Recombinant Bovine RecQ-mediated genome instability protein 2 (RMI2)

Basic Research Questions

• What is the functional role of bovine RMI2 in the BTR complex?

RMI2 functions as an integral component of the BTR complex (BLM-Topo IIIα-RMI1-RMI2), which plays a crucial role in maintaining genomic stability. Within this complex, RMI2 directly interacts with BLM, while RMI1 binds to Topo IIIα . The BTR complex is essential for resolving double Holliday junctions during homologous recombination, thereby preventing aberrant recombination products .

Methodologically, studying RMI2's function requires:

• Protein-protein interaction assays (co-immunoprecipitation, yeast two-hybrid)

• Complex formation analysis using size-exclusion chromatography

• Functional assays measuring homologous recombination efficiency

• DNA damage repair assessment following RMI2 depletion or overexpression

• What are the optimal storage and handling protocols for recombinant bovine RMI2 protein?

For optimal preservation of recombinant bovine RMI2:

• Store at -20°C for regular use, or -80°C for extended storage

• Maintain working aliquots at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as they compromise protein integrity

• Prior to use, briefly centrifuge the vial to bring contents to the bottom

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol to 5-50% final concentration for long-term storage (50% is standard)

The shelf life is approximately 6 months for liquid form and 12 months for lyophilized form when stored at -20°C/-80°C .

• How does bovine RMI2 compare structurally to RMI2 from other species?

Bovine RMI2 (UniProt: A5PJU7) consists of 157 amino acids and shares significant structural conservation with human and mouse orthologs due to its critical role in genomic stability .

Key structural features include:

• The functional protein spans amino acids 2-157

• Contains an OB-fold (oligonucleotide/oligosaccharide binding) domain, crucial for its function in the BTR complex

• The sequence: AAPTDSLSVSGPTAVRLPRSPPIKVLAEQLRRDAEGGPGSWRLSRAAVGREPLELRAVWMQGTVVEAGGGVARLRDPSGSFSVRGLERV PRGRPCLVPGKYVMVMGVIQACSPEPCLQAVKMTDLSDNPLHESLDLEVEDLHRHIYSLDDVGTGD

Methodology for comparative structural analysis includes:

• Sequence alignment tools to identify conserved domains

• Structural prediction using crystallography or NMR data

• Functional complementation studies across species

• What protein expression systems are available for producing recombinant bovine RMI2?

Multiple expression systems exist for producing recombinant bovine RMI2, each with specific advantages:

Source data from

Methodologically, selection should be based on:

• Experimental requirements for post-translational modifications

• Desired purity level (typically >85% by SDS-PAGE)

• Downstream applications (structural studies vs. functional assays)

• How is RMI2 involved in DNA damage repair processes?

RMI2, as part of the BTR complex, plays critical roles in DNA damage repair through several mechanisms:

• Facilitates repair of ionizing radiation-induced DNA damage

• Contributes to homologous recombination (HR) repair of double-strand breaks

• Helps prevent and resolve aberrant recombination products

• Supports recovery from DNA replication stress

• Maintains genomic stability during cell division

Methodological approaches to study this involvement include:

• Measuring DNA damage levels (γH2AX foci) following RMI2 knockdown

• Assessing cell cycle progression after DNA damage in RMI2-deficient cells

• Quantifying HR repair efficiency using reporter assays

• Analyzing chromosomal abnormalities through metaphase spreads

Advanced Research Questions

• What experimental design considerations are crucial when studying RMI2 phosphorylation during mitosis?

