Recombinant Aspergillus niger DNA mismatch repair protein msh3 (msh3), partial

What is the role of MSH3 in fungal DNA mismatch repair?

MSH3 in Aspergillus species functions as an essential component of the highly conserved DNA mismatch repair (MMR) system that maintains genomic stability. In eukaryotes, including fungi, repair is initiated by heterodimeric MutS homolog (Msh) complexes that form sliding clamps on DNA to scan for nucleotide misincorporation events and extrahelical lesions introduced during DNA replication and homologous recombination . Specifically, MSH3 partners with MSH2 to form the MutSβ heteroduplex that primarily recognizes insertion-deletion loops (IDLs) involving one or more unpaired nucleotides and some single-nucleotide mismatches . After lesion recognition, MutL homolog (Mlh) complexes are recruited to the repair site, where they harbor a mismatch-dependent endonuclease activity critical for MMR . This process is followed by exonuclease activity and DNA synthesis to complete the repair.

How does A. niger MSH3 compare structurally with homologs in other organisms?

While the specific structure of A. niger MSH3 has not been completely characterized, comparative analysis with well-studied homologs provides significant insights. Based on human MSH3 data, the protein likely contains conserved DNA binding domains, ATPase domains, and protein interaction regions essential for forming the heterodimer with MSH2 . The human MSH3 protein has a calculated molecular weight of approximately 40.3 kDa and forms a functional MutSβ complex that initiates mismatch repair by binding to a mismatch before forming a complex with MutLα heterodimer . In Aspergillus fumigatus, the MSH2 homolog (mshA) shows significant variation among strains, with 12 strains out of 62 (18.2%) exhibiting variants, suggesting that similar structural diversity might exist in A. niger MSH3 . This structural diversity could contribute to functional differences in repair efficiency and potentially impact phenotypes like antifungal resistance.

What experimental evidence suggests MSH3's importance in genomic stability?

Research in various organisms demonstrates MSH3's critical role in genomic stability. In colorectal cancer studies, MSH3-deficient cells show increased sensitivity to the irinotecan metabolite SN-38 and to oxaliplatin, indicating its role in responding to specific types of DNA damage . These MSH3-deficient cells exhibit higher levels of phosphorylated histone H2AX and Chk2, markers of DNA damage response activation . Additionally, studies in A. fumigatus revealed that mshA null mutants (MSH2 homolog) could influence virulence in both neutropenic murine models of invasive pulmonary aspergillosis and in the moth Galleria mellonella . These findings collectively suggest that MSH3 in A. niger likely plays a similar crucial role in maintaining genomic integrity and may influence pathogenicity and stress responses.

What expression systems are optimal for recombinant A. niger MSH3 production?

For optimal expression of recombinant A. niger MSH3, multiple expression systems should be considered based on experimental requirements:

For E. coli expression systems, fusion tags like His-tag can facilitate purification, as demonstrated with human MSH3 recombinant protein . Expression at lower temperatures (16-18°C) after induction can improve solubility. Based on protocols for similar recombinant proteins, the pET expression system combined with a His-tag for purification represents a good starting point .

What purification strategy yields highest purity and functional protein?

A multi-step purification strategy is essential for obtaining high-purity, functional recombinant A. niger MSH3:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein, with optimized imidazole gradients to minimize non-specific binding .

• Intermediate purification: Ion exchange chromatography (typically anion exchange at pH 8.0) to separate based on charge differences and remove nucleic acid contamination.

• Polishing step: Size exclusion chromatography to achieve final purity and confirm proper oligomeric state.

• Buffer optimization: Final buffer composition significantly impacts stability - typically 20-50 mM Tris-HCl pH 7.5-8.0, 100-200 mM NaCl, 1-5 mM DTT or 0.5-1 mM TCEP, and 5-10% glycerol for storage.

For human MSH3 recombinant protein, lyophilization from sterile PBS (pH 7.4) with 5% trehalose, 5% mannitol, and 0.25M Arginine has proven effective for storage stability . Regular quality control testing should include SDS-PAGE, western blotting, and functional DNA binding assays to confirm protein integrity.

How can researchers verify functional activity of purified MSH3?

