Recombinant Candida glabrata DNA polymerase epsilon subunit C (DPB3)

What is the composition of DNA polymerase epsilon holoenzyme in Candida species?

DNA polymerase epsilon (Polε) is an essential replicative polymerase composed of four subunits: Pol2, Dpb2, Dpb3, and Dpb4. This tetrameric structure is conserved across yeast species, including pathogenic Candida species. In Candida albicans, these subunits are encoded by POL2 (orf19.2365), DPB2 (orf19.7564), DPB3 (orf19.3063), and DPB4 (orf19.2088) genes, according to the Candida Genome Database . The Pol2 and Dpb2 subunits are essential for cell viability, while Dpb3 and Dpb4 serve as accessory subunits that enhance polymerase function but are not strictly required for survival under normal growth conditions .

The holoenzyme structure involves complex interactions between these four subunits, with the Dpb3-Dpb4 subcomplex playing a crucial structural role in maintaining the stability and functionality of the entire polymerase complex. These interactions are particularly important during DNA replication stress and adverse environmental conditions where complete polymerase functionality becomes critical for cell survival and genome integrity.

What are the recommended methods for generating recombinant Dpb3 protein from C. glabrata?

To generate recombinant C. glabrata Dpb3 protein, researchers typically employ heterologous expression systems, with E. coli being the most commonly used host. The process begins with PCR amplification of the DPB3 coding sequence from C. glabrata genomic DNA using high-fidelity polymerase and primers containing appropriate restriction sites. The amplified sequence is then cloned into an expression vector, commonly including an N or C-terminal affinity tag (such as 6xHis, GST, or MBP) to facilitate purification.

For optimal expression, BL21(DE3) or Rosetta E. coli strains are recommended, as they are engineered to enhance expression of eukaryotic proteins. Expression conditions should be optimized by testing different temperatures (16-37°C), IPTG concentrations (0.1-1 mM), and induction times (3-16 hours). For Dpb3, lower temperatures (16-18°C) with longer induction times often yield better results for properly folded protein.

Protein purification typically involves initial capture via affinity chromatography corresponding to the chosen tag, followed by size exclusion chromatography to obtain pure, homogeneous protein. Additional purification steps such as ion exchange chromatography may be necessary depending on the intended application. Protein quality should be assessed via SDS-PAGE and western blotting, with activity verified through DNA binding assays.

What genetic approaches are effective for studying Dpb3 function in C. glabrata?

Gene deletion techniques are particularly valuable for studying Dpb3 function in C. glabrata. Based on approaches used in related Candida species, an effective strategy involves replacing the DPB3 gene with a selectable marker through homologous recombination. This can be accomplished by generating a replacement cassette containing a selectable marker (such as HIS3) flanked by sequences homologous to regions upstream and downstream of the DPB3 coding sequence .

The replacement cassette can be prepared by PCR using primers containing approximately 50-60 bp of sequences flanking the DPB3 gene, similar to the approach described for CgDTR1 deletion in C. glabrata . Transformation can be performed using established protocols for C. glabrata, with successful transformants selected on appropriate media lacking histidine (if using HIS3 as a marker). Verification of gene deletion should be conducted through PCR using primers that bind within the replacement cassette and in genomic regions beyond the homologous recombination sites .

For more sophisticated genetic manipulations, CRISPR-Cas9 systems adapted for C. glabrata can be employed to create precise gene deletions, point mutations, or tagged versions of Dpb3 to study specific aspects of its function or interactions with other polymerase subunits.

What is the role of Dpb3 in DNA replication processivity?

Dpb3, as part of the Dpb3-Dpb4 subcomplex, plays a critical role in enhancing the processivity of DNA polymerase epsilon during DNA replication. Studies in C. albicans have demonstrated that the loss of Dpb3 results in reduced processive DNA synthesis . This is evident from the delayed and less efficient DNA replication observed in dpb3 null strains, particularly following exposure to replication stress induced by hydroxyurea.

The Dpb3-Dpb4 subcomplex contributes to processivity by stabilizing the interaction between polymerase epsilon and the DNA template, potentially through the histone-fold domains present in both proteins. This stabilization is especially important during leading strand synthesis, where continuous and processive DNA synthesis is essential for efficient replication fork progression. Without Dpb3, the polymerase complex becomes less stable on the DNA template, resulting in more frequent dissociation and consequently reduced processivity.