When investigating RMI2 phosphorylation during mitosis, implement this experimental design framework:

• Cell synchronization strategies:

  • Use mechanical methods (centrifugal elutriation) to avoid damage artifacts

  • If using chemical synchronization, compare nocodazole and other agents to identify agent-specific effects

  • Include mitotic shake-off to isolate pure mitotic populations

• Phosphorylation detection:

  • Preserve phosphorylation status with phosphatase inhibitors in all buffers

  • Employ Phos-tag gels for enhanced separation of phospho-species

  • Use mass spectrometry for unbiased phosphosite identification

  • Develop phospho-specific antibodies for key sites (similar to Ser112 in human RMI2)

• Kinase identification:

  • Apply both pharmacological inhibitors and genetic approaches

  • Use in vitro kinase assays with purified components

  • Consider CDK1 as a potential kinase (responsive to roscovitine)

• Functional validation:

  • Generate phospho-mutants (S→A) and phospho-mimetics (S→E/D)

  • Perform rescue experiments in RMI2-depleted backgrounds

  • Assess BTR complex formation with mutant RMI2 variants

• Controls:

  • Include asynchronous cells and other cell cycle phases

  • Use phosphatase treatment to confirm phosphorylation specificity

  • Validate findings across multiple cell lines

• How can researchers effectively assess the functional relationship between RMI2 and the spindle assembly checkpoint (SAC)?

To investigate RMI2's role in SAC function, implement a comprehensive experimental approach:

• Protein manipulation strategies:

  • Generate inducible knockdown/knockout systems for temporal control

  • Create separation-of-function mutations targeting specific interactions

  • Develop fluorescently tagged RMI2 for live imaging during mitosis

• SAC activity assessment:

  • Measure mitotic duration following microtubule disruption (nocodazole treatment)

  • Quantify kinetochore localization of SAC components (Mad1, Mad2, BubR1)

  • Assess formation of the mitotic checkpoint complex through co-immunoprecipitation

  • Analyze APC/C substrate degradation kinetics (Cyclin B1, Securin)

• Phosphorylation analysis:

  • Identify mitosis-specific phosphorylation sites (focus on Ser112 equivalent)

  • Generate phospho-mutants to determine functional significance

  • Investigate relationship with mitotic kinases, particularly MPS1

• Chromosome segregation consequences:

  • Quantify chromosome segregation errors through live imaging

  • Measure micronuclei formation as indicator of mitotic defects

  • Assess genomic instability in cells expressing RMI2 phospho-mutants

• BTR complex contribution:

  • Determine interdependence between RMI2 and other BTR components in SAC function

  • Assess whether RMI2's SAC function is dependent on or independent of the BTR complex

• What approaches should be used to resolve contradictory data about RMI2 function across different experimental systems?

When confronting contradictory findings regarding RMI2 function, apply this systematic reconciliation framework:

• Methodological comparison:

  • Analyze differences in cell types, experimental conditions, and assay sensitivities

  • Consider protein expression levels across studies (endogenous vs. overexpression)

  • Examine knockout/knockdown efficiency and potential compensatory mechanisms

  • Evaluate temporal aspects—different studies may capture distinct time windows

• Context evaluation:

  • Determine whether contradictions reflect cell cycle-specific functions

  • Assess stress conditions used (replication stress, spindle damage, ionizing radiation)

  • Consider species differences when comparing bovine, human, and mouse systems

• Direct vs. indirect effects:

  • Distinguish primary RMI2 functions from secondary consequences of BTR complex disruption

  • Employ rapid protein depletion systems to capture immediate effects

  • Perform BTR complex integrity assessment following RMI2 manipulation

• Resolution strategies:

  • Use multiple independent techniques measuring the same parameter

  • Implement both loss-of-function and gain-of-function approaches

  • Include rescue experiments with RNAi-resistant constructs

  • Validate key findings across multiple cell lines

  • Isolate specific functions through domain-specific mutations

• Statistical considerations:

  • Apply appropriate statistical methods based on experimental design

  • Ensure adequate sample sizes for detecting biological differences

  • Control for multiple testing when appropriate

• What are the key considerations for designing experiments to study the role of RMI2 in DNA replication stress response?

