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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.

International audience; Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway involved in many cellular pathways and crucial for telomere maintenance and embryo development. Core NMD factors Upf1, Upf2 and Upf3 are conserved from yeast to mammals, but a universal NMD model is lacking. We used affinity purification coupled with mass spectrometry and an improved data analysis protocol to obtain the first large-scale quantitative characterization of yeast NMD complexes in yeast (112 experiments). Unexpectedly, we identified two distinct complexes associated with Upf1: Detector (Upf1/2/3) and Effector. Effector contained the mRNA decapping enzyme, together with Nmd4 and Ebs1, two proteins that globally affected NMD and were critical for RNA degradation mediated by the Upf1 C-terminal helicase region. The fact that Nmd4 association to RNA was dependent on Detector components and the similarity between Nmd4/Ebs1 and mammalian Smg5-7 proteins suggest that NMD operates through successive Upf1-bound Detector and Effector complexes in other species. This model can be extended to accommodate steps that are missing in yeast, and thus serve for mechanistic studies of NMD in all eukaryotes. Running title: The Detector/Effector NMD model

Acentrosomal spindle assembly in mouse oocytes depends on chromosomes and acentriolar microtubule-organizing centers (aMTOCs). Manil-Ségalen et al. observe that Plk4-induced perturbation of aMTOCs coupled to Cre-mediated gene editing generates fragile chromosomes that break when subjected to forces exerted by altered meiosis I spindles., Mouse female meiotic spindles assemble from acentriolar microtubule-organizing centers (aMTOCs) that fragment into discrete foci. These are further sorted and clustered to form spindle poles, thus providing balanced forces for faithful chromosome segregation. To assess the impact of aMTOC biogenesis on spindle assembly, we genetically induced their precocious fragmentation in mouse oocytes using conditional overexpression of Plk4, a master microtubule-organizing center regulator. Excessive microtubule nucleation from these fragmented aMTOCs accelerated spindle assembly dynamics. Prematurely formed spindles promoted the breakage of three different fragilized bivalents, generated by the presence of recombined Lox P sites. Reducing the density of microtubules significantly diminished the extent of chromosome breakage. Thus, improper spindle forces can lead to widely described yet unexplained chromosomal structural anomalies with disruptive consequences on the ability of the gamete to transmit an uncorrupted genome.

Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway involved in many cellular pathways and crucial for telomere maintenance and embryo development. Core NMD factors Upf1, Upf2 and Upf3 are conserved from yeast to mammals, but a universal NMD model is lacking. We used affinity purification coupled with mass spectrometry and an improved data analysis protocol to obtain the first large-scale quantitative characterization of yeast NMD complexes in yeast (112 experiments). Unexpectedly, we identified two distinct complexes associated with Upf1: Detector (Upf1/2/3) and Effector. Effector contained the mRNA decapping enzyme, together with Nmd4 and Ebs1, two proteins that globally affected NMD and were critical for RNA degradation mediated by the Upf1 C-terminal helicase region. The fact that Nmd4 association to RNA was dependent on Detector components and the similarity between Nmd4/Ebs1 and mammalian Smg5-7 proteins suggest that in all eukaryotes NMD operates through successive Upf1-bound Detector and Effector complexes.

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.