Recombinant Rat MutS protein homolog 5 (Msh5), partial

What is Rat MutS protein homolog 5 (Msh5) and what is its primary function?

Msh5 is a member of the MutS protein family that bears structural homology to bacterial MutS proteins but has evolved specialized functions in meiosis. Unlike classic MutS proteins involved in DNA mismatch repair (MMR), Msh5 lacks the domain for binding mismatches in DNA and is not associated with MMR activity . Instead, Msh5 forms a heterodimer with Msh4 that promotes meiotic crossovers by stabilizing strand exchange intermediates . In vivo studies demonstrate that Msh5 binds to DSB hotspots, chromosome axes, and centromeres, suggesting multiple roles in meiotic recombination . Its primary function is essential for proper chromosome synapsis and segregation during meiosis, making it critical for fertility .

What are the phenotypic consequences of Msh5 deficiency in model organisms?

Mice carrying disruptions in the Msh5 gene exhibit complete male and female sterility due to meiotic failure . Histological and cytological examination reveals specific defects during prophase I of meiosis, characterized by an extended zygotene stage with impaired and aberrant chromosome synapsis . This synaptic failure is followed by apoptotic cell death of germ cells. In male Msh5-deficient mice, spermatogonial multiplication appears normal with many spermatocytes formed, but these cells undergo massive apoptosis at a specific developmental stage . These findings establish that murine Msh5 promotes the synapsis of homologous chromosomes during early meiotic prophase I, which is essential for fertility .

How does Msh5 localization change during meiotic progression?

The localization of Msh5 shows a dynamic pattern during meiosis with distinctive characteristics compared to other recombination proteins. In normal meiotic progression, Msh5 forms discrete foci on chromosomes with an average steady-state number of approximately 42 foci per nucleus . Time-course experiments in hybrid yeast strains (S-sp/Y) show that Msh5 expression begins around 2 hours after meiotic induction, with peak expression at 6 hours . ChIP-qPCR analysis demonstrates that Msh5 binding at DSB hotspots reaches maximum levels at the 5-hour time point post-induction . This timing correlates with the transition from early recombination intermediates to stable joint molecules that will become crossovers.

What distinguishes Msh5 from other meiotic recombination proteins?

Msh5 exhibits several unique characteristics that distinguish it from other meiotic recombination proteins:

This unique behavior suggests that Msh5 has evolved specialized regulatory mechanisms distinct from other recombination proteins .

What methods are used to study Msh5 binding patterns on chromosomes?

Researchers employ several sophisticated methodologies to characterize Msh5 binding patterns:

• ChIP-seq Analysis: Genome-wide Msh5 binding sites are identified using chromatin immunoprecipitation followed by sequencing. Peak calling algorithms like MACS2 (model-based analysis for ChIP-seq) are employed with statistical significance thresholds (P ≤ 10^-5) to identify reliable binding sites .

• Calibrated ChIP-seq: To control for variable mapping efficiencies between different strain backgrounds, researchers use "spike-in" calibration with heterologous DNA (e.g., S. mikatae meiotic cells) and calculate occupancy ratios to normalize binding data .

• ChIP-qPCR: For targeted analysis of specific loci, quantitative PCR is performed on immunoprecipitated DNA to measure relative enrichment at DSB hotspots compared to coldspots .

• Analysis of Chimeric Reads: In heterozygous backgrounds, researchers analyze both 150 bp paired-end and 300 bp single-end sequencing data to detect chimeric reads (containing SNPs from both homologs) versus non-chimeric reads (containing SNPs from only one homolog) at Msh5 binding sites, providing insights into the nature of the DNA structures bound by Msh5 .

• Cytological Analysis: Immunofluorescence microscopy is used to visualize and quantify Msh5 foci on meiotic chromosome spreads, allowing temporal analysis of focus formation and disappearance .

How does heterozygosity affect Msh5 binding to meiotic chromosomes?

Heterozygosity significantly impacts Msh5 binding patterns on meiotic chromosomes. Calibrated ChIP-seq analysis comparing homozygous (SK1) and heterozygous (S-sp/Y hybrid) yeast strains reveals several key differences:

• Msh5 binding peaks at DSB hotspots are wider in the heterozygous S-sp/Y hybrid (median 0.73 kb, mean 1.16 kb) compared to the homozygous SK1 strain (median 0.55 kb, mean 0.85 kb) .

• Msh5 shows preferential enrichment in genomic regions with lower SNP density. The mean and median SNP densities genome-wide (4.06 and 3.0 SNPs/kb) are significantly higher compared to the mean and median SNP density in Msh5-bound regions (3.77 and 2.53 SNPs/kb) .

