Recombinant Ajellomyces capsulata DNA mismatch repair protein MSH3 (MSH3), partial

What is Ajellomyces capsulata and its relationship to Histoplasma capsulatum?

Ajellomyces capsulatus is the teleomorph (sexual form) of Histoplasma capsulatum, a dimorphic fungus that causes histoplasmosis. Histoplasma capsulatum exists in a filamentous mold form in the environment at temperatures below 35°C and transforms into a yeast form in tissues or when cultured at temperatures above 35°C using brain heart infusion agar or brain heart infusion with blood . This dimorphic nature is critical for researchers working with MSH3 in this organism, as the growth conditions significantly affect protein expression and function. The organism has three variants: var. capsulatum, var. duboissii, and var. farciminosum, with each potentially expressing MSH3 differently . Researchers must specify which variant they are working with to ensure experimental reproducibility across different laboratories.

Understanding this relationship is particularly important when designing expression systems for recombinant MSH3 protein, as the dimorphic nature of the organism may influence protein folding, post-translational modifications, and ultimately functional activity of the recombinant protein. Temperature-sensitive expression systems may be particularly valuable for studying this protein in its native context.

What is the structure and function of DNA mismatch repair protein MSH3 in eukaryotic systems?

MSH3 is a critical component of the DNA mismatch repair (MMR) system that maintains genomic stability. In eukaryotes, MSH3 forms a heterodimer called MutSβ with MSH2, which specializes in recognizing and initiating repair of insertion/deletion loops (IDLs) of 1-15 nucleotides and some base-base mispairs that can arise during DNA replication or recombination . The protein contains several functional domains, including a mismatch-binding domain (MBD) that directly interacts with DNA.

Based on homology modeling studies, the MBD of MSH3 likely adopts a fold similar to that of MSH6 and bacterial MutS, although with critical differences that account for its distinct substrate specificity . During DNA mismatch recognition, the MutSβ complex binds to DNA and induces a significant bend, with the MBD of MSH3 and part of the MBD of MSH2 inserting into the groove formed by this bend at the insertion/deletion loop site .

How does MSH3 contribute to genomic stability and what are the consequences of its dysfunction?

MSH3 contributes to genomic stability through several key mechanisms:

• Recognition and repair initiation for insertion/deletion loops (IDLs): As part of the MutSβ complex, MSH3 recognizes IDLs of 1-15 nucleotides that can occur during DNA replication, with particular efficiency for longer loops compared to the MutSα complex .

• Base-base mispair recognition: MutSβ can also recognize certain base-base mispairs, providing redundancy with the MutSα complex for some types of DNA damage .

• Microsatellite stability maintenance: By repairing IDLs, MSH3 helps prevent instability in repetitive DNA sequences, particularly in longer microsatellites. Deficiency in MSH3 is associated with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) .

The consequences of MSH3 dysfunction can be significant. Both loss of expression and overexpression can lead to genomic instability through different mechanisms. Loss of MSH3 function results in deficient repair of longer insertion/deletion loops, increasing mutation rates particularly in tetranucleotide repeats . This instability can contribute to carcinogenesis, with MSH3 deficiency being identified in approximately 50% of mismatch repair-deficient colorectal cancers .

Conversely, overexpression of MSH3 can disrupt the balance between MutSβ and MutSα complexes, as MSH3 sequesters MSH2 away from MSH6, leading to degradation of unpaired MSH6 proteins . This imbalance reduces repair efficiency for the more common short insertion/deletion loops and base-base mispairs typically handled by MutSα, potentially increasing mutation rates in these contexts .

What experimental approaches are commonly used to study MSH3 expression and localization?

Several complementary techniques are essential for accurately characterizing MSH3 expression and localization:

• Western blotting for protein quantification:

  • Using validated antibodies specific to MSH3

  • Including recombinant protein standards for absolute quantification

  • Normalizing to appropriate loading controls (housekeeping proteins)

  • Applying digital Western blotting for higher quantitative accuracy

• Immunofluorescence microscopy for localization:

  • Employing confocal microscopy for higher resolution localization

  • Performing z-stack imaging to capture the full cellular volume

  • Applying deconvolution to improve spatial resolution

  • Quantifying signal intensity in different cellular compartments

• Subcellular fractionation:

  • Separating nuclear, cytoplasmic, and other fractions using established protocols

  • Verifying fraction purity using compartment-specific markers

  • Quantifying MSH3 in each fraction relative to total cellular MSH3

• Live-cell imaging with fluorescent protein fusions:

  • Creating MSH3-GFP (or similar) fusion proteins, validating function is maintained

  • Using time-lapse imaging to track dynamic changes in localization

  • Applying photoactivatable or photoconvertible tags for pulse-chase experiments

Of particular importance is the finding that MSH3 can shuttle between the nucleus and cytoplasm in response to inflammatory signals, affecting its availability for nuclear DNA repair functions . This dynamic localization necessitates careful experimental design when studying MSH3, including controls for cell state and environmental conditions that might influence its cellular distribution.

