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Recq Helicase Translocates Along Single-Stranded DNA with a Moderate Processivity and Tight Mechanochemical Coupling

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Recq Helicase Translocates Along Single-Stranded DNA with a Moderate Processivity and Tight Mechanochemical Coupling RecQ helicase translocates along single-stranded DNA with a moderate processivity and tight mechanochemical coupling Kata Sarlós, Máté Gyimesi, and Mihály Kovács1 Department of Biochemistry, Eötvös Loránd University - Hungarian Academy of Sciences, “Momentum” Motor Enzymology Research Group, Eötvös Loránd University, H-1117, Budapest, Hungary Edited* by Stephen C. Kowalczykowski, University of California, Davis, CA, and approved May 8, 2012 (received for review September 2, 2011) Maintenance of genome integrity is the major biological role of characterized by diffusion along the DNA strand in alternating RecQ-family helicases via their participation in homologous re- weak and strong binding states, was used to describe the trans- combination (HR)-mediated DNA repair processes. RecQ helicases location of hepatitis C virus NS3 helicase (19). Because of the exert their functions by using the free energy of ATP hydrolysis low coupling between the enzymatic (ATPase) and mechanical for mechanical movement along DNA tracks (translocation). In (translocation) cycles, ratchet mechanisms usually lead to the addition to the importance of translocation per se in recombina- consumption of more than one ATP molecule per nucleotide tion processes, knowledge of its mechanism is necessary for the traveled. In contrast to the above enzymes, although the biological understanding of more complex translocation-based activities, in- functions of E. coli RecQ are well described (20–23), mechanistic cluding nucleoprotein displacement, strand separation (unwind- knowledge of the underlying molecular processes is scarce. ing), and branch migration. Here, we report the key properties of DNA activates the ATPase activity of RecQ, and the ATP the ssDNA translocation mechanism of Escherichia coli RecQ heli- hydrolysis cycle is coupled to DNA unwinding (22, 24). DNA case, the prototype of the RecQ family. We monitored the pre– unwinding by RecQ is driven by translocation along the track steady-state kinetics of ATP hydrolysis by RecQ and the dissocia- strand in a 3′-to-5′ direction (2, 22). Recent studies on the tion of the enzyme from ssDNA during single-round translocation. working mechanism of RecQ focused on its unwinding activity BIOCHEMISTRY We also gained information on the translocation mechanism from (25–27). Here, we report the key parameters of the translocation the ssDNA length dependence of the steady-state ssDNA-activated mechanism of RecQ (Fig. 1). ssDNA enhances the ATPase ac- ATPase activity. We show that RecQ occludes 18 ± 2 nt on ssDNA tivity of RecQ in a length-dependent manner, which allows the during translocation. The hydrolysis of ATP is noncooperative in extraction of mechanistic information from steady-state and the presence of ssDNA, indicating that RecQ active sites work in- transient profiles of translocation-coupled ATP consumption. dependently during translocation. In the applied conditions, the We show that RecQ binding occludes about 18 ± 2 nt on ssDNA. − enzyme hydrolyzes 35 ± 4 ATP molecules per second during ssDNA Translocation is performed at a cycling rate of 35 ± 4s 1 with translocation. RecQ translocates at a moderate processivity, with a mechanochemical coupling of 1.1 ± 0.2 ATP consumed per a mean run length of 100–320 nt on ssDNA. The determined tight nucleotide traveled. RecQ translocates along ssDNA with mechanochemical coupling of 1.1 ± 0.2 ATP consumed per nucle- a moderate processivity, performing a mean of 110–350 ATPase otide traveled indicates an inchworm-type mechanism. cycles while traveling 100–320 nt along ssDNA in a single run, as determined in different conditions. Mechanistic insights into the elicases are NTP-driven nucleic acid motors that couple the translocation mechanism will aid the understanding of trans- Hliberation of chemical energy to mechanical movement location-based activities, including dsDNA unwinding, branch along nucleic acid strands to exert diverse biological activities. migration, nucleoprotein displacement, replication fork restart, Escherichia coli RecQ helicase is the prototype of its family HR regulation, and catenation. [belonging to superfamily (SF) 2], whose members are essential in genome maintenance from bacteria to humans (1–3) by Results playing roles in homologous recombination (HR)-mediated RecQ Active Sites Work Independently both in the Absence and dsDNA break (DSB) repair (4–7). In humans, loss-of-function Presence of ssDNA. We measured the ATP concentration de- mutations of at least three RecQ helicase homologs are linked to pendence of the steady-state ATPase activity of RecQ by using a