Construction and characterization of ribonuclease H2 knockout NIH3T3 cells

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Ribonuclease H (RNase H) enzymes degrade RNA within RNA/DNA hybrids.
• RNase H2 uniquely processes single ribonucleotides in DNA duplexes, distinct from RNase H1.
• Understanding RNase H2's cellular function is critical for DNA repair and genome stability.

Purpose of the Study:

• To investigate the specific role of mammalian type 2 Ribonuclease H (RNase H2) in cellular processes.
• To characterize the impact of RNase H2 deficiency on genomic DNA integrity and cell proliferation.
• To determine if RNase H2 deficiency affects interferon-stimulated gene (ISG) expression.

Main Methods:

• CRISPR/Cas9 gene editing was employed to create RNase H2 knockout NIH3T3 cells.
• Hydrolysis assays were performed to assess RNA degradation in RNA/DNA hybrids and single ribonucleotides.
• Genomic DNA was analyzed for ribonucleotide content using alkaline or RNase H2 treatment.
• Cell growth rates and ISG expression levels were compared between knockout and wild-type cells.

Main Results:

• RNase H2 knockout cells retained the ability to hydrolyze RNA in RNA/DNA hybrids but not single ribonucleotides.
• Genomic DNA from knockout cells exhibited significantly higher susceptibility to hydrolysis, indicating increased ribonucleotide incorporation.
• The proliferation rate of knockout cells was reduced to 60% compared to wild-type cells.
• No significant elevation in interferon-stimulated gene expression was observed in knockout cells.

Conclusions:

• Mammalian RNase H2 plays a vital role in suppressing the accumulation of ribonucleotides within genomic DNA in NIH3T3 cells.
• RNase H2 is crucial for maintaining genomic integrity by removing embedded ribonucleotides.
• RNase H2 deficiency impacts cell growth but does not appear to trigger a significant interferon response in this context.