Role of the Excluded Strand in DNA Unwinding by Hexameric Helicases

Abstract

Hexameric DNA helicases control both the initiation and elongation phases of DNA replication. It is well known that these toroidal enzymes encircle and translocate preferentially on one strand with specific polarities dependent on their families and organismal domains. The question remains as to what if any role the excluded strand has in the unwinding mechanism or coordination of activities at the replication fork. Over the past few years, there has been an increasing amount of evidence to suggest that the excluded‐strand plays a significant role during unwinding by interacting with the helicase's outer surface in a Steric Exclusion and Wrapping (SEW) mode. The implications are that the excluded strand interaction controls the kinetics of unwinding while maintaining the ssDNA product for handoff to other replication components.We have investigated the interaction of the excluded strand with the outer surface of helicases in archaea, mitochondria, and bacteria using a variety of biochemical, biophysical, and genetic techniques. We have not only defined a minimal interaction path on the outer surface of these helicases, but we can also show that this interaction differentially controls the unwinding rate and efficiency. For the archaea MCM helicase, which has 3′‐5′ polarity, disruption of the excluded strand interaction inhibits unwinding consistent with a ratchet mechanism. Conversely, for the bacterial DnaB helicase, which has 5′‐3′ polarity, disruption increases the unwinding rate more consistent with a regulator‐type mechanism. The contrasting effects of the excluded strand interaction coupled with the opposing translocation polarities of these helicases will be discussed within the context of their respective replisomes.Support or Funding InformationNSF‐1613534