Recombinant Candida glabrata DNA polymerase epsilon subunit D (DPB4)

What is the structure and function of DPB4 in Candida glabrata?

DPB4 is a non-catalytic subunit of DNA polymerase epsilon (Polε), a replicative polymerase essential for leading strand synthesis during DNA replication. In C. glabrata, as in other fungi, Polε consists of four subunits: Pol2 (the catalytic subunit), Dpb2, Dpb3, and Dpb4. The Dpb3-Dpb4 subcomplex plays critical roles in maintaining genome stability and replication fidelity .

Structurally, DPB4 contains histone-fold domains that facilitate DNA binding and protein-protein interactions. These domains are evolutionarily conserved across fungal species, though specific sequence variations exist. The histone-fold domain enables DPB4 to interact with Dpb3, forming a stable subcomplex that enhances DNA binding affinity and polymerase processivity.

How does DPB4 contribute to DNA replication fidelity?

DPB4 enhances replication fidelity through several mechanisms:

Research strategies to investigate these functions include in vitro polymerase assays, mutation rate analysis using fluctuation tests, and ChIP-seq to monitor DPB4 localization during replication.

What are the optimal methods for expressing and purifying recombinant C. glabrata DPB4?

Efficient expression and purification of recombinant C. glabrata DPB4 requires careful consideration of expression systems and purification strategies:

Expression Systems:

• E. coli system: Use BL21(DE3) strains with pET vectors containing codon-optimized DPB4. Optimal expression typically occurs at lower temperatures (18-20°C) with 0.1-0.5 mM IPTG induction.

• Yeast expression system: S. cerevisiae or P. pastoris systems may provide proper post-translational modifications.

• Co-expression strategies: Co-expressing DPB4 with DPB3 often improves solubility and stability.

Purification Protocol:

• Cell lysis in buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitors

• Affinity chromatography using His-tag, GST-tag, or TAP-tag approaches

• Ion-exchange chromatography for removing contaminants

• Size-exclusion chromatography for final purification

• Activity verification using DNA binding assays

For interaction studies, tandem affinity purification combined with mass spectrometry has proven effective in identifying protein partners in C. glabrata, as demonstrated in studies of histone H4 interactomes .

How can researchers generate and validate DPB4 knockout strains in C. glabrata?

Creating reliable DPB4 knockout strains requires careful genetic manipulation and thorough validation:

Generation Methods:

• Homologous recombination using selection markers (NAT1, URA3, etc.)

• CRISPR-Cas9 systems adapted for C. glabrata

• Recyclable marker systems for multiple genetic manipulations

Validation Strategy:

• PCR confirmation with primers binding outside the targeted region

• Southern blot analysis to verify single integration

• RT-PCR and western blot to confirm absence of transcript and protein

• Phenotypic analysis including:

  • Sensitivity to genotoxic agents (MMS, UV, H₂O₂)

  • Growth rate determination

  • Microscopic examination for morphological changes

• Complementation with wild-type DPB4 to confirm phenotype specificity

Essential Controls:

• Parent wild-type strain

• Heterozygous mutant (if applicable)

• Complemented strain expressing DPB4 from a plasmid or reintegrated into the genome

How does DPB4 deficiency affect the DNA damage response in C. glabrata?

Based on studies in C. albicans and related findings in C. glabrata, DPB4 deficiency significantly impacts DNA damage response pathways:

Research in C. albicans has shown that dpb4 null strains exhibit sensitivity to genotoxic agents and delayed cell cycle progression . In C. glabrata, the homologous recombination pathway involving CgRad52 is essential for DNA damage repair , suggesting potential interaction between DPB4 and recombination machinery.

Experimental approaches to further investigate this relationship include epistasis analysis between DPB4 and DNA repair genes, ChIP-seq to map DPB4 localization at damaged sites, and quantitative mutation spectrum analysis.

How does DPB4 influence C. glabrata virulence and host interaction?

Research in C. albicans provides insights into how DPB4 might affect C. glabrata virulence:

In C. albicans, DPB4-deficient strains are constitutively filamentous and non-pathogenic in mice models of systemic candidiasis . While C. glabrata does not form true hyphae, DPB4 likely contributes to its virulence through other mechanisms, potentially including its intracellular survival in macrophages, which is a key virulence determinant .

Experimental approaches to study DPB4's role in virulence include macrophage infection assays, Galleria mellonella infection models (as used for CgXbp1 studies ), and mouse models of disseminated candidiasis.

Could DPB4 play a role in antifungal drug resistance mechanisms?

While direct evidence for DPB4's role in antifungal resistance in C. glabrata is limited, several mechanisms are plausible:

• Genomic stability regulation: DPB4 maintains replication fidelity, and its deficiency could increase mutation rates, potentially accelerating the development of resistance mutations.

