Characterization of DNA unwinding properties of three N-terminal fragments of RecQ5β helicase
RecQ5β is one member of the human RecQ family helicases that belong to superfamily 2 (SF2) and are critical for the maintenance of genomic stability. Here, the DNA unwinding kinetics of three N-terminal fragments of RecQ5β helicase, RecQ5β1-467, RecQ5β1-567 and RecQ5β1-662, were studied with stopped-flow method based on fluorescence resonance energy transfer (FRET). Under single-turnover kinetic conditions, we found that both the unwinding amplitude and rate increased with the increase of the 3’-tail length of the DNA substrate for each fragment. The maximum amplitudes were 73.5, 57.6 and 35.5% for RecQ5β1-467, RecQ5β1-567 and RecQ5β1-662, respectively. Obviously, the unwinding amplitude decreased with the increase of the fragment length. For each RecQ5β fragment, when the 3’-tail length of the DNA substrates was short, essentially only one slow unwinding process occurred. When the 3’-tail length was increased, the unwinding amplitude of the fast unwinding process increased obviously; that is, the RecQ5β-catalyzed DNA unwinding depended on the 3’-tail length of the DNA substrate. It indicates that RecQ5β molecules are cooperative in DNA unwinding. This is an interesting new feature for a SF2 helicase.
Key words: RecQ5β helicase, stopped-flow technique, fluorescence resonance energy transfer (FRET), DNA unwinding kinetics.