Researchers have identified and characterized two bacteriophage recombinases derived from extreme environments that may have potential applications in nucleic acid–based diagnostics. The study, published in Nucleic Acids Research, describes the structure and function of the enzymes and evaluates their performance in molecular assays.
The enzymes, named UvsXt and UvsXp, were discovered through metagenomic analysis of viral samples from Icelandic hot springs and Arctic deep-sea environments. UvsXt originated from high-temperature conditions (62 to 88°C), while UvsXp was isolated from near-freezing marine environments.
Recombinases are DNA-binding proteins that facilitate homologous recombination and DNA repair. Proteins in the RecA family are commonly used in molecular diagnostics, including recombinase polymerase amplification, polymerase chain reaction, and loop-mediated isothermal amplification (LAMP), because they bind single-stranded DNA and promote strand exchange.
Structural analysis showed that both UvsXt and UvsXp share features with known RecA-family recombinases, including ATP-binding sites and catalytic residues. Crystal structures confirmed RecA-like folding, with some structural differences in the N-terminal regions. Both enzymes form helical filaments typical of recombinase activity.
Functional testing demonstrated that the enzymes bind single-stranded DNA and exhibit ATPase activity. In strand exchange assays, both promoted DNA strand displacement, with UvsXp showing higher activity than Escherichia coli RecA under certain conditions. However, neither enzyme fully substituted for RecA in bacterial UV-damage repair assays, suggesting functional differences despite structural similarity.
Thermostability testing reflected their environmental origins. UvsXt demonstrated higher thermal stability, with melting temperatures up to 71°C under stabilizing conditions, while UvsXp maintained stability across a broader range of chemical environments.
The researchers also evaluated UvsXt in reverse transcription LAMP (RT-LAMP) assays. When added to viral RNA detection reactions at 65°C, UvsXt reduced time to detection and improved assay sensitivity. In this context, the enzyme appeared to enhance DNA binding rather than rely on ATP-dependent strand exchange.
While additional validation is required before clinical implementation, these extremophile-derived enzymes may contribute to future improvements in rapid molecular diagnostics, particularly in the rapid detection of viral RNA.
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