How is MSH3 expression regulated in fungal and mammalian systems?

MSH3 expression regulation shows both common features and important differences between fungal and mammalian systems:

In mammalian systems, MSH3 is typically expressed at low levels across various tissues and cell types, suggesting it functions as a "housekeeping" gene. Expression has been detected in multiple tissues including spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes, heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas .

Several regulatory mechanisms affect MSH3 expression:

• Genomic context: In humans, the MSH3 gene is located upstream of the dihydrofolate reductase (DHFR) gene, and amplification of the DHFR gene (e.g., in response to methotrexate treatment) can lead to overexpression of MSH3 . This mechanism has been linked to drug resistance in cancer treatment.

• Transcriptional regulation: MSH3 expression is likely regulated by cell cycle-dependent factors and DNA damage response pathways, as is common for DNA repair genes.

• Post-translational regulation: MSH3 protein localization can be regulated through nuclear-cytoplasmic shuttling in response to inflammation, which affects its availability for nuclear DNA repair functions .

In fungal systems, including Ajellomyces capsulata, expression patterns may differ, particularly due to the dimorphic nature of this organism. Temperature-dependent regulation may be especially important given the different growth forms at different temperatures. The shift between yeast and filamentous forms likely involves global transcriptional reprogramming that could affect MSH3 expression.

Research on MSH3 expression specifically in Ajellomyces capsulata would require experimental investigation using techniques such as RT-qPCR, Western blotting, or reporter gene assays, with careful consideration of growth conditions that affect morphological state.

How do mutations in the mismatch-binding domain (MBD) of MSH3 affect its function in DNA repair?

Mutations in the MBD of MSH3 can have diverse effects on DNA repair function, with some key insights coming from studies in Saccharomyces cerevisiae:

Unlike MutS and Msh6, where mutation of a conserved phenylalanine residue severely compromises mismatch repair, mutation of the equivalent position in S. cerevisiae Msh3 (K158) caused only a modest MMR defect . This suggests fundamental differences in how MSH3 recognizes DNA mismatches compared to other MMR proteins.

Interestingly, combining the K158A mutation with K160A created a double mutant with a greater MMR defect than either single mutant alone and caused a loss of specificity for mispaired DNA . This synergistic effect highlights the cooperative nature of residues within the MBD for substrate recognition.

Among various conserved residues and predicted DNA-backbone-contacting residues in S. cerevisiae Msh3 that were mutated to alanine, only the R247A mutation caused a significant defect in the repair of 1-, 2-, and 4-nucleotide-long insertion/deletion mispairs . This pinpoints R247 as a critical residue for MSH3 function.

For researchers studying Ajellomyces capsulata MSH3, these findings suggest several approaches:

• Homology modeling based on known MSH3 structures would help identify potential critical residues in the MBD.

• Site-directed mutagenesis of these residues, followed by functional assays, would confirm their importance for MSH3 function.

• Combinatorial mutations may reveal synergistic effects that single mutations do not show.

• Comparative analysis with other fungal MSH3 proteins could highlight species-specific adaptations in the MBD.

The table below summarizes key mutations studied in S. cerevisiae Msh3 and their effects:

What are the implications of MSH3 shuttling between nucleus and cytoplasm for DNA repair efficiency?

The discovery that MSH3 can shuttle between the nucleus and cytoplasm in response to inflammatory signals has significant implications for DNA repair efficiency and genomic stability . This dynamic localization affects several aspects of MSH3 function:

• Nuclear MSH3 concentration: When MSH3 accumulates in the cytoplasm, its concentration in the nucleus decreases, potentially limiting the formation of functional MutSβ complexes where they are needed for DNA repair . This reduction in nuclear MSH3 creates a functional deficiency even when total cellular MSH3 levels remain unchanged.

• EMAST development: Reduced nuclear MSH3 has been associated with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) . This phenotype indicates compromised repair of specific types of DNA mismatches, particularly affecting longer repetitive sequences.