• Stress response pathway interaction: DPB4 may interact with transcription factors like CgRpn4, which regulates fluconazole resistance genes including ergosterol biosynthesis genes .

• DNA damage response modulation: Similar to histone H4's role in MMS resistance , DPB4 might influence responses to drug-induced DNA damage.

• Morphological adaptation: If DPB4 regulates morphological transitions in C. glabrata as it does in C. albicans , it could affect cell surface properties relevant to drug uptake and efflux.

Research approaches include transcriptome analysis comparing wild-type and DPB4-deficient strains under antifungal exposure, evolution experiments to compare resistance acquisition rates, and epistasis studies with known resistance factors like CgRpn4 .

How can researchers target the Dpb3-Dpb4 interface for antifungal development?

The Dpb3-Dpb4 subcomplex represents a promising antifungal target because:

• It is critical for fungal morphogenesis and virulence

• Strains lacking these subunits are avirulent in animal models

• The interacting interface may contain fungal-specific elements

Development strategy:

• Structural characterization: Obtain high-resolution structures of the Dpb3-Dpb4 interface using X-ray crystallography or cryo-EM

• Hotspot identification: Identify critical residues through alanine scanning mutagenesis

• Virtual screening: Perform in silico screening against the interface

• Fragment-based approach: Identify small molecules that bind at the interface

• Assay development: Create assays to measure complex formation/disruption

  • Fluorescence polarization

  • FRET-based assays

  • Surface plasmon resonance

Advantages of this approach include the potential for fungal selectivity and the targeting of virulence rather than essential functions, which may reduce selection pressure for resistance development.

How do temporal transcriptional responses affect DPB4 function during infection?

The temporal dynamics of gene expression during infection are critical for understanding DPB4's role in pathogenesis. Based on studies of transcriptional responses in C. glabrata :

• Immediate early response (0-30 minutes after phagocytosis):

  • Initial stress response genes activation

  • Potential regulation of DPB4 expression or localization

• Early response (30-120 minutes):

  • Adaptation to intracellular environment

  • Potential role of DPB4 in maintaining replication under stress

• Late response (2-24 hours):

  • Growth and proliferation within macrophages

  • DPB4's role in maintaining genomic integrity during proliferation

Research methodologies include:

• ChIP-seq against elongating RNA Polymerase II to map transcriptional responses during infection

• Time-course transcriptomics comparing wild-type and DPB4-deficient strains

• Monitoring DPB4 protein levels and localization during infection stages

What proteins interact with DPB4 in C. glabrata and how are these interactions modulated by stress?

Understanding DPB4's protein interaction network provides insight into its various functions. Based on studies of protein interactions in C. glabrata :

Expected core interactors:

• Dpb3 (forming the Dpb3-Dpb4 subcomplex)

• Pol2 and Dpb2 (other subunits of Polε)

• Replication factors (PCNA, RFC complex)

• Chromatin remodelers and histones

Potential stress-specific interactors:

• DNA damage response proteins

• Stress-responsive transcription factors (potentially including CgRpn4 and CgXbp1 )

• Cell cycle regulators

Research in C. glabrata has shown that stress conditions significantly alter protein interactomes. For example, histone H4 displayed a substantially smaller interactome in MMS-treated cells . Similar dynamics might apply to DPB4, with its interaction network remodeling under stress conditions.

Methodologies to study these interactions include:

• Tandem affinity purification coupled with mass spectrometry

• Proximity labeling approaches (BioID, APEX)

• Yeast two-hybrid screening

• Co-immunoprecipitation under various stress conditions

How does histone H4 abundance regulation relate to DPB4 function in DNA damage response?

Research in C. glabrata has shown that histone H4 levels are tightly regulated during DNA damage response, with MMS exposure triggering significant downregulation of H4 transcript and protein levels . This regulation is linked to increased homologous recombination and improved survival.

Potential relationships between histone H4 regulation and DPB4 function include:

• DPB4, containing histone-fold domains, may interact directly with H4 or influence H4 deposition during replication and repair

• Reduced H4 levels during DNA damage may alter chromatin structure, affecting DPB4 access to DNA or recruitment of repair factors

• Both H4 and DPB4 may interact with common regulatory factors like CgCmr1, a WD40-repeat protein shown to interact with H4 and regulate HR and MMS stress survival

• Parallel regulatory pathways may exist where both H4 and DPB4 levels are modulated during stress response

Experimental approaches include ChIP-seq to examine co-localization patterns, genetic epistasis studies between H4 and DPB4, and proteomic analysis to identify shared interaction partners.