Recombinant Bovine Origin recognition complex subunit 2 (ORC2), partial

What is Origin Recognition Complex subunit 2 (ORC2) and what is its role in DNA replication?

ORC2 is a key component of the six-subunit Origin Recognition Complex (ORC), which functions as a critical ATPase in eukaryotic DNA replication. The ORC heterohexamer binds to origins of replication and facilitates loading of the MCM2-7 helicase complex to initiate DNA replication. Within the complex, ORC2 forms part of a ring structure that includes ORC1-5, with CDC6 later joining to complete the ring by filling the gap between ORC1 and ORC2 .

For researchers investigating bovine ORC2, it's important to note that while the protein likely serves similar functions across mammalian species, species-specific differences in replication dynamics may exist and should be characterized experimentally.

What experimental methods are effective for studying ORC2 function in bovine cells?

To study ORC2 function in bovine cells, researchers should employ a multi-faceted approach:

• Genetic manipulation techniques:

  • CRISPR-Cas9 gene editing for creating ORC2 knockout or knockdown models

  • Inducible expression systems for controlled ORC2 expression

  • Expression of tagged ORC2 variants for localization studies

• Replication analysis methods:

  • BrdU incorporation assays to measure DNA synthesis

  • DNA fiber analysis to study replication fork progression

  • Origin mapping using nascent strand sequencing or BrdU immunoprecipitation with sequencing (BrIP-seq)

• Protein-interaction studies:

  • Co-immunoprecipitation to identify ORC2 binding partners

  • Chromatin immunoprecipitation to identify genomic binding sites

  • Proximity ligation assays for in situ interaction detection

Researchers should be particularly attentive to the relationship between ORC2 and CDC6, as human ORC2-depleted cells become critically dependent on CDC6 for survival and DNA replication . This dependency suggests a compensatory mechanism that may be conserved in bovine cells.

How should researchers design expression systems for recombinant bovine ORC2 production?

Effective recombinant bovine ORC2 production requires careful consideration of expression systems and conditions:

• Expression vector selection:

  • Include appropriate affinity tags (His, GST, FLAG) for purification

  • Consider inducible promoters for controlled expression

  • For partial ORC2 constructs, carefully define domain boundaries based on structural information

• Expression host selection:

  • Bacterial systems: Suitable for individual domains but may present folding challenges for full-length ORC2

  • Insect cell systems: Preferred for full-length mammalian proteins requiring proper folding

  • Mammalian cell systems: Optimal for functional studies requiring native post-translational modifications

• Optimization parameters:

  • Expression temperature (often lowered to improve solubility)

  • Induction conditions (concentration, timing)

  • Co-expression with chaperones for improved folding

  • Cell lysis conditions to maintain protein stability

For partial ORC2 constructs, it's crucial to select regions that maintain stable folding and relevant functional domains. Consider the structural information showing that ORC2 participates in a ring formation with other ORC subunits and interacts with CDC6 .

What insights can be gained from studying DNA replication in ORC2-deficient cells?

Recent research has challenged the traditional view that ORC2 is absolutely essential for DNA replication. Human ORC2-/- cells can replicate their DNA and proliferate, though with altered replication dynamics . For bovine research, this opens several important lines of inquiry:

• Origin distribution and efficiency:

  • ORC2-/- human cells show approximately 13,000 BrIP-seq origins mapped to unique DNA sequences (compared to ~20,000 in wild-type cells)

  • Inter-origin distances are slightly increased (26 kb vs 25 kb in wild-type cells)

  • Origins remain enriched in gene-rich domains and near transcription start sites

• MCM2-7 chromatin loading:

  • ORC2-depleted cells show decreased chromatin loading of MCM2-7 helicase complex

  • Despite this reduction, origin plasticity persists, suggesting efficient utilization of the limited MCM2-7 loaded onto chromatin

• Compensatory mechanisms:

  • Increased dependency on CDC6 for survival and DNA replication

  • This suggests either that CDC6 can substitute for some ORC2 functions or that the remaining ORC subunits work more efficiently with CDC6 in ORC2's absence

When designing experiments with ORC2-deficient bovine cells, researchers should include comprehensive controls to verify ORC2 depletion and carefully monitor cell cycle progression, replication timing, and genetic stability over multiple generations.

How does the absence of ORC2 affect the structure and function of the ORC complex?

