Single-Molecule Fluorescence Reveals the Unwinding Stepping Mechanism of Replicative Helicase

摘要

class="head no_bottom_margin" id="sec1title">IntroductionDNA helicases are motor enzymes that convert the chemical energy of nucleotide triphosphate hydrolysis into mechanical energy for translocation on single-stranded DNA (ssDNA) and unwinding of double-stranded DNA (dsDNA) (, ). These enzymes encounter DNA sequences of different stabilities, and studying the effect of base pair stability can provide insights into the unwinding mechanisms (, ). Previous studies have shown that the unwinding rates of T7 helicase, T4 helicase, and hepatitis virus C NS3 helicase depend on the stability of nucleic acid base pairs (, , , ).T7 helicase (gp4A′ protein) has served as a good model system for hexameric helicases (, ). It assembles into a ring-shaped hexamer in the presence of deoxythymidine triphosphate (dTTP) and ssDNA (, ), and translocates on ssDNA in the 5′ to 3′ direction. It unwinds dsDNA using a strand-exclusion mechanism whereby it binds and moves along one strand of the dsDNA in the 5′ to 3′ direction while excluding the complementary strand from its central channel (, , href="#bib27" rid="bib27" class=" bibr popnode">Jezewska et al., 1998, href="#bib30" rid="bib30" class=" bibr popnode">Kaplan and O’Donnell, 2002).Using ensemble single-turnover kinetic analysis, href="#bib26" rid="bib26" class=" bibr popnode">Jeong et al. (2004) estimated the kinetic step size of unwinding by T7 helicase to be ∼10 bp. As defined, the kinetic step size provides an estimate of how often a recurrent rate-limiting step takes place during processive unwinding. However, the kinetic step size estimated from ensemble measurements can be inflated if there exists significant heterogeneity in the reaction rate among individual molecules (href="#bib49" rid="bib49" class=" bibr popnode">Park et al., 2010). In addition, the crystal structures of hexameric helicases published thus far have not given detailed information on the unwinding mechanisms because either the nucleic acid substrates were not included in the structures (href="#bib3" rid="bib3" class=" bibr popnode">Bailey et al., 2007, href="#bib19" rid="bib19" class=" bibr popnode">Gai et al., 2004, href="#bib36" rid="bib36" class=" bibr popnode">Li et al., 2003, href="#bib60" rid="bib60" class=" bibr popnode">Singleton et al., 2000, href="#bib65" rid="bib65" class=" bibr popnode">Wang et al., 2008) or only the single-stranded substrates were cocrystallized (href="#bib17" rid="bib17" class=" bibr popnode">Enemark and Joshua-Tor, 2006, href="#bib25" rid="bib25" class=" bibr popnode">Itsathitphaisarn et al., 2012, href="#bib63" rid="bib63" class=" bibr popnode">Thomsen and Berger, 2009). Although single-molecule techniques have provided detailed insights into the mechanisms of various helicases (href="#bib6" rid="bib6" class=" bibr popnode">Bianco et al., 2001, href="#bib8" rid="bib8" class=" bibr popnode">Cheng et al., 2007, href="#bib9" rid="bib9" class=" bibr popnode">Cheng et al., 2011, href="#bib11" rid="bib11" class=" bibr popnode">Dessinges et al., 2004, href="#bib12" rid="bib12" class=" bibr popnode">Dohoney and Gelles, 2001, href="#bib18" rid="bib18" class=" bibr popnode">Fili et al., 2010, href="#bib21" rid="bib21" class=" bibr popnode">Ha et al., 2002, href="#bib24" rid="bib24" class=" bibr popnode">Honda et al., 2009, href="#bib28" rid="bib28" class=" bibr popnode">Johnson et al., 2007, href="#bib31" rid="bib31" class=" bibr popnode">Karunatilaka et al., 2010, href="#bib33" rid="bib33" class=" bibr popnode">Klaue et al., 2013, href="#bib34" rid="bib34" class=" bibr popnode">Lee et al., 2006, href="#bib38" rid="bib38" class=" bibr popnode">Lionnet et al., 2007, href="#bib41" rid="bib41" class=" bibr popnode">Manosas et al., 2009, href="#bib44" rid="bib44" class=" bibr popnode">Myong et al., 2005, href="#bib45" rid="bib45" class=" bibr popnode">Myong et al., 2007, href="#bib46" rid="bib46" class=" bibr popnode">Myong et al., 2009, href="#bib49" rid="bib49" class=" bibr popnode">Park et al., 2010, href="#bib53" rid="bib53" class=" bibr popnode">Perkins et al., 2004, href="#bib54" rid="bib54" class=" bibr popnode">Qi et al., 2013, href="#bib61" rid="bib61" class=" bibr popnode">Spies et al., 2003, href="#bib62" rid="bib62" class=" bibr popnode">Sun et al., 2011), unwinding steps have not been detected for any hexameric helicase. In this report, we used single-molecule fluorescence resonance energy transfer (smFRET) (href="#bib20" rid="bib20" class=" bibr popnode">Ha et al., 1996) to measure real-time DNA unwinding by individual T7 helicase molecules. Taking advantage of the sequence-dependent unwinding rate, we used a designed DNA substrate to find a relation between FRET efficiency and the number of base pairs unwound. From substrates with a high guanine-cytosine (GC) content, we could detect the individual steps of DNA unwinding and analyze their kinetics.