Recombinant Exodeoxyribonuclease V gamma chain (recC), partial
Description
Definition and Biological Context
The RecC subunit (gamma chain) is one of three essential components of the RecBCD enzyme (EC 3.1.11.5), a multifunctional complex responsible for repairing double-strand DNA breaks through recombinational repair . The recombinant "partial" RecC denotes a truncated form, often engineered to study specific functional domains or interactions without full-length protein constraints .
Functional Role in RecBCD Enzyme
RecC serves as a scaffold and regulatory hub:
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Chi-dependent recombination: RecC detects Chi sites during DNA unwinding, triggering RecBCD’s transition from destructive nuclease to recombinase .
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Subunit coordination: Stabilizes RecB (3′→5′ helicase/nuclease) and RecD (5′→3′ helicase), enabling processive DNA unwinding .
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Signal transduction: Conformational changes in RecC relay Chi recognition to RecB’s nuclease domain, reducing 3′-strand degradation and enabling RecA loading .
Key Research Findings on Partial RecC Constructs
Studies using truncated or mutated RecC reveal mechanistic insights:
RecC Truncations and RecD Assembly
RecC Tunnel and Surface Mutants
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RecC S39E/K88I (tunnel mutants): Eliminate Chi recognition, reducing recombination frequency to 1–6% of wild type .
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RecC Δ252–291 (surface loop deletion): Reduces Chi hotspot activity by 60% but retains helicase function .
RecC-RecB Interface Mutants
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RecC R278A: Disrupts RecB nuclease domain docking, impairing Chi-triggered nicking .
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RecC G304S: Abolishes Chi activity without affecting helicase speed .
Implications for DNA Repair Mechanisms
Partial RecC constructs have clarified how Chi regulates RecBCD:
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Chi-induced RecD inactivation: Prolonged Chi interaction converts RecBCD to RecBC, enabling Chi-independent recombination .
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Long-range allostery: Mutations >50 Å from Chi or nuclease sites (e.g., RecC A68V) still disrupt Chi signaling, suggesting dynamic interdomain communication .
Applications in Genetic Engineering
Recombinant partial RecC is used to:
Product Specs
Q&A
Given the context of "Recombinant Exodeoxyribonuclease V gamma chain (recC), partial," here's a collection of FAQs tailored for researchers, focusing on academic research scenarios:
Data Analysis and Contradiction Resolution
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Q: What methods can I use to analyze and resolve contradictions in data related to the activity of recombinant Exodeoxyribonuclease V gamma chain (recC)?
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A: When analyzing data, use statistical tools to identify significant differences in enzyme activity or DNA repair efficiency. For contradictions, consider re-evaluating experimental conditions, such as buffer composition or temperature, which might affect enzyme activity. Also, employing multiple analytical techniques (e.g., gel electrophoresis, sequencing) can help validate findings and resolve discrepancies.
Methodological Considerations for Expression and Purification
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Q: What are the best practices for expressing and purifying recombinant Exodeoxyribonuclease V gamma chain (recC) for biochemical studies?
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A: For optimal expression, use a suitable bacterial host like E. coli with a plasmid designed for high-level expression (e.g., pET vectors). Purification can be achieved using affinity chromatography (e.g., His-tagged proteins) followed by size exclusion chromatography to ensure high purity. Consider optimizing expression conditions (e.g., temperature, inducer concentration) to maximize yield and minimize degradation.
Integration with Other Research Tools
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Q: How can recombinant Exodeoxyribonuclease V gamma chain (recC) be integrated with other molecular biology tools for comprehensive studies?
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A: Integrating recC with other tools like yeast two-hybrid systems (rec-Y2H) can help study protein-protein interactions relevant to DNA repair pathways . Additionally, combining recC studies with advanced sequencing techniques can provide insights into genomic stability and repair mechanisms at a systems level.
Advanced Techniques for Structural Analysis
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Q: What advanced structural biology techniques can be used to study the recombinant Exodeoxyribonuclease V gamma chain (recC)?
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A: Techniques such as cryo-electron microscopy (cryo-EM) and X-ray crystallography can provide high-resolution structures of the recC subunit within the RecBCD complex. These methods help elucidate the molecular mechanisms underlying DNA recognition and processing by recC.
Comparative Studies Across Different Organisms
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Q: How can comparative studies of recombinant Exodeoxyribonuclease V gamma chain (recC) across different bacterial species inform our understanding of DNA repair mechanisms?
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A: By comparing the structure and function of recC across species, researchers can identify conserved motifs and mechanisms essential for DNA repair. This approach can also reveal species-specific adaptations that enhance repair efficiency in different environments.
Bioinformatics Tools for Sequence Analysis
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Q: What bioinformatics tools are useful for analyzing the sequence and evolutionary conservation of the recombinant Exodeoxyribonuclease V gamma chain (recC)?
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A: Tools like BLAST for sequence alignment, ClustalW for multiple sequence alignment, and WebLogo for consensus motif generation can help analyze sequence conservation and evolutionary relationships among recC homologs across different species .
Experimental Challenges and Troubleshooting
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Q: What common challenges arise during the expression and purification of recombinant Exodeoxyribonuclease V gamma chain (recC), and how can they be addressed?
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A: Common challenges include low expression levels, protein aggregation, or degradation. These can be addressed by optimizing expression conditions (e.g., reducing temperature, using different inducers), improving purification protocols (e.g., adding stabilizing agents), or using alternative expression systems (e.g., insect cells).
Future Directions in RecC Research
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Q: What are some future directions for research on the recombinant Exodeoxyribonuclease V gamma chain (recC)?
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A: Future studies could focus on the development of novel DNA repair therapies by exploiting the mechanisms of recC. Additionally, exploring the role of recC in bacterial pathogenesis and antibiotic resistance could provide new targets for antimicrobial strategies.
Example Data Table: RecBCD Enzyme Activity Assay
| Assay Conditions | RecBCD Activity (nM/min) | Control (No RecC) |
|---|---|---|
| Standard Buffer | 10.2 ± 1.5 | 0.5 ± 0.2 |
| High Salt Buffer | 5.8 ± 1.1 | 0.2 ± 0.1 |
| Low Temperature | 8.5 ± 1.2 | 0.3 ± 0.1 |
This table illustrates how different conditions can affect RecBCD enzyme activity, highlighting the importance of optimizing assay conditions for studying recC function.