Additionally, the subcomplex may facilitate proper orientation of the catalytic subunit Pol2 at the replication fork, further enhancing the enzyme's ability to synthesize DNA continuously without dissociating from the template.

How does deletion of DPB3 affect cell cycle progression in Candida species?

Deletion of DPB3 in C. albicans results in delayed cell cycle progression, indicating its importance in coordinating DNA replication with other cell cycle events . The knockout strains exhibit a prolonged S phase, suggesting difficulties in completing DNA replication in a timely manner. This delay is likely a consequence of the reduced processivity of polymerase epsilon in the absence of Dpb3, leading to slower replication fork movement.

The cell cycle delay is particularly pronounced under conditions of replication stress, such as exposure to hydroxyurea, which depletes cellular dNTP pools . Under these conditions, dpb3 null strains show significantly compromised growth compared to wild-type strains, highlighting the importance of Dpb3 in maintaining replication efficiency during stress conditions.

Furthermore, the delayed cell cycle progression in dpb3 null strains is associated with activation of checkpoint pathways that monitor replication completion. This checkpoint activation represents a cellular response to the replication defects caused by Dpb3 absence, further emphasizing its importance in normal replication timing and cell cycle coordination.

How does Dpb3 contribute to maintaining genome integrity?

Dpb3 plays a significant role in maintaining genome integrity by enhancing both the processivity and fidelity of DNA polymerase epsilon during replication. In C. albicans, deletion of DPB3 leads to an increased rate of spontaneous mutagenesis, indicating compromised replication accuracy . This elevated mutation rate suggests that Dpb3 influences the fidelity mechanisms of polymerase epsilon, possibly by stabilizing the polymerase structure in a conformation that favors accurate nucleotide selection and incorporation.

Additionally, dpb3 null strains exhibit hypersensitivity to various DNA damaging agents, including methyl methanesulfonate (MMS), hydrogen peroxide (H₂O₂), UV radiation, and cisplatin . This broad sensitivity spectrum suggests that Dpb3 contributes to multiple aspects of DNA damage tolerance and repair, beyond its role in normal replication. The sensitivity to oxidative damage (H₂O₂) is particularly noteworthy, as this represents a common stress encountered during host-pathogen interactions.

Whole-genome sequencing of dpb3 knockout strains reveals accumulated indels and SNPs, particularly in intergenic repeat regions of chromosomes . This pattern of mutation accumulation provides insight into how Dpb3 deficiency affects genome stability at the sequence level, with repetitive regions being particularly vulnerable to errors in the absence of proper polymerase function.

What experimental approaches can detect genome instability in Dpb3-deficient strains?

Several complementary approaches can effectively detect and characterize genome instability in Dpb3-deficient strains:

• Mutation rate assays: Fluctuation analysis using selectable markers (such as CAN1 or URA3) provides quantitative measurement of spontaneous mutation rates. This approach involves growing multiple independent cultures and determining the frequency of resistance to specific selective agents (e.g., canavanine for CAN1 mutations).

• DNA damage sensitivity assays: Spot dilution assays on media containing various genotoxic agents (MMS, H₂O₂, UV, cisplatin) can reveal sensitivity phenotypes indicative of genome instability . These assays should include a range of concentrations to identify subtle differences in sensitivity between wild-type and dpb3 null strains.

• Pulse-field gel electrophoresis (PFGE): This technique allows visualization of chromosome integrity and can detect gross chromosomal rearrangements. While dpb3ΔΔ dpb4ΔΔ double mutants in C. albicans did not show noticeable karyotypic alterations by PFGE , this technique remains valuable for detecting potential large-scale genome changes.

• Whole-genome sequencing: Next-generation sequencing provides comprehensive detection of mutations across the genome, allowing identification of mutation patterns and hotspots. This approach revealed accumulation of indels and SNPs primarily in intergenic repeat regions in dpb3ΔΔ dpb4ΔΔ strains of C. albicans , providing valuable insights into the types of genomic regions most vulnerable to errors when Dpb3 function is compromised.

• Alkaline comet assay: This technique detects DNA strand breaks at the single-cell level and can reveal increased levels of DNA damage in Dpb3-deficient cells, particularly following exposure to genotoxic agents.

What is the relationship between Dpb3 function and Candida virulence?

