Escherichia coli RecQ Is a Rapid, Efficient, and Monomeric Helicase

Abstract

RecQ family helicases play a key role in chromosome maintenance. Despite extensive biochemical, biophysical, and structural studies, the mechanism by which helicase unwinds double-stranded DNA remains to be elucidated. Using a wide array of biochemical and biophysical approaches, we have previously shown that the Escherichia coli RecQ helicase functions as a monomer. In this study, we have further characterized the kinetic mechanism of the RecQ-catalyzed unwinding of duplex DNA using the fluorometric stopped-flow method based on fluorescence resonance energy transfer. Our results show that RecQ helicase binds preferentially to 3-flanking duplex DNA. Under the pre-steady-state conditions, the burst amplitude reveals a 1:1 ratio between RecQ and DNA substrate, suggesting that an active monomeric form of RecQ helicase is involved in the catalysis. Under the single-turnover conditions, the RecQ-catalyzed unwinding is independent of the 3-tail length, indicating that functional interactions between RecQ molecules are not implicated in the DNA unwinding. It was further determined that RecQ unwinds DNA rapidly with a step size of 4 bp and a rate of ϳ21 steps/s. These kinetic results not only further support our previous conclusion that E. coli RecQ functions as a monomer but also suggest that some of the Superfamily 2 helicases may function through an "inchworm" mechanism.Helicases are molecular motor proteins that use the energy of nucleotide triphosphate hydrolysis to translocate along and separate the complementary strands of a nucleic acid duplex. These enzymes play essential roles in most aspects of the DNA metabolic pathway, such as replication, repair, recombination, and transcription (1-5). A large number of helicases have been identified; however, the mechanisms by which helicases unwind double-stranded DNA (dsDNA) 3 remain obscure.One of the major concerns in studying the unwinding mechanism of a DNA helicase is its oligomeric state. This is because an oligomeric structure could provide multiple potential binding sites for the DNA substrate and nucleotide cofactors, which are absolutely required for the helicase to translocate along the DNA track during the processive DNA unwinding. Indeed, the biochemical and structural studies have shown that some helicases assemble into stable cooperative hexameric rings (3). These helicases include the Escherichia coli DnaB (6, 7) and Rho (8, 9), bacteriophage T4 gp41 (10, 11), and T7 gp4 (12)(13)(14). DNA passes through such a central channel and is unwound with a high processivity. For the non-ring helicases, the enzymes could be assembled into oligomers, and the DNA binding sites may be located on the separate subunits. On the basis of quantitative analyses of DNA binding properties, a "rolling model" was proposed to explain factorial DNA unwinding (15). This model suggests that each monomer of the Rep dimer binds alternatively to ssDNA and dsDNA. This process is regulated by repeated binding and hydrolysis of ATP and release of ADP. In this way, Rep dimer rolls along ...

RecQ helicases: Multiple structures for multiple functions?

“…The crystal structure of a RecQ 1-516 variant, lacking the HRDC domain, indicates that the catalytic core of E. coli RecQ is monomeric in its nucleotide bound and unbound forms . Although a previous study suggested that the full-length E. coli RecQ helicase might exist as a multimer comprising at least three subunits in solution (Harmon and Kowalczykowski, 2001), more recent biochemical and biophysical studies provided evidence that this enzyme remains in a monomeric form also when bound to single-stranded DNA and when unwinding DNA, in agreement with the crystallographic results (Xu et al, 2003;Zhang et al, 2006).…”

G-quadruplexes Significantly Stimulate Pif1 Helicase-catalyzed Duplex DNA Unwinding

“…Stopped-flow Fluorescence Measurements-The stopped-flow assays were carried out using a Bio-Logic SFM-400 mixer with a 1.5 ϫ 1.5-mm cell (FC-15, Bio-Logic), and the Bio-Logic MOS450/AF-CD optical system was equipped with a 150-watt mercury-xenon lamp (39). Fluorescein was excited at 492 nm (2-nm slit width), and its emission was monitored at 525 nm using a high pass filter with a 20-nm bandwidth (D525/20; Chroma Technology Co.).…”

Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

“…Paradoxically, at limiting concentrations, most RecQ helicases nonetheless efficiently unwind several kilobases of dsDNA in the course of their normal functions (9,16,(24)(25)(26), suggesting a dynamic unwinding process. Although RecQ can unwind DNA as a monomer, it also shows a functional cooperativity when unwinding DNA with an ssDNA tail (27)(28)(29). Thus, multiple monomers can bind to ssDNA to unwind long dsDNA regions.…”