the AEP family. The quaternary structure of these polymerases also varies depending upon whether they are composed of one or more subunits. Therefore, in this review, we used a quaternary structure-based classification approach to group DNA polymerases as either monomeric or multimeric and highlighted functional significance of their accessory subunits. Additionally, we have briefly summarized various DNA polymerase discoveries from a historical perspective, emphasized unique catalytic mechanism of each DNA polymerase and highlighted recent advances in understanding their cellular functions.

'PIPs' in DNA polymerase: PCNA interaction affairs.

Interaction of PCNA with DNA polymerase is vital to efficient and processive DNA synthesis. PCNA being a homotrimeric ring possesses three hydrophobic pockets mostly involved in an interaction with its binding partners. PCNA interacting proteins contain a short sequence of eight amino acids, popularly coined as PIP motif, which snuggly fits into the hydrophobic pocket of PCNA to stabilize the interaction. In the last two decades, several PIP motifs have been mapped or predicted in eukaryotic DNA polymerases. In this review, we summarize our understandings of DNA polymerase-PCNA interaction, the function of such interaction during DNA synthesis, and emphasize the lacunae that persist. Because of the presence of multiple ligands in the replisome complex and due to many interaction sites in DNA polymerases, we also propose two modes of DNA polymerase positioning on PCNA required for DNA synthesis to rationalize the tool-belt model of DNA replication.

Virulence and pathogenicity of a Candida albicans mutant with reduced filamentation.

Deletion of DNA polymerase eta (Rad30/Polη) in pathogenic yeast Candida albicans is known to reduce filamentation induced by serum, ultraviolet, and cisplatin. Because nonfilamentous C. albicans is widely accepted as avirulent form, here we explored the virulence and pathogenicity of a rad30Δ strain of C. albicans in cell-based and animal systems. Flow cytometry of cocultured fungal and differentiated macrophage cells revealed that comparatively higher percentage of macrophages was associated with the wild-type than rad30Δ cells. In contrast, higher number of Polη-deficient C. albicans adhered per macrophage membrane. Imaging flow cytometry showed that the wild-type C. albicans developed hyphae after phagocytosis that caused necrotic death of macrophages to evade their clearance. Conversely, phagosomes kill the fungal cells as estimated by increased metacaspase activity in wild-type C. albicans. Despite the morphological differences, both wild-type and rad30∆ C. albicans were virulent with a varying degree of pathogenicity in mice models. Notably, mice with Th1 immunity were comparatively less susceptible to systemic fungal infection than Th2 type. Thus, our study clearly suggests that the modes of interaction of morphologically different C. albicans strains with the host immune cells are diverged, and host genetic background and several other attributing factors of the fungus could additionally determine their virulence.

TLS dependent and independent functions of DNA polymerase eta (Polη/Rad30) from Pathogenic Yeast Candida albicans.

Polη, a unique TLS DNA polymerase that promotes efficient bypass of UV-induced CPDs and cisplatin adducts, has not been explored in Candida species yet. Here, we show that CaPolη plays a vital role in protecting Candida albicans genome from diverse array of DNA damaging agents, not limited to UV and cisplatin. Polη deficient strain did not exhibit any hyphal development in the presence of UV and cisplatin while the wild type strain profusely developed DNA damage induced filamentation. The polarized growth induced by HU and MMS was found to be Polη independent. No common regulatory pathway of morphogenesis operates in C. albicans due to genomic stress, rather Polη branches away from RAD53 dependent pathway to be specific to UV/cisplatin. Interestingly, serum that does not inflict any DNA damage also induces hyphal growth in C. albicans, and requires a functionally active Polη. Importantly, deletion of RAD30 sensitized the strain to amphotericin B; but its presence resulted in azole drug tolerance only in DNA damaging conditions. We suggest that the roles of CaPolη in genome stability and genotoxins induced filamentation are due to its TLS activities; whereas its TLS independent functions play a vital role in serum induced morphogenesis and amphotericin B resistance.