The six-subunit ORC is traditionally described as forming a ring-shaped heterohexamer, with ORC2 positioned between ORC1 and ORC3. The absence of ORC2 raises fundamental questions about complex structure and function:

• Complex integrity:

  • Without ORC2, the remaining subunits likely form an incomplete ring structure

  • This challenges models proposing that a complete ORC ring is necessary to surround DNA and load MCM2-7

• Functional consequences:

  • Despite the structural gap, the incomplete ORC complex can still function in origin licensing

  • This suggests either greater flexibility in ORC architecture than previously thought or alternative mechanisms for MCM2-7 loading

• Interaction with CDC6:

  • Normally, CDC6 joins the ORC ring by filling the gap between ORC1 and ORC2

  • In ORC2's absence, CDC6 interaction with the complex would be altered

  • This may explain the increased CDC6 dependency observed in ORC2-deficient cells

Research indicates that knockdown of ORC5 still affects DNA synthesis in ORC2-/- cells, suggesting that the remaining ORC subunits continue to function in replication initiation even without ORC2 . This challenges our understanding of the minimal requirements for a functional pre-replication complex.

What methodological approaches can identify replication origins in ORC2-deficient bovine cells?

To map and characterize replication origins in ORC2-deficient bovine cells, researchers should employ multiple complementary techniques:

• BrdU immunoprecipitation sequencing (BrIP-seq):

  • Incorporates BrdU into nascent DNA strands during replication

  • Immunoprecipitates BrdU-labeled fragments

  • Sequences and maps these fragments to identify replication initiation sites

  • Allows quantification of origin efficiency and distribution

• Molecular combing:

  • Labels replicating DNA with nucleotide analogs

  • Stretches DNA fibers on glass slides

  • Directly visualizes labeled tracks to measure inter-origin distances

  • In human ORC2-/- cells, this revealed slightly longer inter-origin distances (inter-origin distance of 26 kb vs 25 kb in wild-type)

• Comparative analysis:

  • Assess origin overlap between wild-type and ORC2-deficient cells

  • In human cells, 40% of BrIP-seq peaks in ORC2-/- cells overlapped with wild-type origins

  • This partial overlap reflects plasticity in origin usage observed in normal cells

• Origin characterization:

  • Analyze genomic features of origins (GC content, chromatin state)

  • Map origins relative to transcriptional features

  • In human cells, origins remain enriched near transcription start sites even in ORC2-deficient conditions

These methods should be combined with chromatin immunoprecipitation of other pre-replication complex components to understand how origin recognition and licensing occur in the absence of ORC2.

How does CDC6 dependency change in ORC2-deficient cells, and how can this be studied?

ORC2-deficient human cells show critical dependency on CDC6 for survival and DNA replication . This relationship can be investigated through several methodological approaches:

• Synthetic lethality testing:

  • Use siRNA or inducible shRNA to deplete CDC6 in ORC2-deficient cells

  • Compare viability and replication capacity to similarly treated wild-type cells

  • Quantify differential sensitivity to establish dependency relationship

• CDC6 chromatin association:

  • Perform chromatin fractionation to measure CDC6 loading

  • Compare timing and quantity of CDC6 association between wild-type and ORC2-deficient cells

  • Use ChIP-seq to map CDC6 binding sites genome-wide

• Protein-protein interaction studies:

  • Immunoprecipitate CDC6 and analyze associated proteins by mass spectrometry

  • Identify potential novel interactions that may compensate for ORC2 absence

  • Compare CDC6 interactome between wild-type and ORC2-deficient conditions

• Functional complementation:

  • Express CDC6 variants with mutations in specific domains

  • Assess which CDC6 functions are critical in ORC2-deficient background

  • Test whether CDC6 overexpression can further enhance viability or replication efficiency

Understanding this dependency relationship may reveal novel mechanisms of replication initiation and potential therapeutic targets for conditions with dysregulated replication.

What functional assays can validate recombinant bovine ORC2 activity?

To confirm that recombinant bovine ORC2 (either full-length or partial) is functionally active, researchers should implement these validation assays:

• DNA binding assays:

  • Electrophoretic mobility shift assays with origin DNA sequences

  • Surface plasmon resonance for binding kinetics

  • DNA footprinting to identify protected regions

• Protein-protein interaction assays:

  • Pull-down assays with other recombinant ORC subunits

  • Size exclusion chromatography to assess complex formation

  • Isothermal titration calorimetry for binding thermodynamics

• Complementation studies:

  • Introduce recombinant ORC2 into ORC2-depleted cells

  • Measure rescue of phenotypes:

    • MCM2-7 chromatin loading

    • Origin firing efficiency

    • Cell cycle progression

    • CDC6 dependency

• In vitro pre-RC assembly:

  • Reconstitute pre-replication complex with purified components

  • Measure MCM2-7 loading onto template DNA

  • Compare efficiency with and without ORC2

  • Assess cooperation with CDC6

• ATPase activity assays:

  • While ORC2 itself lacks ATPase activity (only ORC1 has this function in the complex)

  • Test how ORC2 affects the ATPase activity of the complete complex

These functional assays should include appropriate controls such as known inactive mutants and denatured protein to validate specific activity.