Recombinant Drosophila virilis MAU2 chromatid cohesion factor homolog (GJ10962), partial

What is the function of MAU2 chromatid cohesion factor homolog in Drosophila virilis?

MAU2 is a critical component of the kollerin complex that regulates sister chromatid cohesion. It forms a heterodimeric complex with NIPBL (Nipped-B-like protein) to mediate the loading of cohesin onto chromosomes. In Drosophila virilis, MAU2 plays essential roles in chromosome dynamics during both mitosis and meiosis. Research has demonstrated that MAU2 interacts with NIPBL through its tetratricopeptide repeat (TPR) domain to perform its biological functions . This interaction is crucial for maintaining genomic stability during cell division, as proper chromatid cohesion prevents chromosome segregation errors and subsequent genomic instability. Functional studies indicate that MAU2 is more than just a passive stabilizer of NIPBL; it actively participates in the mechanoregulatory functions of the kollerin complex.

How is recombinant Drosophila virilis MAU2 typically expressed and purified?

Recombinant MAU2 from Drosophila virilis can be expressed in multiple host systems, each offering different advantages:

For initial structural and functional studies, E. coli expression is often preferred due to its simplicity and high yield . For functional assays requiring proper protein folding and activity, insect cell expression using baculovirus vectors may be more appropriate. The purification process typically involves affinity chromatography (using His-tag, FLAG-tag, or GST-tag), followed by size exclusion and/or ion exchange chromatography to achieve high purity.

What is the relationship between MAU2 and NIPBL in Drosophila species?

MAU2 and NIPBL form a heterodimeric complex called kollerin that is essential for loading cohesin onto chromosomes. Their relationship is characterized by:

• Structural interdependence: MAU2 envelops the N-terminus of NIPBL through its TPR array domain

• Protein stability regulation: RNA interference experiments have demonstrated that depletion of NIPBL greatly reduces cellular levels of MAU2

• Functional cooperation: The complex acts as a cohesin loader with both proteins contributing to the process

Research using in silico analysis of the co-crystal structure of the S. cerevisiae cohesin loader complex (Scc2/NIPBL-Scc4/MAU2) has revealed detailed molecular interactions between these proteins. Even small deletions in MAU2 can significantly impair the MAU2-NIPBL interaction. In quantitative mammalian two-hybrid interaction assays, a seven amino acid deletion in MAU2 reduced heterodimerization activity to 46% compared to wild type . This highlights the precise molecular requirements for proper complex formation and function.

How does recombinant MAU2 expression differ across Drosophila species?

Comparative analysis across Drosophila species reveals conservation of MAU2 with species-specific variations:

Recombinant expression protocols need to account for these species-specific characteristics, particularly when conducting comparative functional studies. The phylogenetic relationships within the virilis group, which diversified approximately 9 million years ago , provide a framework for understanding the evolution of MAU2 structure and function across species.

What expression vectors are most effective for recombinant Drosophila virilis MAU2?

For optimal expression of recombinant D. virilis MAU2, several vector systems have proven effective:

• Bacterial expression: pET vectors with T7 promoter systems provide high-level expression in E. coli. The pET28a vector with an N-terminal His-tag facilitates purification while minimizing interference with protein function.

• Insect cell expression: The pFastBac vector system with Baculovirus expression is highly effective for obtaining properly folded MAU2 with post-translational modifications. This system is particularly valuable when studying MAU2-NIPBL interactions that depend on proper protein folding.

• Mammalian expression: pcDNA3.1-FLAG-N has been successfully used for MAU2 expression in mammalian cells, as evidenced in studies examining MAU2-NIPBL interactions .

When designing cloning strategies, researchers should consider:

• Inclusion of optimal Kozak sequences for efficient translation initiation

• Codon optimization for the host expression system

• Careful selection of fusion tags that minimize interference with protein function

• Incorporation of protease cleavage sites for tag removal

A typical cloning strategy involves PCR amplification with high-fidelity polymerase (such as Phanta® Max Super-Fidelity DNA Polymerase) and one-step cloning kits (like ClonExpress® II) .