Multifaceted activities of DNA polymerase η: beyond translesion DNA synthesis.

DNA polymerases are evolved to extend the 3'-OH of a growing primer annealed to a template DNA substrate. Since replicative DNA polymerases have a limited role while replicating structurally distorted template, translesion DNA polymerases mostly from Y-family come to the rescue of stalled replication fork and maintain genome stability. DNA polymerase eta is one such specialized enzyme whose function is directly associated with casual development of certain skin cancers and chemo-resistance. More than 20 years of extensive studies are available to support TLS activities of Polη in bypassing various DNA lesions, in addition, limited but crucial growing evidence also exist to suggest Polη possessing TLS-independent cellular functions. In this review, we have mostly focused on non-TLS activities of Polη from different organisms including our recent findings from pathogenic yeast Candida albicans.

Identification of two functional PCNA-binding domains in human DNA polymerase κ.

Previously, we have shown that human DNA polymerase (Pol) η has two functional PCNA-binding motifs, PIP1 and PIP2, and that a C-terminal deletion of Polη that lacks the ubiquitin-binding UBZ domain and the PIP2 domain but retains the PIP1 domain promotes normal levels of translesion synthesis (TLS) opposite a cis-syn TT dimer in human cells. Here, we identify two PIP domains in Polκ and show that TLS occurs normally in human fibroblast cells in which the pip1 or pip2 mutant Polκ is expressed, but mutational inactivation of both PIP domains renders Polκ nonfunctional in TLS opposite the thymine glycol lesion. Thus, the two PIP domains of Polκ function redundantly in TLS opposite this DNA lesion in human cells. However, and surprisingly, whereas mutational inactivation of the PIP1 domain completely inhibits the stimulation of DNA synthesis by Polκ in the presence of proliferating cell nuclear antigen (PCNA), replication factor C, and replication protein A, mutations in PIP2 have no adverse effect on PCNA-dependent DNA synthesis. This raises the possibility that activation of Polκ PIP2 as a PCNA-binding domain occurs during TLS in human cells and that protein-protein interactions and post-transcriptional modifications are involved in such activation.

Characterization of proliferating cell nuclear antigen (PCNA) from pathogenic yeast Candida albicans and its functional analyses in S. cerevisiae.

BACKGROUND: Proliferating cell nuclear antigen (PCNA/POL30) an essential protein forms a homotrimeric ring encircling dsDNA and serves as a molecular scaffold to recruit various factors during DNA replication, repair and recombination. According to Candida Genome Database (CGD), orf19.4616 sequence is predicted to encode C. albicans PCNA (CaPCNA) that has not been characterized yet. RESULTS: Molecular modeling studies of orf19.4616 using S. cerevisiae PCNA sequence (ScPCNA) as a template, and its subsequent biochemical characterizations suggest that like other eukaryotic PCNAs, orf19.4616 encodes for a conventional homotrimeric sliding clamp. Further we showed by surface plasmon resonance that CaPCNA physically interacted with yeast DNA polymerase eta. Plasmid segregation in genomic knock out yeast strains showed that CaPCNA but not its G178S mutant complemented for cell survival. Unexpectedly, heterologous expression of CaPCNA in S. cerevisiae exhibited slow growth phenotypes, sensitivity to cold and elevated temperatures; and showed enhanced sensitivity to hydroxyurea and various DNA damaging agents in comparison to strain bearing ScPCNA. Interestingly, wild type strains of C. albicans showed remarkable tolerance to DNA damaging agents when compared with similarly treated yeast cells. CONCLUSIONS: Despite structural and physiochemical similarities; we have demonstrated that there are distinct functional differences between ScPCNA and CaPCNA, and probably the ways both the strains maintain their genomic stability. We propose that the growth of pathogenic C. albicans which is evolved to tolerate DNA damages could be controlled effectively by targeting this unique fungal PCNA.

PCNA binding domains in all three subunits of yeast DNA polymerase δ modulate its function in DNA replication.

