DUT Pyrococcus Fruriosus

Pyrococcus furiosus is a hyperthermophilic archaeon, known for thriving in extremely high-temperature environments, such as hydrothermal vents. The thermostable dUTPase enzyme is expressed recombinantly in Escherichia coli (E. coli) to ensure high yield and purity .

The primary function of dUTPase is to hydrolyze dUTP (deoxyuridine triphosphate) into dUMP (deoxyuridine monophosphate) and pyrophosphate. This reaction is critical for maintaining the fidelity of DNA replication and repair. By reducing the levels of dUTP, the enzyme prevents its incorporation into DNA, which can lead to mutations and DNA damage .

In PCR, the presence of dUTPase is essential for several reasons:

• Prevention of dUTP Misincorporation: dUTPase removes contaminating dUTP from PCR reactions and dNTP solutions, preventing its incorporation into the DNA strand. This is particularly important when using proofreading DNA polymerases, as dUTP incorporation can cause the polymerase to stall .
• Enhanced PCR Yield and Fidelity: By maintaining low levels of dUTP, dUTPase ensures higher yield and fidelity of PCR products. This is crucial for applications such as cloning, gene expression analysis, and other downstream applications where accuracy is paramount .
• Stability and Storage: The enzyme is supplied in a sterile filtered liquid formulation and can be stored at -20°C for long-term use. It is recommended to avoid multiple freeze-thaw cycles to maintain its activity .

dUTPase is specific for dUTP and is critical for the fidelity of DNA replication and repair. The enzyme’s activity ensures that dUTP levels remain below inhibitory concentrations, despite the constant generation of dUTP through the spontaneous deamination of dCTP. This regulation is vital for maintaining genomic stability and preventing mutations .