Structure, function, and applications of two novel phage recombinases from extreme environments.

Area of Science:

• Molecular Biology
• Biotechnology
• Structural Biology

Background:

• DNA recombinases are crucial for genome integrity and DNA repair across all life forms.
• Their ability to bind and stabilize single-stranded DNA (ssDNA) enables diverse molecular biology applications.
• Extremophilic enzymes offer unique properties for biotechnological innovation.

Purpose of the Study:

• To identify and characterize novel extremophilic phage recombinases.
• To explore the biochemical and structural properties of UvsXt and UvsXp.
• To evaluate their potential biotechnological applications, including diagnostic tools.

Main Methods:

• Metagenomic analysis for enzyme discovery.
• Biochemical assays to assess DNA strand-exchange activity.
• High-resolution X-ray crystallography for structural determination.

Main Results:

• Identified and characterized two new extremophilic recombinases, UvsXt and UvsXp, with homology to RecA/UvsX.
• Demonstrated superior DNA strand-exchange activity compared to E. coli RecA.
• Determined crystal structures revealing conserved RecA-like folds with distinct N-terminal variations.
• Confirmed that neither enzyme can functionally replace RecA in E. coli.
• Showcased UvsXt's ability to enhance viral RNA amplification by stabilizing ssDNA.

Conclusions:

• UvsXt and UvsXp are robust, thermostable recombinases with significant biotechnological potential.
• Their unique properties expand the toolkit of available DNA recombinases.
• UvsXt shows promise for enhancing diagnostic applications, such as isothermal amplification assays.