• Increased DNA damage: The shuttling and resulting reduction in nuclear MSH3 can lead to increased DNA damage accumulation, as repair of insertion/deletion loops becomes less efficient . This accumulated damage may contribute to genomic instability and mutagenesis.

• Inflammation-genomic instability connection: The link to inflammation suggests a mechanism by which inflammatory conditions might indirectly affect genomic stability through MSH3 mislocalization . This connection could be particularly relevant in diseases with chronic inflammation components.

For researchers studying Ajellomyces capsulata MSH3, it would be valuable to investigate whether similar shuttling occurs in this organism and under what conditions. This investigation could involve:

• Creating fluorescently tagged MSH3 constructs to track localization in living cells

• Examining MSH3 localization under various stress conditions relevant to fungal physiology

• Correlating MSH3 localization with measures of DNA repair efficiency

• Identifying the specific signals or modifications that regulate MSH3 localization

Understanding these dynamics could provide insights into how environmental or pathological conditions might affect DNA repair in Ajellomyces capsulata and potentially reveal novel regulatory mechanisms of the MMR system in fungi.

How does the interaction between MSH3 and MSH2 influence specificity for different types of DNA mismatches?

The interaction between MSH3 and MSH2 forming the MutSβ complex is crucial for determining which types of DNA mismatches are recognized and repaired. This heterodimerization creates a functional complex with unique substrate specificity distinct from the MutSα complex (MSH2-MSH6):

• Substrate specificity: MutSβ recognizes insertion/deletion loops (IDLs) of 1-15 nucleotides with particular efficiency for longer loops, while also recognizing some base-base mispairs . In contrast, MutSα specializes in base-base mispairs and short IDLs.

• Structural basis for recognition: During recognition of IDLs, DNA is severely bent, and the mismatch-binding domain of MSH3 and part of the mismatch-binding domain of MSH2 insert into the groove formed by this bend . This cooperative interaction between MSH3 and MSH2 enables recognition of specific DNA structures.

• Molecular recognition mechanism: Unlike MSH6, which uses a conserved phenylalanine residue to recognize base-base mispairs, MSH3 employs different key residues for substrate recognition . This fundamental difference in recognition mechanism contributes to the distinct substrate preferences of MutSβ versus MutSα.

• Balance between complexes: The relative levels of MutSβ and MutSα are critical for comprehensive mismatch repair. Overexpression of MSH3 leads to increased formation of MutSβ at the expense of MutSα, potentially reducing repair efficiency for base-base mispairs and short IDLs .

• Domain swapping evidence: Replacement of the Msh6 MBD with the Msh3 MBD generated a functional chimera possessing Msh3 substrate specificity, confirming that the MBD is the primary determinant of substrate preference .

For researchers studying Ajellomyces capsulata MSH3-MSH2 interaction, several approaches would be valuable:

• Co-immunoprecipitation or yeast two-hybrid assays to confirm and characterize the interaction

• In vitro binding assays with various mismatched DNA substrates to determine specificity

• Structure-function studies using chimeric proteins to identify specificity-determining regions

• Analysis of the relative expression levels of MSH3 and MSH2 under different conditions

The unique properties of the MSH3-MSH2 interaction highlight the specialized role of MutSβ in maintaining genomic stability through recognition of specific DNA lesions that might otherwise escape repair.

What experimental approaches can be used to study the kinetics of MSH3-dependent DNA repair?

Studying the kinetics of MSH3-dependent DNA repair requires sophisticated experimental approaches that can capture both biochemical and cellular aspects of the repair process:

• In vitro biochemical assays:

  • Gel shift assays with purified recombinant MSH3 and MSH2 proteins to measure binding kinetics to various DNA substrates

  • ATPase assays to determine the rate of ATP hydrolysis during the repair process

  • Reconstituted repair assays with purified components to measure complete repair reaction kinetics

  • Surface plasmon resonance (SPR) or bio-layer interferometry (BLI) to measure real-time binding and dissociation rates

• Cellular assays:

  • Fluorescence recovery after photobleaching (FRAP) to measure the dynamics of fluorescently tagged MSH3 in living cells

  • DNA damage induction followed by time-course sampling and repair quantification

  • Pulsed-field gel electrophoresis to monitor repair of specific lesions over time

  • Comet assays to measure DNA damage resolution kinetics in single cells

• Advanced imaging techniques:

