RNA helicases are a diverse group of RNA-dependent ATPases known to play a large number of biological roles inside the cell, such as RNA unwinding, remodeling, export and degradation. Understanding how helicases mediate changes in RNA structure is therefore of fundamental interest. The advent of single-molecule spectroscopic techniques has unveiled with unprecedented detail the interplay of RNA helicases with their substrates. In this review, we describe the characterization of helicase-RNA interactions by single-molecule approaches. State-of-the-art techniques are presented, followed by a discussion of recent advancements in this exciting field.

Macroautophagy, or simply autophagy, is a catabolic program for restoring cellular resources and energy balance as cells recognize damaged organelles, protein aggregates, and invading pathogens. 1,2 As new studies emerge, the role of autophagy has been expanded to include cell growth, cell development and aging, tumor suppression, and immunity among others. 2 Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel present in the lung and intestinal epithelial cells and forms a key transporter to regulate fluid transit across the epithelium. Deficiency of CFTR dependent function due to genetic alterations in the Cftr gene causes cystic fibrosis (CF). CF is a multi-organ disorder, while the lung remains the most impacted organ due to recurrent episodes of infection that leads to inflammation. The role of autophagy in CF is now becoming increasingly recognized with some evidence of impaired autophagic clearance in CF macrophages causing low bactericidal activity, aggresome formation, and therapeutic benefit of autophagy modulators in the CF epithelial cells. [3][4][5] Autophagy can be stimulated by nutritional deprivation and pathogen insult. 6 Autophagy begins with the formation of an isolation membrane (phagophore) at the endoplasmic reticulum (ER)-mitochondrial interface, 7 with subsequent sequestering of a portion of cytoplasm and damaged organelles as autophagic intermediates continue to grow and form enclosed, double-membraned structures called

Translesion DNA synthesis is an essential process that helps resume DNA replication at forks stalled near bulky adducts on the DNA. Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon (PAH) that can be metabolically activated to benzo[a]pyrene diol epoxide (BPDE), which then can react with DNA to form carcinogenic DNA adducts. Here, we have used single-molecule florescence resonance energy transfer (smFRET) experiments, classical molecular dynamics simulations, and nucleotide incorporation assays to investigate the mechanism by which the model Y-family polymerase, Dpo4, bypasses a (+)-cis-B[a]P-N 2-dG adduct in DNA. Our data show that when (+)-cis-B[a]P-N 2-dG is the templating base, the B[a]P moiety is in a non-solvent exposed conformation stacked within the DNA helix, where it effectively blocks nucleotide incorporation across the adduct by Dpo4. However, when the media contains a small amount of dimethyl sulfoxide (DMSO), the adduct is able to move to a solvent-exposed conformation, which enables error-prone DNA replication past the adduct. When the primer terminates across from the adduct position, the addition of DMSO leads to the formation of an insertion complex capable of accurate nucleotide incorporation.

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