DNA polymerase δ (Polδ) plays an essential role in replication from yeast to humans. Polδ in Saccharomyces cerevisiae is comprised of three subunits, the catalytic subunit Pol3 and the accessory subunits Pol31 and Pol32. Yeast Polδ exhibits a very high processivity in synthesizing DNA with the proliferating cell nuclear antigen (PCNA) sliding clamp; however, it has remained unclear how Polδ binds PCNA to achieve its high processivity. Here we show that PCNA interacting protein (PIP) motifs in all three subunits contribute to PCNA-stimulated DNA synthesis by Polδ, and mutational inactivation of all three PIP motifs abrogates its ability to synthesize DNA with PCNA. Genetic analyses of mutations in these PIPs have revealed that in the absence of functional Pol32 PIP domain, PCNA binding by both the Pol3 and Pol31 subunits becomes essential for cell viability. Based on our biochemical and genetic studies we infer that yeast Polδ can simultaneously utilize all three PIP motifs during PCNA-dependent DNA synthesis, and suggest that Polδ binds the PCNA homotrimer via its three subunits. We consider the implications of these observations for Polδ's role in DNA replication.

A chemically induced attenuated strain of Candida albicans generates robust protective immune responses and prevents systemic candidiasis development.

Despite current antifungal therapy, invasive candidiasis causes >40% mortality in immunocompromised individuals. Therefore, developing an antifungal vaccine is a priority. Here, we could for the first time successfully attenuate the virulence of Candida albicans by treating it with a fungistatic dosage of EDTA and demonstrate it to be a potential live whole cell vaccine by using murine models of systemic candidiasis. EDTA inhibited the growth and biofilm formation of C. albicans. RNA-seq analyses of EDTA-treated cells (CAET) revealed that genes mostly involved in metal homeostasis and ribosome biogenesis were up- and down-regulated, respectively. Consequently, a bulky cell wall with elevated levels of mannan and ß-glucan, and reduced levels of total monosomes and polysomes were observed. CAET was eliminated faster than the untreated strain (Ca) as found by differential fungal burden in the vital organs of the mice. Higher monocytes, granulocytes, and platelet counts were detected in Ca- vs CAET-challenged mice. While hyper-inflammation and immunosuppression caused the killing of Ca-challenged mice, a critical balance of pro- and anti-inflammatory cytokines-mediated immune responses are the likely reasons for the protective immunity in CAET-infected mice.

Hsp90-Mediated Multi-Drug Resistance in DNA Polymerase-Defective Strains of Candida albicans.

The incidence of infections caused by Candida species, specifically by drug-resistant isolates, is a major health concern as they can disseminate to and colonize most vital organs, enhancing morbidity and mortality. Several molecular mechanisms have been reported to be involved in drug resistance. These are mostly drug- and isolate-specific. Here, we characterized three different genetically modified strains of C. albicans that were multi-drug-resistant (MDR) and deciphered a uniform mechanism responsible for resistance. DNA polymerase epsilon (Polε) is a leading strand-specific polymerase consisting of four subunits, namely, Pol2, Dpb2, Dpb3, and Dpb4. The deletion of one or both of the Dpb3 and Dpb4 subunits in C. albicans rendered multi-drug resistance. A detailed characterization of these strains revealed that acquired mutagenesis, drug efflux pumps, and other known mechanisms did not play a significant role because the complemented strain showed drug sensitivity. More importantly, the function of heat shock protein 90 (Hsp90) in these knockout strains is critical for reducing susceptibility to several antifungal drugs. Cell wall deformity and composition in these strains can add to such a phenotype. The inhibition of Hsp90 function by geldanamycin and tricostatin A sensitized the MDR strains to antifungals. Considering our earlier research and this report, we suggest that replication stress induces Hsp90 expression and activity in order to orchestrate a cellular stress response circuit and thus develop fungal drug resistance. Thus, Hsp90 is an important drug target for use in combinatorial therapy.

Immunogenicity and efficacy of CNA25 as a potential whole-cell vaccine against systemic candidiasis.

