Recombinant Neosartorya fumigata Postreplication repair E3 ubiquitin-protein ligase rad18 (rad18)

What is Neosartorya fumigata and how does it relate taxonomically to Aspergillus species?

Neosartorya fumigata belongs to the Aspergillus section fumigati subgenus fumigati. Genetic-based methods have revealed that organisms phenotypically identified as Aspergillus fumigatus actually constitute a mold complex. While morphologically similar to A. fumigatus, Neosartorya species have distinct genetic profiles and growth characteristics. Multilocus DNA sequence analysis, particularly of the internal transcribed spacer 1 and 2 regions of ribosomal DNA (rDNA), β-tubulin, and rodlet A genes, can distinguish Neosartorya species from A. fumigatus sensu stricto . Under microscopy with lactophenol cotton-blue staining, Neosartorya udagawae (a related species) demonstrates morphologic features remarkably similar to A. fumigatus, highlighting the challenge in distinguishing these organisms without molecular techniques .

What is the fundamental role of RAD18 in postreplication repair pathways?

RAD18 plays a pivotal role in postreplication repair, which functions in gap-filling of daughter strands during replication of damaged DNA. In Saccharomyces cerevisiae, RAD18 works in conjunction with RAD6, a ubiquitin-conjugating enzyme (E2) . When DNA lesions induced by mutagens are not removed by base or nucleotide excision repair, or when the replication machinery encounters the lesion before repair occurs, replication stalls at the lesion. This results in gaps in newly synthesized strands across from damaged sites, which postreplication repair mechanisms must fill . RAD18's conserved ring-finger motif is essential for its interaction with RAD6/HR6 proteins, while its zinc-finger motif facilitates DNA binding, both critical for its function in DNA damage response .

How do RAD18 homologs differ across fungal species?

Homologs of RAD18 have been identified across various species with varying degrees of sequence conservation. The Neurospora crassa homolog, UVS-2, shares 25.5% amino acid identity with S. cerevisiae RAD18 and interacts with the RAD6 homolog MUS8 . Unlike the yeast rad18 mutant, the N. crassa uvs-2 mutant shows high mutation frequencies under both spontaneous and induced conditions, indicating functional divergence despite sequence similarity . The human homolog, hRAD18, maintains functional conservation with yeast RAD18 despite sequence divergence, suggesting core functional domains are maintained across evolutionary distance . While specific information about Neosartorya fumigata RAD18 is limited in the provided research, its function can be inferred to share similarities with these homologs based on the conservation of DNA repair mechanisms across fungal species.

What techniques are most effective for expressing and purifying recombinant Neosartorya fumigata RAD18?

Methodological approach for expression and purification of recombinant RAD18:

• Molecular cloning: Identify the RAD18 gene from Neosartorya fumigata genome using PCR with primers designed based on conserved regions across fungal RAD18 homologs.

• Expression system selection: Based on research with other RAD18 homologs, a bacterial expression system using E. coli BL21(DE3) with a pET vector containing a 6x-His tag for purification is often effective for initial characterization.

• Protein solubility optimization: RAD18 contains multiple domains including ring-finger and zinc-finger motifs that require proper folding. Expression at lower temperatures (16-18°C) after IPTG induction, and inclusion of zinc in the growth medium can improve proper folding of these domains.

• Purification strategy: A two-step purification using nickel affinity chromatography followed by size exclusion chromatography typically yields protein of sufficient purity for biochemical assays.

• Protein activity verification: After purification, verify E3 ligase activity through in vitro ubiquitination assays using recombinant RAD6/HR6 as the E2 enzyme partner.

This methodology draws from techniques used for human RAD18 expression, which demonstrated functional conservation with yeast homologs despite sequence divergence .

How can researchers effectively analyze RAD18 interaction with binding partners?

Multiple complementary techniques should be employed to characterize RAD18 interactions:

• Yeast two-hybrid assays: This approach successfully demonstrated the interaction between human RAD18 and HR6A/HR6B, and revealed that mutations in the ring-finger motif (C28F) abolished this interaction while mutations in the zinc-finger motif (C207F) did not .

• Co-immunoprecipitation: For validating interactions in fungal systems, epitope-tagged versions of RAD18 can be expressed in Neosartorya fumigata, and binding partners can be identified through mass spectrometry analysis of co-precipitated proteins.

• Biolayer interferometry or surface plasmon resonance: These techniques provide quantitative binding kinetics and can determine binding constants between purified RAD18 and its partners under various conditions (e.g., presence of damaged DNA).

• Fluorescence microscopy: Localization studies can reveal how RAD18 colocalizes with potential binding partners in response to DNA damage. As demonstrated with human RAD18, wild-type and zinc-finger mutants localized predominantly in the nucleus, while the ring-finger mutant showed different localization patterns .