How does MAU2 contribute to meiotic recombination regulation in Drosophila virilis?

MAU2's role in meiotic recombination in D. virilis appears to be connected to chromosome cohesion and the regulation of double-strand break (DSB) repair. During hybrid dysgenesis in D. virilis, which involves the activation of transposable elements, the meiotic recombination landscape remains surprisingly robust despite increased DNA damage .

Research findings indicate:

• MAU2 may function in the selection of DSB repair pathways, influencing whether breaks are repaired as crossovers or non-crossovers

• The interaction between MAU2 and other recombination factors could be critical during periods of genomic stress, such as hybrid dysgenesis

• MAU2's function in chromosome cohesion provides structural support for the recombination machinery

Interestingly, while meiotic recombination remains stable during hybrid dysgenesis, mitotic recombination increases significantly . This suggests that the regulation of meiotic recombination, potentially involving MAU2, is robust to germline DNA damage, whereas mitotic recombination mechanisms may be more sensitive to transposon-induced double-strand breaks.

What molecular techniques are most effective for analyzing MAU2-NIPBL interactions in Drosophila virilis?

Several complementary approaches have proven effective for studying MAU2-NIPBL interactions:

• Mammalian two-hybrid assays: Quantitative assessment of protein-protein interactions using constructs encoding NIPBL fragments coupled to DNA binding domains and MAU2 (wild-type or mutant) bound to activation domains

• Yeast two-hybrid assays: Provides an alternative system for measuring interaction strength, with slower growth on selective medium indicating weaker interactions

• Co-immunoprecipitation: Detects native complexes in D. virilis cells or tissues

• Molecular dynamics (MD) simulations: Utilizes software like Gromacs 5.14 with molecular force fields (Gromos53a6) to analyze the interaction dynamics between MAU2 and NIPBL over time (typically 50 ns simulations)

• Structural prediction and analysis: AlphaFold2 and Rosetta software can predict protein structures and binding modes, with visualization through PyMOL v2.5, VMD, and Origin v8.5

In studies of mutations affecting MAU2-NIPBL interactions, point mutations can be introduced using mutagenesis kits (such as Mut Express® MultiS Fast Mutagenesis Kit V2) followed by transfection into cells (like human 293T cells) for functional assessment .

How can CRISPR-Cas9 be used to study MAU2 function in Drosophila virilis?

CRISPR-Cas9 genome editing offers powerful approaches for investigating MAU2 function in D. virilis:

• Knockout studies: Complete gene deletion to assess null phenotypes, revealing essential functions in development and chromosome dynamics

• Precise mutations: Introduction of specific mutations to mimic those found in conditions like Cornelia de Lange syndrome (CdLS) to study functional consequences

• Domain analysis: Targeted modification of specific protein domains (such as the TPR array) to assess their contribution to MAU2-NIPBL interaction and cohesin loading

• Endogenous tagging: Insertion of fluorescent or affinity tags at the endogenous locus for live imaging or biochemical studies

A methodological approach includes:

• Design of guide RNAs targeting specific regions of the MAU2 gene

• Creation of donor templates containing desired modifications with appropriate homology arms

• Microinjection into D. virilis embryos

• Screening for successful editing events

• Establishment of stable lines

• Phenotypic and molecular characterization

CRISPR editing in D. virilis has been successfully implemented, though with lower efficiency than in D. melanogaster, requiring optimization of injection mix components and screening approaches.

How do mutations in MAU2 affect chromosome cohesion and recombination in Drosophila virilis?

Mutations in MAU2 can have significant impacts on chromosome cohesion and recombination, with effects dependent on the nature and location of the mutation:

The functional consequences of MAU2 mutations highlight the complex regulatory networks governing chromosome dynamics during cell division. Further studies using precise genome editing approaches will help elucidate the specific roles of different MAU2 domains in these processes.