• Analysis of chromosome-wide binding patterns shows a weak negative association between Msh5 binding and regions of high heterozygosity .

These findings suggest that sequence differences between homologous chromosomes influence Msh5 binding dynamics during meiotic recombination, potentially affecting crossover distribution patterns in hybrid organisms.

What is the mechanism by which Msh4-Msh5 promotes crossover formation?

The Msh4-Msh5 heterodimer promotes crossover formation through several molecular mechanisms:

• Holliday Junction Stabilization: In vitro studies demonstrate that the Msh4-Msh5 complex binds and stabilizes D-loops and Holliday junction intermediates, preventing their premature dissolution .

• Bidirectional Binding: Analysis of chimeric reads in heterozygous backgrounds reveals that Msh5 binds to DNA containing SNPs/alleles from both homologs (chimeric) as well as DNA containing SNPs from only one homolog (non-chimeric) . The distribution patterns of chimeric and non-chimeric reads appear as mirror images of each other, suggesting that Msh5 binds to both strands of recombination intermediates like double Holliday junctions .

• Localization to Multiple Chromosomal Elements: Msh5 localizes not only to DSB hotspots but also to chromosome axes and centromeres, suggesting coordination between recombination intermediates and chromosome structural elements .

• ATP-Dependent Activity: The protein contains a putative ATPase domain essential for its function, as demonstrated by the infertility phenotype observed when this domain is disrupted in mouse models .

This multi-faceted mechanism ensures the stable progression of recombination intermediates toward crossover resolution, which is essential for proper chromosome segregation during meiosis.

What are the optimal conditions for ChIP-seq analysis of Msh5?

Successful ChIP-seq analysis of Msh5 requires careful optimization of several experimental parameters:

For heterozygous backgrounds, additional considerations include using both paired-end (150 bp) and longer single-end (300 bp) sequencing to detect chimeric reads containing SNPs from both homologs, which provides insights into the nature of the DNA structures bound by Msh5 .

How can researchers distinguish between functional and non-functional recombinant Msh5?

Distinguishing functional from non-functional recombinant Msh5 requires multiple validation approaches:

• Structural Integrity: The recombinant protein should contain the putative ATPase domain, which has been shown to be essential for Msh5 function in vivo . Disruption of this domain in mouse models results in complete sterility .

• Heterodimerization: Functional Msh5 forms a heterodimer with Msh4. Co-immunoprecipitation or size-exclusion chromatography can be used to verify heterodimerization of recombinant Msh5 with its partner protein .

• DNA Binding Activity: In vitro assays using substrate DNA structures that mimic recombination intermediates (D-loops, Holliday junctions) should demonstrate specific binding by the recombinant Msh5-Msh4 complex .

• Complementation Testing: The ability of recombinant Msh5 to rescue the phenotype of Msh5-deficient cells provides the most stringent test of functionality .

• ATP Hydrolysis: Since Msh5 contains an ATPase domain, functional protein should exhibit ATP hydrolysis activity, particularly when bound to DNA substrates .

What are the best model systems for studying Msh5 function?

Several model systems offer complementary advantages for studying Msh5 function:

The choice of model system depends on the specific research question. Yeast models like SK1 and S-sp/Y hybrid strains are particularly valuable for mechanistic studies of Msh5 binding and recombination dynamics, providing insights into how heterozygosity affects these processes . Mouse models offer the advantage of studying mammalian-specific aspects of Msh5 function and its impact on fertility .

How should researchers analyze chimeric versus non-chimeric DNA binding by Msh5?

Analysis of chimeric versus non-chimeric DNA binding by Msh5 provides insights into its interaction with recombination intermediates. Researchers should:

• Employ Both Sequencing Approaches:

  • Use 150 bp paired-end sequencing to detect SNPs from both ends of Msh5-bound DNA fragments

  • Use 300 bp single-end sequencing to detect closely spaced SNP markers on a single read

• Define Classification Criteria:

  • Classify reads as "chimeric" if they contain SNPs/alleles from both homologs

  • Classify reads as "non-chimeric" if they contain SNPs from only one homolog

• Analyze Distribution Patterns:

  • Examine if chimeric and non-chimeric reads appear as mirror images of each other, which suggests binding to double Holliday junctions

  • Compare patterns at DSB hotspots versus coldspots as a control

• Correlate with Recombination Intermediates:

  • Analyze the distribution of chimeric reads in relation to known recombination intermediates

  • Consider the timing of appearance of chimeric reads during meiotic progression

What controls are essential when studying recombinant Rat Msh5?