Functional validation of purified recombinant A. niger MSH3 requires multiple complementary approaches:

When studying MSH3-DNA interactions, researchers should use DNA substrates containing specific mismatches or insertion-deletion loops (IDLs) . It's also crucial to test MSH3 in complex with MSH2 to form the functional MutSβ complex for complete activity assessment, as MSH3 forms a heterodimer with MSH2 to initiate mismatch repair .

How does MSH3 function influence antifungal resistance development?

Research with A. fumigatus provides compelling evidence that mismatch repair proteins like MSH3 can significantly impact antifungal resistance development. Studies demonstrated that loss of mshA (the MSH2 homolog) function can provide increased azole resistance when selected for . The mechanism likely involves increased genomic instability and mutation rates in the absence of proper mismatch repair, accelerating the development of resistance-conferring mutations.

To investigate MSH3's role in A. niger antifungal resistance, researchers should:

• Generate MSH3 knockout or knockdown strains using CRISPR-Cas9 or RNAi techniques

• Perform susceptibility testing against multiple antifungal classes (azoles, echinocandins, polyenes)

• Conduct evolution experiments with sub-inhibitory antifungal concentrations

• Analyze mutation spectra and rates in wild-type versus MSH3-deficient strains

• Sequence resistant isolates to identify resistance-conferring mutations

The finding that A. fumigatus mshA null mutants demonstrated enhanced virulence attributes after sequential mitotic passages suggests that similar phenotypic evolution might occur with A. niger MSH3 mutants .

What role does MSH3 play in fungal virulence and pathogenicity?

The connection between mismatch repair deficiency and virulence has been established in A. fumigatus, where researchers demonstrated that mshA null mutants influenced virulence in both a neutropenic murine model of invasive pulmonary aspergillosis and in the moth Galleria mellonella . Different populations of the null mshA mutants grown through 10 sequential mitotic passages evolved virulence attributes in the G. mellonella model of infection .

For A. niger MSH3, researchers investigating its role in virulence should consider:

• Creating targeted MSH3 gene deletions or mutations

• Evaluating phenotypic changes in stress resistance (oxidative, pH, temperature)

• Assessing adhesion to host cells and tissues

• Measuring secretion of hydrolytic enzymes and secondary metabolites

• Conducting in vivo virulence studies in appropriate animal models

The hypermutator phenotype typically associated with mismatch repair deficiency may allow more rapid adaptation to host environments, potentially enhancing virulence through accelerated evolution.

How can site-directed mutagenesis reveal MSH3 structure-function relationships?

Site-directed mutagenesis represents a powerful approach to dissect the functional domains of A. niger MSH3. Based on conserved domains in homologous proteins, researchers should target:

When designing mutagenesis experiments, researchers should refer to the amino acid sequence of homologous proteins like human MSH3, which has been well-characterized . Single amino acid substitutions can provide insights into critical residues, while domain swapping or deletion constructs can reveal the function of larger protein regions. The systematic mutational analysis should progress from conserved motifs to less conserved regions to build a comprehensive structure-function map.

What are the most effective methods for studying MSH3-DNA interactions?

Several complementary techniques can effectively characterize MSH3-DNA interactions:

For initial characterization, EMSA using fluorescently labeled oligonucleotides containing specific mismatches or IDLs provides a straightforward approach . For quantitative studies, SPR or fluorescence anisotropy can determine binding constants. To reconstitute the complete mismatch recognition process, it's crucial to include both MSH3 and MSH2 to form the functional MutSβ complex, as MSH3 forms a heterodimer with MSH2 to initiate mismatch repair .

How should researchers troubleshoot expression and purification challenges?