Implement this experimental design framework to investigate RMI2's function in replication stress:

• Stress induction approaches:

  • Compare hydroxyurea, aphidicolin, and other replication inhibitors

  • Titrate stress agent concentrations to distinguish acute vs. chronic effects

  • Include pulse-chase experiments to examine recovery dynamics

• RMI2 manipulation strategies:

  • Use inducible systems for temporal control of RMI2 depletion/overexpression

  • Generate domain-specific mutations targeting particular functions

  • Create tagged versions for monitoring localization to stressed replication forks

• Phenotypic assessments:

  • Measure fork progression rates using DNA fiber analysis

  • Quantify replication fork restart efficiency after stress removal

  • Assess formation of DNA double-strand breaks during replication stress

  • Monitor recruitment of DNA repair factors to stalled replication forks

  • Evaluate cell survival following replication stress

• Molecular mechanisms:

  • Investigate RMI2 recruitment to stalled replication forks

  • Examine interactions with replication fork components (RPA, PCNA)

  • Assess BTR complex formation and activity under replication stress

  • Determine whether RMI2's role is dependent on its incorporation into the BTR complex

• Experimental design optimization:

  • Include appropriate controls (non-targeting siRNA, wild-type reconstitution)

  • Perform time-course experiments to capture dynamic responses

  • Compare results across different cell types to assess generalizability

  • Use synchronized cells to examine cell cycle-specific effects

• How should researchers design experiments to investigate potential post-translational modifications of RMI2 beyond phosphorylation?

To comprehensively study RMI2 post-translational modifications (PTMs):

• Discovery phase:

  • Employ mass spectrometry-based proteomics under various conditions:

    • Different cell cycle stages

    • DNA damage responses

    • Replication stress

    • Mitotic checkpoint activation

  • Use enrichment strategies for specific modifications:

    • Phospho-peptide enrichment (TiO2, IMAC)

    • Ubiquitin remnant motif antibodies

    • Anti-SUMO antibodies

• Validation approaches:

  • Generate site-specific PTM antibodies

  • Perform site-directed mutagenesis of modified residues

  • Use specific inhibitors of modification enzymes

  • Apply PTM-specific detection methods (Phos-tag gels, ubiquitin pulldowns)

• Functional assessment:

  • Conduct rescue experiments with non-modifiable mutants

  • Employ PTM-mimetic mutations when feasible

  • Assess protein interactions, localization, and stability of PTM mutants

  • Determine cell cycle-specific consequences of preventing modifications

• Regulatory enzyme identification:

  • Perform candidate-based screens of enzymes (kinases, E3 ligases)

  • Use proximity-based labeling to identify proteins in RMI2's vicinity

  • Conduct co-immunoprecipitation under conditions promoting modification

  • Implement in vitro modification assays with purified components

• Crosstalk analysis:

  • Investigate interdependence between different modifications

  • Examine sequential modification patterns during cellular processes

  • Assess competition between modifications at shared or adjacent sites

• What methodological approaches are most effective for investigating the relationship between RMI2 and cancer development?

To study RMI2's role in cancer biology, particularly breast cancer where it's been identified as a prognostic marker :

To effectively investigate interactions within the BTR complex (BLM-Topo IIIα-RMI1-RMI2):

• Protein expression and purification strategies:

  • Express individual components with different tags for differential purification

  • Consider co-expression systems to improve complex stability

  • Optimize buffer conditions to maintain complex integrity

  • Use insect or mammalian expression systems for full-length BLM

• Interaction detection approaches:

  • Implement multiple complementary techniques:

    • Co-immunoprecipitation with antibodies against each component

    • Pull-down assays using tagged recombinant proteins

    • Yeast two-hybrid or mammalian two-hybrid assays

    • Fluorescence resonance energy transfer (FRET)

    • Bioluminescence resonance energy transfer (BRET)

  • Validate interactions under physiological conditions

• Interaction domain mapping:

  • Generate truncation mutants to identify minimal interaction domains

  • Perform alanine scanning mutagenesis of conserved residues

  • Use peptide arrays to identify specific binding sequences

  • Apply hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• Complex assembly analysis:

  • Monitor sequential assembly using size-exclusion chromatography

  • Apply analytical ultracentrifugation to determine stoichiometry

  • Use native mass spectrometry to analyze intact complex

  • Employ single-molecule techniques to observe assembly dynamics

• Functional consequences:

  • Assess effects of mutations on complex formation and stability

  • Determine functional activity of subcomplexes lacking individual components

  • Investigate how post-translational modifications affect interactions

  • Examine complex formation under different cellular conditions (cell cycle, DNA damage)