RNASEH2A E.Coli
Description
Production and Biochemical Applications
RNASEH2A E. coli is produced via bacterial expression systems and purified using affinity chromatography. Applications span structural studies, disease modeling, and enzymatic assays:
Mechanistic Roles
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RNA-DNA Hybrid Resolution: RNase H2 resolves R-loops during transcription, preventing replication stress and genome instability .
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Ribonucleotide Excision: Removes ribonucleotides incorporated into DNA during replication, critical for genomic fidelity .
Disease Links
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Aicardi-Goutières Syndrome: Mutations in RNASEH2A disrupt RNA-DNA hybrid processing, triggering autoimmune responses and neuroinflammation .
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Cancer and Genomic Instability: Deficient RNase H2 activity correlates with increased R-loops and chromosomal fragmentation .
Comparative Analysis of RNase H2 Complexes
The E. coli-produced RNASEH2A differs from bacterial RNase HI/HII in substrate specificity and subunit dependency:
| Feature | Human RNase H2 (RNASEH2A) | Bacterial RNase HI/HII |
|---|---|---|
| Subunits | Requires RNASEH2B/C for activity | Single-subunit enzymes |
| Catalytic Core | RNASEH2A (catalytic) | Autocatalytic |
| Primary Substrate | RNA-DNA hybrids, ribonucleotides | RNA primers in DNA replication |
Future Directions
Product Specs
Product Science Overview
Ribonuclease H2A (RNASEH2A) is a crucial enzyme involved in the maintenance of genomic stability. It is a member of the RNase HII family and plays a significant role in DNA replication by mediating the removal of RNA primers from Okazaki fragments during lagging-strand synthesis . The recombinant form of this enzyme, produced in Escherichia coli (E. coli), is widely used in research due to its high purity and activity.
The RNASEH2A recombinant produced in E. coli is a single, non-glycosylated polypeptide chain consisting of 322 amino acids, with a molecular mass of approximately 35.8 kDa . This recombinant protein is fused to a 23 amino acid His-tag at the N-terminus, which facilitates its purification through chromatographic techniques .
RNASEH2A catalyzes the endonucleolytic cleavage of RNA to a 5’-phosphomonoester. This activity is essential for the removal of RNA primers during DNA replication, ensuring the proper synthesis of the lagging strand . The enzyme requires magnesium or manganese ions as cofactors for its catalytic activity .
The recombinant RNASEH2A enzyme is extensively used in molecular biology and genetic research. Its ability to specifically target and cleave RNA-DNA hybrids makes it a valuable tool for studying DNA replication, repair, and recombination processes. Additionally, it is used in the investigation of diseases such as Aicardi-Goutieres syndrome type 4 (AGS4), which is caused by defects in the RNASEH2A gene .
The production of RNASEH2A recombinant protein involves the expression of the RNASEH2A gene in E. coli cells. The expressed protein is then purified using proprietary chromatographic techniques to achieve a purity greater than 85%, as determined by SDS-PAGE . The final product is a sterile, filtered, colorless solution formulated in a buffer containing 20 mM Tris-HCl (pH 8.0), 0.4 M urea, and 10% glycerol .
The RNASEH2A recombinant protein should be stored at 4°C if it will be used within 2-4 weeks. For longer storage periods, it is recommended to freeze the protein at -20°C. To prevent degradation, it is advisable to add a carrier protein such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA) and avoid multiple freeze-thaw cycles .