What role does MAU2 play in the hybrid dysgenesis phenomenon in Drosophila virilis?

The hybrid dysgenesis syndrome in D. virilis provides an excellent model for understanding how MAU2 functions under genomic stress conditions:

Hybrid dysgenesis in D. virilis occurs when males with multiple active transposable element (TE) families fertilize females lacking active copies of the same families . This results in:

• Germline activation of diverse transposable elements

• Reduced fertility

• Male recombination (normally absent in Drosophila)

• Occasional gonadal atrophy driven by germline stem cell death

While MAU2's exact role hasn't been directly characterized in this phenomenon, several connections can be inferred:

• Chromosome stability maintenance: MAU2's function in cohesin loading may become critical when transposable elements create DNA damage and chromosomal rearrangements

• Recombination regulation: During hybrid dysgenesis, meiotic recombination patterns remain remarkably stable despite increased DNA damage , suggesting robust regulatory mechanisms potentially involving MAU2

• Mitotic recombination: Hybrid dysgenesis increases transmission of chromosomes with mitotic recombination , which might involve altered MAU2 function during early development when transposons are most active

The most intriguing finding is that despite the genomic upheaval of hybrid dysgenesis, the landscape of meiotic recombination appears robust . This suggests that either transposition is ameliorated in the adult female germline or that the regulation of meiotic recombination (potentially involving MAU2) is resilient to ongoing transposition.

How does the evolutionary history of MAU2 in the Drosophila virilis species group inform functional studies?

The evolutionary history of MAU2 in the D. virilis species group provides valuable context for functional studies:

The D. virilis group diverged approximately 9 million years ago and consists of three subgroups: the virilis, montana, and littoralis phylads . This evolutionary framework offers opportunities to examine how MAU2 function has been conserved or diverged:

• Sequence evolution: Comparative genomics approaches can identify conserved domains critical for MAU2 function across the virilis group

• Functional divergence: The virilis group shows variation in recombination rates, with D. virilis having significantly higher recombination rates than D. melanogaster , suggesting potential differences in cohesin regulation

• Adaptation to transposon dynamics: The virilis group species differ in their transposable element profiles , potentially driving adaptive evolution in genes like MAU2 that maintain chromosome stability

• Natural selection signatures: Analysis of molecular evolution at meiosis genes across Drosophila species groups (melanogaster, obscura, and virilis) has revealed recurrent positive selection at several meiosis genes , providing a framework for examining selection on MAU2

The virilis species group originated in East Asia and spread into North America via Beringia, with members of each phylad present in both Nearctic and Palearctic regions . This biogeographic history may have influenced the evolution of MAU2 function in response to different environmental challenges.

What structural features of MAU2 are critical for its function in Drosophila virilis?

The structural analysis of MAU2 reveals several critical features essential for its function:

• TPR (tetratricopeptide repeat) array: This domain forms a structure that envelops the N-terminus of NIPBL . Deletion of even seven amino acids from a helix within this array can significantly impair interaction with NIPBL

• N-terminal region: Based on structural homology with the yeast Scc4/MAU2 protein, this region is likely critical for initial complex formation with NIPBL/Scc2

• Protein-protein interaction surfaces: Specific helices directly contact NIPBL's N-terminus, with mutations in these regions potentially leading to distortion of the MAU2-NIPBL interaction

Molecular dynamics simulations using the Gromacs 5.14 software with the Gromos53a6 molecular force field have been employed to analyze the structural dynamics of MAU2-NIPBL interactions . These simulations typically run for around 50 ns and use methods like:

• Particle Mesh Ewald for calculating electrostatic interactions

• Leapfrog algorithm for calculating atomic motion

• Steepest descent energy method for energy minimization

• 50 ps position constraint simulation for each simulation system

Visualization and analysis tools like PyMOL v2.5, VMD, and Origin v8.5 can generate graphical representations of these structural features and their dynamics .

What are the best approaches for studying MAU2 function in meiotic versus mitotic recombination in Drosophila virilis?

