Mechanistic Insights of Hexameric Helicase Function Provided by Single-Molecule FRET

Abstract

DNA replication is an essential process across all domains of life. Replicative helicases play an integral part in this process by unwinding the duplex DNA to make single-strand template strands available for duplication. While the steric exclusion model of unwinding, where one strand is encircled by the hexameric helicase and the other excluded from the central channel, is widely accepted, the complexities of this process remain unclear. Details of the helicases’ loading and unwinding mechanism(s) are continually being revealed. One such detail is the interaction and role played by the excluded strand.Our group has recently shown that a wrapping interaction between the excluded single-strand of DNA and the outer surface of the helicase is crucial for the unwinding activity of the 3′-5’ MCM helicase from Sulfolobus solfataricus (Sso). Using single-molecule FRET (smFRET), we can now show that this interaction also exists for the hexameric E.coli DnaB (EcDnaB) helicase, which has 5′-3’ polarity, and that similar dynamics are exhibited by both helicases. This suggests that the interaction may be an important component of hexameric helicase unwinding across various helicase superfamilies independent of polarity.We have also investigated the interaction between EcDnaB's inner channel and the encircled-strand using smFRET. A compaction of the encircled strand by the helicase has been suggested based on several crystal structures of hexameric helicases bound to single-strand DNA that exhibits a significant decrease in rise per base. Using EcDnaB, we show that the helicase's binding induces a ‘scrunching’ of the DNA, and that in the absence of ATP, this is a stable interaction. Both excluded-strand wrapping and encircled-strand scrunching are likely critical aspects of replicative helicase unwinding.