  • Single-molecule techniques to track individual repair events in real-time

  • Super-resolution microscopy to visualize repair complex assembly and progression

  • FRET-based sensors to detect conformational changes during repair

  • Live-cell imaging with damage-specific fluorescent probes

• Genetic approaches:

  • Creation of temperature-sensitive MSH3 mutants for rapid inactivation studies

  • Inducible expression systems to determine how changing MSH3 levels affect repair kinetics

  • Site-directed mutagenesis of key domains to identify rate-limiting steps

• Mathematical modeling:

  • Kinetic modeling based on experimental data to understand rate-limiting steps

  • Systems biology approaches to integrate multiple levels of regulation

  • Stochastic modeling to account for cell-to-cell variability in repair efficiency

Data from these approaches should be integrated to develop a comprehensive model of MSH3-dependent repair kinetics. When studying Ajellomyces capsulata MSH3, researchers should consider the dimorphic nature of the organism and how temperature and morphological changes might affect repair kinetics. Temperature-controlled experiments are particularly important to distinguish between direct kinetic effects and indirect effects due to morphological changes.

How does MSH3 function differ between fungal and mammalian systems?

Comparing MSH3 function between fungal systems like Ajellomyces capsulata and mammalian systems reveals important similarities and differences that reflect evolutionary adaptations to different cellular environments:

• Sequence and structural variations:

  • While the core MMR machinery is conserved across eukaryotes, fungal MSH3 proteins show distinct sequence features compared to their mammalian counterparts

  • Homology modeling studies highlight that key functional residues may differ between species, affecting substrate specificity and protein interactions

  • Fungal MSH3 proteins may have adapted to recognize specific types of DNA damage common in their genomic context

• Regulation and expression:

  • Mammalian MSH3 is expressed at low levels across many tissues as a "housekeeping" gene

  • In humans, MSH3 expression can be influenced by amplification of the nearby DHFR gene

  • Fungal MSH3 expression may be more tightly linked to cell cycle and growth conditions

  • The dimorphic nature of Ajellomyces capsulata likely necessitates specialized regulation of DNA repair systems during morphological transitions

• Protein interactions and complex formation:

  • The fundamental MSH2-MSH3 (MutSβ) interaction is conserved across eukaryotes

  • Fungal systems may have evolved specific interacting partners reflecting their unique DNA repair requirements

  • The relative importance of MutSβ versus MutSα may differ between fungi and mammals

• Subcellular localization:

  • Mammalian MSH3 can shuttle between nucleus and cytoplasm in response to inflammation

  • Whether similar shuttling occurs in fungal systems and what signals might regulate it remains to be determined

  • Temperature-dependent localization could be especially relevant in dimorphic fungi

• Role in genome maintenance:

  • In mammals, MSH3 deficiency is associated with cancer development, particularly colorectal cancer

  • In fungi, the consequences of MSH3 deficiency may manifest differently, potentially affecting adaptation to environmental stresses

  • The higher mutation rates in some fungi may be reflected in specialized functions of their MMR machinery

For researchers, these differences highlight the importance of species-specific studies rather than simply extrapolating findings across evolutionary distant organisms. Understanding the unique aspects of fungal MSH3 function could provide insights into fungal genome evolution and potentially reveal novel aspects of DNA repair mechanisms not evident in mammalian systems.

How should researchers interpret changes in microsatellite stability in relation to MSH3 function?

Interpreting changes in microsatellite stability requires careful consideration of MSH3's specific role in DNA mismatch repair and the distinct patterns of instability associated with deficiencies in different MMR proteins:

• Specificity of microsatellite alterations:

  • MSH3 deficiency is particularly associated with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST)

  • Changes in dinucleotide microsatellites are more commonly associated with MSH2/MSH6 deficiencies

  • This specificity reflects the substrate preferences of MutSβ (MSH2-MSH3) for longer insertion/deletion loops

• Quantitative assessment:

  • The frequency of microsatellite alterations should be quantified across multiple loci

  • Statistical comparison to appropriate controls is essential

  • The degree of instability may correlate with the severity of MSH3 dysfunction

• Correlation with MSH3 status:

  • Microsatellite instability should be correlated with MSH3 expression levels

  • Nuclear versus cytoplasmic localization of MSH3 should be considered, as cytoplasmic accumulation can reduce nuclear repair activity

  • The status of other MMR proteins (especially MSH2) should be assessed in parallel

The table below provides guidance for interpreting microsatellite instability patterns in relation to different MMR protein deficiencies:

When conducting microsatellite stability studies in Ajellomyces capsulata, researchers should:

• Select appropriate microsatellite markers, emphasizing tetranucleotide repeats

• Include both wild-type and known MMR-deficient controls

• Consider the growth phase and morphological state of the organism

• Correlate microsatellite stability with functional assays of MMR activity

• Account for potential environmental influences on MSH3 localization and function

This integrated approach will provide a more comprehensive understanding of how MSH3 function affects genomic stability in this fungal system.