Common challenges in recombinant MSH3 production and their solutions include:

• Poor expression levels:

  • Optimize codon usage for the expression host

  • Try different promoters (T7, tac, AOX1)

  • Adjust induction conditions (temperature, inducer concentration)

  • Consider autoinduction media for E. coli expression

• Protein insolubility:

  • Lower expression temperature (16-18°C)

  • Use solubility-enhancing tags (MBP, SUMO, TrxA)

  • Add solubility enhancers to lysis buffer (0.1% Triton X-100, 5-10% glycerol)

  • Try co-expression with MSH2 partner

• Degradation during purification:

  • Include protease inhibitor cocktail

  • Maintain samples at 4°C throughout purification

  • Minimize purification time with optimized protocols

  • Add stabilizing agents (ATP, ADP, DNA oligonucleotides)

• Loss of activity:

  • Verify proper folding by circular dichroism

  • Optimize buffer conditions with thermal shift assays

  • Test different storage conditions (glycerol percentage, flash freezing vs. slow cooling)

  • Consider storage with stabilizing ligands

For MSH3 specifically, the formation of a complex with MSH2 is critical for its natural function and may enhance stability . Purification protocols similar to those used for human recombinant MSH3 protein, which include lyophilization from sterile PBS with stabilizing agents like trehalose and mannitol, may be adapted for A. niger MSH3 .

What experimental design is optimal for studying MSH3's role in genomic stability?

To comprehensively investigate A. niger MSH3's role in genomic stability, a multi-faceted experimental approach is required:

• Mutation rate analysis:

  • Fluctuation analysis to determine spontaneous mutation rates

  • Specific reporter systems for different mutation types (point mutations, insertions/deletions)

  • Whole-genome sequencing to determine mutation spectra

• DNA damage response:

  • Sensitivity assays to different DNA-damaging agents

  • Immunofluorescence for DNA damage markers (γH2AX)

  • Analysis of checkpoint activation (Chk1/Chk2 phosphorylation)

• Genetic interaction studies:

  • Double mutant analysis with other DNA repair pathways

  • Synthetic genetic arrays to identify functionally related genes

  • High-throughput drug sensitivity profiling

• In vitro reconstitution:

  • Biochemical assays with purified components

  • Single-molecule approaches to study repair dynamics

  • Structure determination of protein-DNA complexes

When designing these experiments, appropriate controls are essential. For knockout studies, complementation with wild-type MSH3 should restore normal phenotypes. Time-course studies can reveal the kinetics of mutation accumulation in MSH3-deficient strains. Comparative analysis with MSH3 mutants from other fungal species, such as the well-studied A. fumigatus mshA mutants that showed increased azole resistance and virulence, can provide valuable context .

How might CRISPR-Cas9 technology advance MSH3 research in A. niger?

CRISPR-Cas9 technology offers revolutionary approaches for studying A. niger MSH3 function:

• Precise genome editing:

  • Introduction of specific point mutations at endogenous locus

  • Domain deletions or swaps to assess functional regions

  • Tagged versions for localization and interaction studies

• High-throughput functional screening:

  • CRISPR interference (CRISPRi) for conditional knockdown

  • CRISPR activation (CRISPRa) for overexpression studies

  • CRISPR-Cas9 screens with guide RNA libraries for genetic interactions

• Mechanistic studies:

  • CRISPR base editors for introducing specific mismatches

  • CRISPR-X for targeted mutagenesis of specific genomic regions

  • CRISPR-mediated fluorescent tagging for live-cell imaging

For A. niger specifically, optimization of CRISPR-Cas9 protocols should focus on efficient transformation methods, appropriate promoters for guide RNA and Cas9 expression, and careful selection of target sites to minimize off-target effects.

What comparative genomics approaches could reveal MSH3 evolution in fungi?

Comparative genomics can provide insights into MSH3 evolution and function across fungal species:

• Phylogenetic analysis:

  • Construct phylogenetic trees of MSH3 across diverse fungi

  • Identify lineage-specific adaptations and selection pressures

  • Correlate MSH3 sequence variations with ecological niches

• Structural comparisons:

  • Identify conserved domains and critical residues

  • Model species-specific structural differences

  • Predict functional differences based on structural variations

• Population genomics:

  • Analyze natural variation in MSH3 within A. niger populations

  • Identify potential adaptive mutations

  • Correlate genotypic variation with phenotypic differences

The observation that 18.2% of A. fumigatus strains examined showed variants in the mshA gene suggests significant natural variation might also exist in A. niger MSH3 . This variation could contribute to differences in mutation rates, DNA repair efficiency, and potentially antifungal susceptibility among different strains.