Disseminated fungal infections account for ~1.5 million deaths per year worldwide, and mortality may increase further due to a rise in the number of immunocompromised individuals and drug-resistance fungal species. Since an approved antifungal vaccine is yet to be available, this study explored the immunogenicity and vaccine efficacy of a DNA polymerase mutant strain of Candida albicans. CNA25 is a pol32ΔΔ strain that exhibits growth defects and does not cause systemic candidiasis in mice. Immunized mice with live CNA25 were fully protected against C. albicans and C. parapsilosis but partially against C. tropicalis and C. glabrata infections. CNA25 induced steady expression of TLR2 and Dectin-1 receptors leading to a faster recognition and clearance by the immune system associated with the activation of protective immune responses mostly mediated by neutrophils, macrophages, NK cells, B cells, and CD4+ and CD8+ T cells. Molecular blockade of Dectin-1, IL-17, IFNγ, and TNFα abolished resistance to reinfection. Altogether, this study suggested that CNA25 collectively activates innate, adaptive, and trained immunity to be a promising live whole-cell vaccine against systemic candidiasis.

DNA polymerase eta lacking the ubiquitin-binding domain promotes replicative lesion bypass in humans cells.

The Rad6-Rad18 mediated monoubiquitylation of proliferating cell nuclear antigen (PCNA) at lys 164 plays a crucial role in promoting the access of translesion synthesis (TLS) DNA polymerases (Pols) to PCNA in the replication fork stalled at a lesion site. Although a number of genetic and biochemical observations have provided strong evidence that TLS Pols bind PCNA at its interdomain connector loop (IDCL) via their PCNA-interacting protein (PIP) domain, a more recent proposal formulates that TLS Pols bind PCNA at two sites, to the IDCL via their PIP domain and to lys-164 linked ubiquitin (Ub) via their ubiquitin-binding domain. To ascertain the relative contributions of the PIP and Ub-binding zinc finger (UBZ) domains of human Poleta in TLS, we have determined whether the C-terminal truncations of hPoleta that contain the PIP1 domain but lack the UBZ and PIP2 domains can still function in TLS in human cells. Our observations that such C-terminally truncated proteins promote efficient TLS opposite a cis-syn TT dimer and confer a high degree of UV resistance to XPV cells provide unambiguous evidence that the binding of PCNA via its PIP domain is essential as well as sufficient for providing hPoleta the ability to carry out TLS in human cells.

Cell Cycle Analysis of Candida albicans by Flow Cytometry.

The cell cycle is a vital process of cell division that is required to sustain life. Since faithful cell division is critical for the proper growth and development of an organism, the study of the cell cycle becomes a fundamental research objective. Saccharomyces cerevisiae has been an excellent unicellular system for unraveling the secrets of cell division, and the process of synchronization in budding yeast has been standardized. Cell synchronization is a crucial step of cell cycle analysis, where cells in a culture at different stages of the cell cycle are arrested to the same phase and, upon release, they progress synchronously. The cellular synchronization of S. cerevisiae is easily achieved by a pheromone or other chemicals like hydroxyurea treatment; however, such methodologies seem to be ineffective in synchronizing cells of multimorphic fungi such as Candida albicans. C. albicans is a human pathogen that can grow in yeast, pseudohyphal, and hyphal forms; these forms differ in morphology as well as cell cycle progression. More importantly, upon subjecting to DNA replication inhibitors for synchronization, C. albicans develops hyphal structures and grows asynchronously. Therefore, here we describe a simple and easy method to synchronize C. albicans cells in the G1 phase and the subsequent analysis of cell cycle progression by using flow cytometry.

Spot Assay and Colony Forming Unit (CFU) Analyses-based sensitivity test for Candida albicans and Saccharomyces cerevisiae.

