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

Abstract

DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40-60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.recombination | fluorescence | TIRF microscopy | DNA repair | BLM

Dissociation of Rad51 Presynaptic Complexes and Heteroduplex DNA Joints by Tandem Assemblies of Srs2

“…In many cases, there is evidence that helicases can behave as tandem assemblies while acting upon nucleic acids, and that the changes in their oligomeric state regulate their activities (Lohman et al, 2008). Specific examples of tandem helicase assemblies include the bacteriophage T4 SF1 helicase DdaA (Byrd and Raney, 2004), the hepatitis C virus Sf2 helicase NS3 (Levin et al, 2004), and E. coli RecQ (Rad et al, 2015). In addition, recent studies have shown that E. coli UvrD translocates either as a monomer or as two tandem monomers, and that the tandem monomers are more processive than a single monomer (Comstock et al, 2015; Lee et al, 2013).…”

Processivity, velocity and universal characteristics of nucleic acid unwinding by helicases

“…Helicases are enzymes that play an important role in almost every aspect of RNA and DNA metabolism [1][2][3][4][5][6][7]. This class of molecular motors, which has been grouped into six super families, utilize the free energy from ATP hydrolysis to translocate directionally over hundreds of bases on single-strand (ss) nucleic acids.…”

Direct Fluorescent Imaging of Translocation and Unwinding by Individual DNA Helicases

“…A complete understanding requires direct measurement of individual events that make up the distribution of behaviors that comprise the ensemble. In recent decades, we have used single-molecule imaging to visualize the movement and actions of DNA motor proteins (Amitani, Baskin, & Kowalczykowski, 2006; Bianco et al, 2001; Handa, Bianco, Baskin, & Kowalczykowski, 2005; Liu, Baskin, & Kowalczykowski, 2013; Nimonkar, Amitani, Baskin, & Kowalczykowski, 2007; Rad, Forget, Baskin, & Kowalczykowski, 2015; Spies, Amitani, Baskin, & Kowalczykowski, 2007; Spies et al, 2003; Amitani, Liu, Dombrowski, Baskin, & Kowalczykowski, 2010; Forget, Dombrowski, Amitani, & Kowalczykowski, 2013; Hilario & Kowalczykowski, 2010; Sun & Wang, 2016). Here, we limit ourselves to a description of some of those methods.…”

“…However, the study of protein–DNA interactions at the single-molecule level is supported by a wide range of complementary techniques, including, but not limited to, atomic force microscopy, electron microscopy, force spectroscopy, fluorescence correlation spectroscopy, Förster resonance energy transfer, magnetic tweezers, optical traps combined with fluorescence microscopy, and total internal reflection fluorescence (TIRF) microscopy (Bustamante, Bryant, & Smith, 2003; Greenleaf, Woodside, & Block, 2007; Ha, Kozlov, & Lohman, 2012; Kapanidis & Strick, 2009; Lionnet et al, 2012; Moffitt, Chemla, Smith, & Bustamante, 2008; Neuman & Nagy, 2008; Qi & Greene, 2016; Roy, Hohng, & Ha, 2008; Spies, 2013; Yodh, Schlierf, & Ha, 2010). Mechanical manipulation of individual DNA molecules by optical trapping of attached microspheres or by attachment to a surface has been a useful platform for studying the mechanisms of DNA helicases and translocases (Amitani et al, 2006, 2010; Bianco et al, 2001; Forget et al, 2013; Hilario & Kowalczykowski, 2010; Liu et al, 2013; Rad et al, 2015; Sun & Wang, 2016). …”