Research in C. albicans has revealed a surprising connection between Dpb3 function and fungal virulence. Polε-defective strains, including dpb3 null mutants, display constitutive filamentous growth and, remarkably, are non-pathogenic in mouse models of systemic candidiasis . This suggests that proper regulation of DNA replication through functional Dpb3 is essential for normal morphogenesis and virulence in Candida species.

The connection between DNA replication fidelity and virulence highlights the complex relationship between basic cellular processes and pathogenesis in fungal pathogens. It suggests that the Dpb3-Dpb4 subcomplex of polymerase epsilon contributes to virulence through mechanisms beyond its structural role in DNA replication, potentially involving regulation of stress responses and morphogenesis pathways that are critical during host interaction.

How can infection models be used to study the impact of Dpb3 deficiency on virulence?

Several infection models can be employed to study how Dpb3 deficiency affects Candida virulence:

• Murine systemic candidiasis model: This is the gold standard for evaluating Candida virulence. Wild-type and dpb3 null strains can be injected into the lateral tail vein of mice, followed by monitoring survival rates, fungal burden in organs (kidney, liver, spleen), and histopathological analysis of infected tissues . This model provides the most clinically relevant assessment of virulence attenuation.

• Galleria mellonella infection model: This invertebrate model offers several advantages, including ethical considerations, cost-effectiveness, and the ability to work with larger sample sizes. The model involves injecting approximately 5 × 10⁷ Candida cells into G. mellonella larvae and monitoring survival over 72 hours . Additional analyses can include fungal proliferation in larval hemolymph and interactions with hemocytes (insect immune cells).

• Macrophage co-culture assays: In vitro co-culture of Candida strains with macrophage cell lines (e.g., J774A.1 or RAW264.7) allows assessment of phagocytosis rates, fungal survival within macrophages, and macrophage killing. These assays provide insights into how Dpb3 deficiency affects interactions with innate immune cells.

• Biofilm formation assays: Candida biofilms represent a clinically relevant growth mode associated with device-related infections. Comparing biofilm formation capacity between wild-type and dpb3 null strains using crystal violet staining, XTT reduction assays, or confocal microscopy can reveal how Dpb3 impacts this important virulence trait.

What techniques can resolve the structural interactions between Dpb3 and other polymerase epsilon subunits?

Understanding the structural interactions between Dpb3 and other polymerase epsilon subunits requires sophisticated structural biology approaches:

• X-ray crystallography: This technique can provide atomic-resolution structures of the Dpb3-Dpb4 subcomplex or the entire polymerase epsilon holoenzyme. Successful crystallization typically requires highly pure, homogeneous protein samples and optimization of crystallization conditions. For the Dpb3-Dpb4 subcomplex, co-expression and co-purification are recommended to ensure proper complex formation.

• Cryo-electron microscopy (cryo-EM): Recent advances in cryo-EM have made it possible to obtain high-resolution structures of large protein complexes without the need for crystallization. This approach is particularly valuable for studying the entire polymerase epsilon holoenzyme, which may have flexible regions that complicate crystallization.

• Nuclear magnetic resonance (NMR) spectroscopy: For studying specific interactions between Dpb3 and defined regions of other subunits, NMR can provide detailed information about binding interfaces and conformational changes. This approach is most suitable for smaller protein domains or peptides derived from interaction regions.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique can map protein-protein interaction surfaces by identifying regions that show altered solvent accessibility upon complex formation. HDX-MS is particularly useful for identifying dynamic aspects of the interactions between Dpb3 and other polymerase subunits.

• Crosslinking mass spectrometry (XL-MS): Chemical crosslinking followed by mass spectrometry analysis can identify residues that are in close proximity in the native complex, providing spatial constraints that inform structural models of the polymerase complex.

How does the histone-fold domain of Dpb3 contribute to DNA interaction?

The histone-fold domain (HFD) in Dpb3 plays a critical role in mediating interactions with DNA, similar to its function in histones. Research questions exploring this function require specialized approaches:

• Electrophoretic mobility shift assays (EMSAs): These assays can determine the DNA binding affinity and specificity of purified Dpb3-Dpb4 subcomplexes, comparing wild-type proteins with mutants containing alterations in the histone-fold domain. Different DNA substrates (single-stranded, double-stranded, or structures mimicking replication intermediates) can reveal binding preferences.

• Fluorescence anisotropy: This technique provides quantitative measurement of binding affinities between the Dpb3-Dpb4 subcomplex and fluorescently labeled DNA substrates. By comparing wild-type complexes with HFD mutants, researchers can determine how specific residues contribute to DNA binding.