When studying recombinant Rat Msh5, several critical controls must be included:

• Knockout/Deletion Controls: Include samples from Msh5-deficient systems (msh5Δ) to confirm antibody specificity and establish baseline signals in functional assays .

• DSB Hotspot vs. Coldspot Comparisons: Include analysis of known DSB coldspots (YCR093W in yeast) as negative controls when assessing Msh5 binding at hotspots .

• Temporal Controls: Analyze multiple time points during meiotic progression to establish the dynamics of Msh5 expression and localization .

• Genetic Background Comparisons: Include both homozygous (e.g., SK1) and heterozygous (e.g., S-sp/Y) backgrounds when studying binding patterns to assess the impact of sequence polymorphisms .

• Other ZMM Protein Comparisons: Compare Msh5 behavior with other ZMM proteins (Zip3, Mer3, Spo22/Zip4) under identical conditions to highlight Msh5-specific properties .

These controls provide essential reference points for interpreting experimental results and distinguishing specific Msh5-related phenomena from background effects or general recombination protein behaviors.

How can researchers resolve contradictory data about Msh5 function?

When faced with contradictory data about Msh5 function, researchers should consider several approaches:

• Genetic Background Effects: Msh5 behavior shows significant variation between different genetic backgrounds, particularly regarding binding peak width and distribution in homozygous versus heterozygous contexts . Researchers should explicitly account for these effects when comparing results across studies.

• Temporal Dynamics: Contradictions may arise from analyzing different time points during meiosis. The temporal pattern of Msh5 expression and localization should be carefully considered when interpreting results .

• Technical Variations: Different methodologies (e.g., uncalibrated versus calibrated ChIP-seq) can yield apparently contradictory results. Implementing calibration approaches with spike-in controls can help resolve such discrepancies .

• Functional Redundancy: Partial redundancy between Msh5 and other recombination proteins may explain variable phenotypes under different conditions. Careful genetic analysis with multiple mutant combinations can help clarify these relationships.

• Model-Specific Effects: Contradictions between results in different model organisms may reflect genuine biological differences. Cross-species comparative approaches can help distinguish conserved versus species-specific aspects of Msh5 function.

By systematically exploring these potential sources of contradiction, researchers can develop more nuanced and accurate models of Msh5 function across different biological contexts.

What are the emerging techniques for studying Msh5 dynamics during recombination?

Several emerging techniques offer new opportunities for understanding Msh5 dynamics:

• Live-Cell Imaging: Development of fluorescently tagged Msh5 compatible with live-cell imaging could reveal real-time dynamics of Msh5 localization during meiotic progression.

• Single-Molecule Approaches: Techniques like single-molecule FRET (Förster Resonance Energy Transfer) could provide insights into the conformational changes of Msh5-Msh4 complexes upon binding to recombination intermediates.

• Cryo-EM Structural Analysis: High-resolution structural studies of the Msh5-Msh4 complex bound to various DNA substrates would advance understanding of its mechanism of action.

• Hi-C and Chromosome Conformation Capture: These approaches could reveal how Msh5 binding affects chromosome architecture during meiosis.

• CRISPR-Based Tagging: Endogenous tagging of Msh5 using CRISPR-Cas9 in mammalian systems would enable more physiologically relevant studies of its dynamics.

These emerging techniques promise to provide deeper insights into the molecular mechanisms by which Msh5 promotes crossover formation and ensures proper chromosome segregation during meiosis.

How might understanding Msh5 function contribute to fertility research?

Understanding Msh5 function has significant implications for fertility research:

• Meiotic Failure Mechanisms: Since Msh5 deficiency causes complete sterility in mice due to meiotic failure , understanding its function could illuminate mechanisms underlying certain forms of human infertility.

• Crossover Regulation: Msh5's role in crossover formation and distribution affects chromosome segregation, with implications for aneuploidy and related fertility disorders.

• Genetic Background Effects: The impact of heterozygosity on Msh5 binding suggests that genetic variation may influence recombination patterns and potentially fertility in mixed genetic backgrounds.

• Therapeutic Targets: Detailed understanding of Msh5 function could identify potential targets for interventions in certain forms of infertility.

• Diagnostic Applications: Characterization of Msh5 function could lead to improved diagnostic approaches for identifying specific causes of infertility related to meiotic recombination defects.

By elucidating the fundamental mechanisms by which Msh5 promotes proper meiotic recombination, researchers can contribute to addressing fertility challenges with more targeted and effective approaches.