What are the optimal experimental approaches for characterizing the functional domains of Neosartorya fumigata RAD18?

To characterize functional domains of Neosartorya fumigata RAD18, researchers should implement a systematic mutagenesis approach:

The combination of these approaches provides comprehensive insights into domain functionality while establishing structure-function relationships specific to Neosartorya fumigata RAD18.

How does RAD18 dysfunction affect genome stability and DNA damage response in filamentous fungi?

RAD18 dysfunction significantly impacts genome stability through multiple mechanisms:

• Increased mutation rates: In S. cerevisiae, rad18 mutations cause increased spontaneous mutation frequency, though they don't affect UV-induced mutagenesis . Interestingly, in N. crassa, the uvs-2 mutant (RAD18 homolog) shows high mutation frequencies under both spontaneous and induced conditions, suggesting divergent functions in different fungal species .

• Defective postreplication repair: When RAD18 is dysfunctional, cells cannot efficiently fill gaps in newly synthesized DNA strands opposite damaged templates, leading to persistent single-strand gaps that may be converted to double-strand breaks during subsequent replication .

• Hypersensitivity to DNA-damaging agents: Human cells expressing RAD18 with mutations in the ring-finger motif show increased sensitivity to UV, methyl methanesulfonate, and mitomycin C, and demonstrate defects in the replication of UV-damaged DNA . This phenotype is likely conserved in filamentous fungi including Neosartorya species.

• Altered DNA damage tolerance: Without functional RAD18, cells cannot properly activate the translesion synthesis pathway, which normally allows replication past DNA lesions using specialized polymerases, including RAD30 (Polη) .

For Neosartorya species specifically, genome stability is particularly relevant given their clinical importance. N. udagawae has been implicated in invasive aspergillosis with distinct clinical features compared to A. fumigatus infections, including longer disease duration and resistance to antifungal treatments . Understanding RAD18's role in maintaining genome stability could provide insights into pathogenicity mechanisms and adaptive responses to host environments.

What techniques can effectively identify and measure ubiquitination targets of RAD18 in fungal systems?

Researchers can employ several complementary approaches to identify and characterize RAD18's ubiquitination targets:

• Proteome-wide analysis:

  • Tandem affinity purification of ubiquitinated proteins followed by mass spectrometry

  • SILAC-based quantitative proteomics comparing wild-type and RAD18-deficient cells after DNA damage

  • Proximity-dependent biotin identification (BioID) with RAD18 as bait

• Candidate protein analysis:

  • In vitro ubiquitination assays with purified recombinant RAD18, RAD6/HR6, E1, ubiquitin, and candidate substrates

  • Site-directed mutagenesis of potential ubiquitination sites on candidate proteins

  • Western blotting with ubiquitin-specific antibodies after immunoprecipitation of target proteins

• Functional validation:

  • Creation of non-ubiquitinatable mutants of target proteins by lysine-to-arginine substitutions

  • Analysis of mutant phenotypes under DNA-damaging conditions

  • Rescue experiments with wild-type versus mutant proteins in RAD18-deficient backgrounds

The combination of these approaches provides robust identification and validation of RAD18 ubiquitination targets while establishing their functional significance in DNA damage response pathways.

How do Neosartorya species' RAD18-mediated repair mechanisms contribute to antifungal resistance?

The connection between RAD18-mediated DNA repair and antifungal resistance represents an important research frontier:

• Stress-induced mutagenesis: DNA repair deficiencies can alter mutation rates, which may accelerate the development of resistance mutations in stress conditions, such as exposure to antifungal agents. N. udagawae clinical isolates have demonstrated resistance to amphotericin B, itraconazole, and voriconazole compared with typical A. fumigatus isolates . This resistance correlates with the clinical observation that N. udagawae infections are more chronic and less responsive to therapy .

• Experimental approach to investigate this connection:

  • Generate RAD18 knockout or functionally impaired mutants in Neosartorya fumigata

  • Compare mutation rates and resistance development under antifungal pressure

  • Analyze genomic stability in resistant isolates

• Potential mechanisms:

  • RAD18 dysfunction may lead to error-prone repair, increasing genetic diversity and accelerating adaptive evolution

  • Alternatively, fully functional RAD18 may help maintain genomic integrity during antifungal stress, allowing cells to survive and develop resistance through other mechanisms

Evidence from clinical isolates supports potential connections between DNA repair and resistance. N. udagawae caused 4 (11%) of 36 invasive aspergillosis cases at one center over an 8-year period, with disease characteristics distinct from typical A. fumigatus infections . These N. udagawae infections were chronic (median duration 35 weeks vs 5.5 weeks for A. fumigatus), and antifungal susceptibility testing demonstrated that N. udagawae was relatively resistant to amphotericin B, itraconazole, and voriconazole .