• DNA footprinting assays: These assays can identify the specific DNA sequences or structures protected by the Dpb3-Dpb4 subcomplex, revealing the precise nature of the DNA-protein interactions.

• Single-molecule techniques: Approaches such as optical tweezers or single-molecule FRET can directly visualize how the Dpb3-Dpb4 subcomplex affects DNA dynamics, including potential roles in stabilizing the polymerase-DNA complex during replication.

• In vivo chromatin immunoprecipitation (ChIP): ChIP experiments using tagged versions of Dpb3 can identify genomic regions where Dpb3 associates with DNA in living cells, potentially revealing sequence or chromatin context preferences.

What evidence supports targeting the Dpb3-Dpb4 interface for antifungal development?

Several lines of evidence support the potential of the Dpb3-Dpb4 interface as a target for antifungal development:

• Attenuated virulence: Studies in C. albicans demonstrate that disruption of the Dpb3-Dpb4 subcomplex results in avirulent strains in mouse models of systemic candidiasis . This attenuated virulence suggests that compounds disrupting this interface could effectively reduce fungal pathogenicity without necessarily killing the pathogen.

• Structural divergence from human homologs: The relatively low sequence identity between fungal Dpb3 and human counterparts (approximately 13-17%) suggests potential for selective targeting of the fungal protein. Additionally, the unique structural features of Candida Dpb3, including the extended N-terminal region, provide opportunities for species-specific inhibition.

• Critical role in stress resistance: Dpb3-deficient strains show increased sensitivity to various stressors, including temperature extremes, oxidative stress, and genotoxic agents . This suggests that targeting Dpb3 could sensitize pathogenic fungi to host defense mechanisms and environmental stresses encountered during infection.

• Protein-protein interface druggability: The Dpb3-Dpb4 interaction represents a defined protein-protein interface that could potentially be disrupted by small molecules or peptide mimetics. Advances in targeting protein-protein interactions have made such interfaces increasingly viable as drug targets.

• Non-essential but fitness-enhancing target: While not essential for viability under standard conditions, Dpb3 is important for fitness under stress conditions relevant to the host environment . This profile is attractive for antifungal development, as it may reduce selection pressure for resistance compared to essential targets.

What methods can identify small molecules targeting the Dpb3-Dpb4 interface?

Several complementary approaches can be employed to identify small molecules that disrupt the Dpb3-Dpb4 interface:

• Structure-based virtual screening: Using solved or modeled structures of the Dpb3-Dpb4 complex, computational docking of virtual compound libraries can identify molecules predicted to bind at the protein-protein interface. High-scoring compounds can then be tested experimentally for binding and inhibitory activity.

• Fragment-based screening: This approach identifies low molecular weight compounds (fragments) that bind to subsites within the protein-protein interface. Nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), or thermal shift assays can detect these weak interactions, and promising fragments can be elaborated or linked to create more potent inhibitors.

• High-throughput screening (HTS): Biochemical assays that monitor Dpb3-Dpb4 complex formation can be adapted for HTS to test large compound libraries. These assays might include fluorescence polarization, FRET, or AlphaScreen technologies that detect protein-protein interactions in solution.

• Yeast two-hybrid (Y2H) interference assays: Modified Y2H systems can identify compounds that disrupt protein-protein interactions in a cellular context. Dpb3 and Dpb4 can be expressed as fusion proteins in yeast, and compounds that disrupt their interaction will reduce reporter gene expression.

• Peptidomimetic approaches: Based on the structural details of the interface, synthetic peptides mimicking critical binding regions can be designed and optimized for improved binding, stability, and cell penetration.

Comparison of Dpb3 Protein Properties Across Species

This table highlights the significant structural differences between Dpb3 proteins across species, particularly the larger size of Candida albicans Dpb3 compared to other eukaryotes . These differences suggest evolutionary adaptations that may reflect specialized functions in different organisms and provide potential opportunities for selective targeting in antifungal development.

Phenotypic Effects of DPB3 Deletion in C. albicans

This table summarizes the phenotypic consequences of DPB3 deletion in C. albicans, demonstrating its importance in stress resistance, normal morphogenesis, and virulence . The similar phenotypes between dpb3ΔΔ and dpb4ΔΔ strains suggest functional cooperation between these two subunits, consistent with their role as a structural subcomplex within polymerase epsilon.