DNA Unwinding Mechanism of a RecQ Helicase from Arabidopsis Thaliana Investigated with Magnetic Tweezers

Abstract

Helicases are ATP hydrolysis driven molecular motors that processively unwind double stranded DNA. In this study we investigate RecQ2 helicase from Arabidopsis thaliana (AtRecQ2) which plays an important role in genomic maintenance. We use high resolution magnetic tweezers in order to probe the unwinding of a DNA hairpin by this enzyme in real time under different external forces. We find an unwinding rate of 7-9 bp/s which is slow compared to many prokaryotic helicases. Applied forces between 5 and 12 pN only weakly affect this parameter, while the AT versus GC content of the unwound DNA has a significant impact. The weak force- but the relatively strong sequence dependence of DNA unwinding is in disagreement with a passive ratchet unwinding mechanism. High-resolution measurements reveal that AtRecQ2 unwinds the DNA in 3-4 bp steps. Beyond the behavior of AtRecQ2 during unwinding we analyze its behavior on single-strand DNA. While failing to detect single-strand DNA translocation, the data in contrary suggests that AtRecQ2 diffuses on single stranded DNA. Such a weak contact to single-strand DNA is supported by the observation that even on a stretched hairpin configuration the enzyme is capable of repetitive shuffling, i.e. the instantaneous restart of DNA unwinding after a terminated event. Based on this we hypothesize that AtRecQ2 switches between a processive unwinding-mode on double-stranded DNA and a diffusive-mode on single-stranded DNA in order to keep a target hairpin constantly open or to search for a new distant target fork.