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

This study describes the identification and characterization of two new extremophilic phage recombinases, UvsXt and UvsXp, discovered through metagenomic analysis within the Virus-X project, and explores their potential applications in biotechnology. DNA recombinases are essential for maintaining genome integrity across all kingdoms of life by facilitating homologous recombination and repairing double-stranded DNA breaks. Their capacity to bind and stabilize single-stranded DNA (ssDNA) has led to wide-ranging applications in molecular biology. UvsXt and UvsXp show homology with known bacterial RecA and viral UvsX recombinases, including conservation of key catalytic residues and DNA-binding motifs. Biochemical assays reveal that both enzymes exhibit superior DNA strand-exchange activity compared to Escherichia coli RecA. High-resolution crystal structures of UvsXt (2.0 Å) and UvsXp (2.6 Å) confirm a conserved RecA-like core fold, with distinct structural variation at the N-terminus responsible for oligomerization. However, in spite of their similarities, we show that neither enzyme is capable to functionally replace RecA in E. coli. Their remarkable thermostability and functionality across diverse chemical environments highlights their robustness for biotechnological use. Notably, UvsXt enhances loop-mediated isothermal amplification of viral RNA by stabilizing ssDNA intermediates. These findings expand the repertoire of thermostable recombinases with potential utility in diagnostic applications.