Different methodological approaches are optimal for investigating MAU2's role in meiotic versus mitotic recombination:

For meiotic recombination:

• Fine-scale genetic mapping: Using multiplexed shotgun genotyping to map crossover events provides high-resolution data on recombination landscapes

• Cytological techniques: Immunofluorescence microscopy with antibodies against MAU2, NIPBL, and recombination proteins (e.g., RAD51) can visualize their dynamic localization during meiosis

• Tetrad analysis: Though challenging in Drosophila, this approach can provide comprehensive data on both crossover and non-crossover events

For mitotic recombination:

• Twin-spot analysis: This genetic method can detect mitotic recombination events in developing tissues

• Hybrid dysgenesis model: The D. virilis hybrid dysgenesis system offers a unique opportunity to study mitotic recombination, as it increases transmission of chromosomes with mitotic recombination while leaving meiotic recombination patterns largely unaffected

• Molecular approaches: PCR-based methods can detect loss of heterozygosity resulting from mitotic recombination

A comparative study revealed that during hybrid dysgenesis, meiotic recombination patterns remain robust, whereas mitotic recombination increases significantly . This finding suggests that studying MAU2 function in the context of hybrid dysgenesis may reveal different roles in these two recombination processes. The clusters of mitotic recombination events in dysgenic females are associated with genomic regions containing transposons implicated in hybrid dysgenesis .

How can comparative genomics approaches reveal evolutionary conservation of MAU2 across Drosophila species?

Comparative genomics provides powerful insights into MAU2 evolution across Drosophila species:

• Sequence analysis: Alignment of MAU2 sequences from multiple Drosophila species can identify conserved domains and residues likely critical for function

• Phylogenetic reconstruction: Building gene trees for MAU2 and comparing with species trees can detect unusual evolutionary patterns, such as ancient introgression events between distant lineages or recent gene flow between closely related species

• Selection analysis: Calculating dN/dS ratios across different regions of the MAU2 gene can identify domains under purifying selection (conserved function) versus positive selection (potentially new functions)

• Synteny analysis: Examining the genomic context of MAU2 across species can reveal conserved gene neighborhoods that might indicate co-evolution of functionally related genes

Recent genomic analyses of the virilis species group revealed pervasive phylogenetic discordance caused by ancient introgression events between distant lineages and more recent gene flow between closely related species . This complex evolutionary history may have implications for understanding MAU2 function across species.

The virilis group originated approximately 9 million years ago, with the littoralis phylad radiating earliest, followed by radiation of the virilis and montana phylads around 3.8 and 4.7 million years ago, respectively . This timeframe provides context for understanding when functional innovations in MAU2 might have emerged.

What are the implications of MAU2 dysfunction for understanding genome stability in Drosophila virilis?

MAU2 dysfunction has significant implications for genome stability in D. virilis:

• Cohesin loading defects: Impaired MAU2-NIPBL interaction can compromise cohesin loading , potentially leading to premature sister chromatid separation and aneuploidy

• Recombination alterations: MAU2 dysfunction may affect the regulation of meiotic recombination, particularly in response to DNA damage from sources like transposable elements

• Transposable element interactions: The D. virilis genome contains numerous transposable elements, including Penelope and Ulysses, which are nonrandomly distributed and associated with chromosomal rearrangement breakpoints . MAU2 dysfunction could exacerbate genomic instability when these elements are active

• Mitotic versus meiotic effects: Research on hybrid dysgenesis suggests differential sensitivity of mitotic and meiotic processes to genomic stress , with implications for how MAU2 functions in these contexts

In the hybrid dysgenesis syndrome of D. virilis, transposable element activation in the germline leads to increased mitotic recombination while meiotic recombination patterns remain stable . This suggests that genome stability mechanisms in meiosis, potentially involving MAU2, are more robust than those in mitotic cells. Understanding these differential responses could provide insights into fundamental mechanisms of genome maintenance across different cell division types.