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Helicases are a broad family of enzymes that separate nucleic acid double strand structures (DNA/DNA, DNA/RNA or RNA/RNA) and thus are essential to DNA replication and the maintenance of nucleic acid integrity. We review the picture that has emerged from single molecule studies of the mechanisms of DNA and RNA helicases and their interactions with other proteins. Many features have been uncovered by these studies that were obscured by bulk studies, such as DNA strands switching, mechanical (rather than biochemical) coupling between helicases and polymerases, helicase-induced re-hybridization and stalled fork rescue. This article is protected by copyright. All rights reserved.

The RNA helicase UPF1 is a key component of the nonsense mediated mRNA decay (NMD) pathway. Previous X-ray crystal structures of UPF1 elucidated the molecular mechanisms of its catalytic activity and regulation. In this study, we examine features of the UPF1 core and identify a structural element that adopts different conformations in the various nucleotide- and RNA-bound states of UPF1. We demonstrate, using biochemical and single molecule assays, that this structural element modulates UPF1 catalytic activity and thereby refer to it as the regulatory loop. Interestingly, there are two alternatively spliced isoforms of UPF1 in mammals which differ only in the lengths of their regulatory loops. The loop in isoform 1 (UPF11) is 11 residues longer than that of isoform 2. We find that this small insertion in UPF11 leads to a two-fold increase in its translocation and ATPase activities. To determine the mechanistic basis of this differential catalytic activity, we have determined the X-ray crystal structure of the helicase core of UPF11 in its apo-state. Our results point toward a novel mechanism of regulation of RNA helicases, wherein alternative splicing leads to subtle structural rearrangements within the protein that are critical to modulate enzyme movements and catalytic activity.

Helicases are ubiquitous enzymes that are involved in all kinds of DNA/RNA metabolisms including transcription, recombination and replication. Although the most basic function of most helicases is to unwind a double stranded nucleic acid substrate, their properties vary a lot from one helicase family to another. Sometimes even within the same subfamily, the mechanistic properties of two helicases can be very different. S. cerevisiae Upf1 (yUpf1) and human Smubp-2 are two helicases from Upf1-like family that belong to SF1B. In this work we show that despite having a similar helicase core, the helicase domain (HD) of these two enzymes work in a very different manner. yUpf1-HD has a strong binding affinity to ssDNA and a very high processivity, whereas Smubp-2-HD has week binding to ssDNA and almost unrecognizable processivity. Helicase cores of Smubp2 and Upf1 have similar folds: they contain two RecA-like domains (1A and 2A) and two sub-domains, 1B and 1C, inserted in domain 1A. Rec-A like domains are believed to act as a motor in a helicase. Hence it is puzzling why the two helicases behave so differently despite having highly conserved domains 1A and 2A. We try to identify the role of sub-domains 1B and 1C in this respect. We have utilized a magnetic tweezers based single molecule assay, acting in parallel on tens of molecules at the same time, which allows detecting the unwinding of DNA hairpin substrates. Using this system, we have studied various chimeras of yUpf1-HD and Smubp-2-HD. We show that domains 1B and 1C play an equally important role in processivity of these helicases.