What statistical approaches are most appropriate for analyzing MSH3 mutation data?

Analyzing MSH3 mutation data requires careful selection of statistical methods appropriate for the specific experimental questions and data types:

• Mutation frequency analysis:

  • Fisher's exact test or chi-square test for comparing mutation frequencies between experimental groups

  • Poisson distribution models for rare mutation events

  • Confidence intervals for mutation frequencies to account for sampling variability

  • Multinomial logistic regression for comparing multiple mutation types simultaneously

• Mutational spectrum analysis:

  • Multiple sequence alignment to identify conserved versus variable regions

  • Clustering algorithms to identify mutation hotspots

  • Shannon entropy calculations to quantify the diversity of mutations at specific positions

  • Principal component analysis to visualize relationships between different mutation patterns

• Structure-function correlations:

  • Regression models to correlate mutation positions with functional outcomes

  • ANOVA for comparing functional effects of different mutation categories

  • Bayesian approaches to incorporate prior knowledge about protein structure

  • Multivariate analysis for complex datasets with multiple outcome measures

• Evolutionary analysis:

  • Ka/Ks ratio analysis to detect selective pressure on different regions of MSH3

  • Phylogenetic methods to trace the evolution of specific mutations across species

  • Ancestral sequence reconstruction to infer the functional importance of specific residues

• Sample size and power considerations:

  • Power analysis to determine adequate sample size for detecting mutations of different effect sizes

  • Correction for multiple testing when screening many potential mutation sites

  • Meta-analysis approaches when combining data from multiple studies

When reporting results, researchers should clearly state:

• The null hypothesis being tested

• The chosen significance level (typically α = 0.05)

• Whether corrections for multiple comparisons were applied

• Effect sizes in addition to p-values

• Confidence intervals where appropriate

For Ajellomyces capsulata MSH3 specifically, researchers should consider the genomic context and potential strain variations when interpreting mutation data. Comparing mutation patterns between different morphological states (yeast versus filamentous) may also provide insights into environment-specific selection pressures on MSH3 function.

How can homology modeling be used to predict the functional impact of novel MSH3 mutations?

Homology modeling provides a powerful approach for predicting the functional impact of novel MSH3 mutations, especially when experimental structural data is limited. Based on previous studies of MSH3 , a systematic workflow can be developed:

• Template selection and alignment:

  • Identify suitable template structures (e.g., human MSH6 MBD has been used as a template for modeling MSH3 MBD)

  • Perform careful sequence alignment between Ajellomyces capsulata MSH3 and the template

  • Pay special attention to conserved functional domains and motifs

• Model building and refinement:

  • Generate multiple models using software like MODELLER, Rosetta, or I-TASSER

  • Refine models using energy minimization and molecular dynamics

  • Validate model quality using tools like PROCHECK or MolProbity

• Identification of functionally important regions:

  • Analyze the model to identify residues at protein-protein interfaces (e.g., MSH3-MSH2 interaction)

  • Identify residues at the DNA-binding interface

  • Locate residues involved in ATPase activity or conformational changes

• Mutation impact prediction:

  • Introduce mutations in silico and assess local structural changes

  • Calculate stability changes (ΔΔG) using tools like FoldX or Rosetta

  • Analyze electrostatic and hydrophobic property changes

The table below outlines a systematic prediction workflow for novel mutations:

Previous studies have successfully used homology modeling to identify critical residues in the Msh3 MBD that mediate mispair recognition . For instance, modeling identified that while the K158 residue in Saccharomyces cerevisiae Msh3 (equivalent to a critical phenylalanine in Msh6) was not essential individually, combining the K158A mutation with K160A created a significant functional defect .

For Ajellomyces capsulata MSH3, researchers should validate model predictions with experimental approaches such as site-directed mutagenesis and functional assays. This iterative process of modeling, prediction, and experimental validation provides the most robust approach for understanding the structural basis of MSH3 function.