Cellular sensitivity is an approach to inhibit the growth of certain cells in response to any non-permissible conditions, as the presence of a cytotoxic agent or due to changes in growth parameters such as temperature, salt, or media components. Sensitivity tests are easy and informative assays to get insight into essential gene functions in various cellular processes. For example, cells having any functionally defective genes involved in DNA replication exhibit sensitivity to non-permissive temperatures and to chemical agents that block DNA replication fork movement. Here, we describe a sensitivity test for multiple strains of Saccharomyces cerevisiae and Candida albicans of diverged genetic backgrounds subjected to several genotoxic chemicals simultaneously. We demonstrate it by testing the sensitivity of DNA polymerase defective yeast mutants by using spot analysis combined with colony forming unit (CFU) efficiency estimation. The method is very simple and inexpensive, does not require any sophisticated equipment, can be completed in 2-3 days, and provides both qualitative and quantitative data. We also recommend the use of this reliable methodology for assaying the sensitivity of these and other fungal species to antifungal drugs and xenobiotic factors.

Escherichia coli, but Not Staphylococcus aureus, Functions as a Chelating Agent That Exhibits Antifungal Activity against the Pathogenic Yeast Candida albicans.

Humans are colonized by diverse populations of microbes. Infections by Candida albicans, an opportunistic fungal pathogen, are a result of imbalances in the gut microbial ecosystem and are due to the suppressed immunity of the host. Here, we explored the potential effects of the polymicrobial interactions of C. albicans with Staphylococcus aureus, a Gram-positive bacterium, and Escherichia coli, a Gram-negative bacterium, in dual and triple in vitro culture systems on their respective growth, morphology, and biofilms. We found that S. aureus promoted the fungal growth and hyphal transition of C. albicans through cell-to-cell contacts; contrarily, both the cell and cell-free culture filtrate of E. coli inhibited fungal growth. A yet to be identified secretory metabolite of E. coli functionally mimicked EDTA and EGTA to exhibit antifungal activity. These findings suggested that E. coli, but not S. aureus, functions as a chelating agent and that E. coli plays a dominant role in regulating excessive growth and, potentially, the commensalism of C. albicans. Using animal models of systemic candidiasis, we found that the E. coli cell-free filtrate suppressed the virulence of C. albicans. In general, this study unraveled a significant antimicrobial activity and a potential role in the nutritional immunity of E. coli, and further determining the underlying processes behind the E. coli-C. albicans interaction could provide critical information in understanding the pathogenicity of C. albicans.

Pol32, an accessory subunit of DNA polymerase delta, plays an essential role in genome stability and pathogenesis of Candida albicans.

Candida albicans is a pathobiont that inflicts serious bloodstream fungal infections in individuals with compromised immunity and gut dysbiosis. Genomic diversity in the form of copy number alteration, ploidy variation, and loss of heterozygosity as an adaptive mechanism to adverse environments is frequently observed in C. albicans. Such genomic variations also confer a varied degree of fungal virulence and drug resistance, yet the factors propelling these are not completely understood. DNA polymerase delta (Polδ) is an essential replicative DNA polymerase in the eukaryotic cell and is yet to be characterized in C. albicans. Therefore, this study was designed to gain insights into the role of Polδ, especially its non-essential subunit Pol32, in the genome plasticity and life cycle of C. albicans. PCNA, the DNA clamp, recruits Polδ to the replication fork for processive DNA replication. Unlike in Saccharomyces cerevisiae, the PCNA interaction protein (PIP) motif of CaPol32 is critical for Polδ's activity during DNA replication. Our comparative genetic analyses and whole-genome sequencing of POL32 proficient and deficient C. albicans cells revealed a critical role of Pol32 in DNA replication, cell cycle progression, and genome stability as SNPs, indels, and repeat variations were largely accumulated in pol32 null strain. The loss of pol32 in C. albicans conferred cell wall deformity; Hsp90 mediated azoles resistance, biofilm development, and a complete attenuation of virulence in an animal model of systemic candidiasis. Thus, although Pol32 is dispensable for cell survival, its function is essential for C. albicans pathogenesis; and we discuss its translational implications in antifungal drugs and whole-cell vaccine development.