The highly conserved Superfamily 1 (SF1) and Superfamily 2 (SF2) nucleic acid-dependent ATPases, are ubiquitous motor proteins with central roles in DNA and RNA metabolism (Jankowsky & Fairman, 2007). These enzymes require RNA or DNA binding to stimulate ATPase activity, and the conformational changes that result from this coupled behavior are linked to a multitude of processes that range from nucleic acid unwinding to the flipping of macromolecular switches (Pyle, 2008, 2011). Knowledge about the relative affinity of nucleic acid ligands is crucial for deducing mechanism and understanding biological function of these enzymes. Because enzymatic ATPase activity is directly coupled to RNA binding in these proteins, one can utilize their ATPase activity as a simple reporter system for monitoring functional binding of RNA or DNA to an SF1 or SF2 enzyme. In this way, one can rapidly assess the relative impact of mutations in the protein or the nucleic acid and obtain parameters that are useful for setting up more quantitative direct binding assays. Here, we describe a routine method for employing NADH-coupled enzymatic ATPase activity to obtain kinetic parameters reflecting apparent ATP and RNA binding to an SF2 helicase. First, we provide a protocol for calibrating an NADH-couple ATPase assay using the well-characterized ATPase enzyme hexokinase, which a simple ATPase enzyme that is not coupled with nucleic acid binding. We then provide a protocol for obtaining kinetic parameters (KmATP, Vmax and KmRNA) for an RNA-coupled ATPase enzyme, using the double-stranded RNA binding protein RIG-I as a case-study. These approaches are designed to provide investigators with a simple, rapid method for monitoring apparent RNA association with SF2 or SF1 helicases.

Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The N-terminal motor domain of the AdnB subunit hydrolyzes ATP to drive rapid and processive 3\' to 5\' translocation of AdnAB on the tracking DNA strand. ATP hydrolysis is mechanically productive when oscillating protein domain motions synchronized with the ATPase cycle propel the DNA tracking strand forward by a single-nucleotide step, in what is thought to entail a pawl-and-ratchet-like fashion. By gauging the effects of alanine mutations of the 16 amino acids at the AdnB-DNA interface on DNA-dependent ATP hydrolysis, DNA translocation, and DSB resection in ensemble and single-molecule assays, we gained key insights into which DNA contacts couple ATP hydrolysis to motor activity. The results implicate AdnB Trp325, which intercalates into the tracking strand and stacks on a nucleobase, as the singular essential constituent of the ratchet pawl, without which ATP hydrolysis on ssDNA is mechanically futile. Loss of Thr663 and Thr118 contacts with tracking strand phosphates and of His665 with a nucleobase drastically slows the AdnAB motor during DSB resection. Our findings for AdnAB prompt us to analogize its mechanism to that of an automobile clutch.

Replicative helicases are ring-shaped hexamers that encircle DNA for duplex unwinding. The currently accepted view of hexameric helicase function is by steric exclusion, where the helicase encircles one DNA strand and excludes the other, acting as a wedge with an external DNA unwinding point during translocation. Accordingly, strand-specific blocks only affect these helicases when placed on the tracking strand, not the excluded strand. We examined the effect of blocks on the eukaryotic CMG and, contrary to expectations, blocks on either strand inhibit CMG unwinding. A recent cryoEM structure of yeast CMG shows that duplex DNA enters the helicase and unwinding occurs in the central channel. The results of this report inform important aspects of the structure, and we propose that CMG functions by a modified steric exclusion process in which both strands enter the helicase and the duplex unwinding point is internal, followed by exclusion of the non-tracking strand.

RecQ helicases feature multiple domains in their structure, of which the helicase domain, the RecQ-Ct domain and the HRDC domains are well conserved among the SF2 helicases. The helicase domain and the RecQ-Ct domain constitute the catalytic core of the enzyme. The domain interfaces are the DNA binding sites which display significant conformational changes in our molecular dynamics simulation studies. The preferred conformational states of the DNA bound and unbound forms of RecQ appear to be quite different from each other. DNA binding induces inter-domain flexibility leading to hinge mobility between the domains. The divergence in the dynamics of the two structures is caused by changes in the interactions at the domain interface, which seems to propagate along the whole protein structure. This could be essential in ssDNA binding after strand separation, as well as aiding translocation of the RecQ protein like an inch-worm.