diseases (mostly Bloom, Werner, and Rothmund–Thomson pyruvate kinase-lactate dehydrogenase (PK-LDH)–linked en- syndromes), which are generally characterized by genome in- zyme assay both in the absence and presence of thymidine oli- stability resulting in cancer predisposition and premature aging gonucleotide of 54-nt length (dT54)(Fig. S1). Based on Hill (8). E. coli RecQ suppresses illegitimate recombination (9) and analysis, RecQ showed no cooperativity [the Hill coefficient (n) regulates HR-mediated DSB repair via initiating HR and pro- was 1.2 ± 0.2 and 1.0 ± 0.1 in the absence and presence of dT54, cessing HR intermediates (2). Moreover, RecQ stabilizes stalled respectively]. These data suggest that RecQ active sites work replication forks as part of the RecF pathway (10, 11). independently in these conditions. The steady-state KATP Different types of translocation mechanisms were proposed (Michaelis constant for ATP) and saturating DNA concentration for SF1 and SF2 helicases. An inchworm mechanism was pro- (kcat) data were in line with previous results (28, 29) (Fig. S1). posed for bacterial PcrA (12, 13), UvrD (14), and human RecQ- family Bloom syndrome (BLM) (15) helicases, which perform unidirectional translocation based on the consumption of one Author contributions: K.S., M.G., and M.K. designed research; K.S. performed research; ATP per kinetic cycle (dictated by a single rate-limiting step), K.S., M.G., and M.K. analyzed data; and K.S., M.G., and M.K. wrote the paper. leading to a single nucleotide translocation along ssDNA. A The authors declare no conflict of interest. more complex, nonuniform inchworm stepping mechanism was *This Direct Submission article had a prearranged editor. suggested for UvrD (16), NS3 (17), and RecBCD (18) helicases. Freely available online through the PNAS open access option. In this model, multiple rapid ATP hydrolysis-coupled trans- 1To whom correspondence should be addressed. E-mail: [email protected]. location steps occur during a single kinetic cycle between two This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. successive rate-limiting steps. A Brownian ratchet mechanism, 1073/pnas.1114468109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1114468109 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 Fig. 1. Model for RecQ translocation along ssDNA. (Top) RecQ (red triangle) binds to an ssDNA strand of track length L (expressed in nucleotides) and occludes a site of b nucleotides in length. (Middle) On ATP-driven translocation, the enzyme travels s nucleotides per ATP molecule consumed, defining the mechanochemical coupling ratio C (= 1/s; number of ATP molecules hydrolyzed per nucleotide traveled). RecQ performs ATPase activities of kATP,trans and kATP,end during translocation and on reaching the end of the DNA track, respectively. (Bottom) Enzyme dissociates at rate constants koff,int and koff,end from internal sites on DNA and the 5′-end, respectively. Processivity, expressed as the probability of performing the oncoming ATP-consuming translocation cycle as opposed to dissociation, is defined as P = kATP,trans/(kATP,trans + koff,int). The mean number of ATP molecules hydrolyzed (single-round ATP consumption amplitude) and nucleotides traveled (run length) in a processive run will thus be <nATP> = P/(1 − P) and <nnt> = <nATP>/C, respectively (15, 30, 31). In this study, we determined the above-mentioned parameters by using optical signals reporting the appearance of ATP hydrolysis products ADP (by NADH ab- sorbance using a PK-LDH linked assay, orange) and Pi (by using a fluorescently labeled Pi binding protein, MDCC-PBP, yellow), as well as the intrinsic Trp fluorescence of the enzyme (green), which increases on dissociation from DNA. In single-round pre–steady-state experiments, we applied DxSO4 (blue) as a protein trap to prevent rebinding of RecQ to DNA after finishing the first processive run on ATP addition. Single-Round Translocation Experiments Reveal Moderate Processivity by using the fluorescent signal of a bacterial Pi binding protein and Tight Mechanochemical Coupling. Precise monitoring of the (MDCC-PBP) (15, 30) (Fig. 2A). In these experiments we applied – pre steady-state kinetics of ATP hydrolysis in single-round dextran sulfate (DxSO4) as a protein trap to ensure single-round translocation conditions allows the determination of mechanistic conditions. RecQ was preincubated with oligo-dT substrates of parameters of translocation (Fig. 1). Single-round conditions can different length (Fig. 2 A–C) or M13 phage circular ssDNA (Fig. be achieved by using a protein trap that inhibits the rebinding of 2C), and it was then rapidly mixed with ATP plus DxSO4 in the the enzyme to DNA after dissociation. As discussed earlier, the stopped-flow apparatus. The time courses of the reaction showed amplitude of ATP hydrolysis during a processive run (moles of two phases with a
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