Characterizing the SHARP PCR Helicase

Helicases are replicative enzymes that unwind duplex DNA (dsDNA) that are powered by Adenosine Tri-Phosphate (ATP) hydrolysis. Plasmid Copy Number Reduction (PcrA) is one such helicase. Recently, a super helicase called PcrA M6 was engineered from the thermostable PcrA of Geobacillus stearothermophilus. This helicase features a stabilized active conformation due to the translation of a disulfide bond. The speed of this enzyme makes it compatible with novel diagnostic and sequencing techniques, namely, SSB-Helicase Assisted Rapid Polymerase Chain Reaction (SHARP PCR). While it was initially reported that disulfide bond stabilization is spontaneous, some researchers, including Dr. Taekjip Ha, have questioned this and suggested an additional crosslinker is needed. To address this uncertainty and the lack of clear protocols, this study presents an optimized method for purifying PcrA M6 using nickel affinity and ssDNA affinity chromatography. To confirm PcrA M6 activity, a Fluorescence Resonance Energy Transfer (FRET) DNA unwinding assay was used to visualize dsDNA unwinding.