Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10051-10056, September 1996 Biochemistry ATPase activity of Escherichia coli Rep helicase crosslinked to single-stranded DNA: Implications for ATP driven helicase translocation (kinetics/mechanism/protein-DNA crosslinking/protein oligomerization/energy transduction) ISAAC WONG AND TIMOTHY M. LOHMANt Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, Box 8231, St. Louis, MO 63110 Communicated by Carl Frieden, Washington University School of Medicine, St. Louis, MO, May 22, 1996 (received for review March 24, 1996) ABSTRACT To examine the coupling ofATP hydrolysis to translocate unidirectionally along ssDNA, although direct evi- helicase translocation along DNA, we have purified and dence in support of this is lacking. On the other hand, we (14) characterized complexes of the Escherichia coli Rep protein, a have shown that unidirectional translocation along ssDNA is not dimeric DNA helicase, covalently crosslinked to a single- essential for DNA unwinding by the E. coli Rep helicase, a stranded hexadecameric oligodeoxynucleotide (S). Crosslinked 3' ->5' DNA helicase. Furthermore, we have proposed an active, Rep monomers (PS) as well as singly ligated (P2S) and doubly rolling model for Rep-catalyzed DNA unwinding (13) based on ligated (P2S2) Rep dimers were characterized. The equilibrium our finding that one subunit ofthe Rep dimer binds directly to the and kinetic constants for Rep dimerization as well as the duplex region to be unwound while the other subunit binds to steady-state ATPase activities of both PS and P2S crosslinked ssDNA (13, 14), and we noted that such a rolling model could also complexes were identical to the values determined for un- be envisioned as a mechanism for translocation of the Rep dimer crosslinked Rep complexes formed with dT16. Therefore, ATP along ssDNA. hydrolysis by both PS and P2S complexes are not coupled to DNA To distinguish between these mechanisms for helicase trans- dissociation. This also rules out a strictly unidirectional sliding location along ssDNA, we reasoned that any directional bias in mechanism for ATP-driven translocation along single-stranded translocation requires coupling of ATP hydrolysis to movement. DNA by either PS or the P2S dimer. However, ATP hydrolysis by In particular, a strictly unidirectional sliding mechanism (no the doubly ligated P2S2 Rep dimer is coupled to single-stranded "slippage"), in which the same protein subunit maintains contact DNA dissociation from one subunit ofthe dimer, although loosely with the ssDNA during translocation, would require tight cou- (low efficiency). These results suggest that ATP hydrolysis can pling to hydrolysis, while a "rolling" mechanism without direc- drive translocation of the dimeric Rep helicase along DNA by a tional bias would not. Toward this end, we characterized the "rolling" mechanism where the two DNA binding sites of the ATPase activities of covalently crosslinked Rep-single-stranded- dimer alternately bind and release DNA. Such a mechanism is oligodeoxynucleotide complexes, reasoning that severe inhibition biologically important when one subunit binds duplex DNA, of the ATPase activities of such complexes would result if followed by subsequent unwinding. ATP-driven translocation occurs by a strictly unidirectional slid- ing mechanism. Such crosslinked Rep-ssDNA complexes will DNA helicases are motor proteins that use the chemical energy also provide a useful means for studying putative intermediates of nucleoside 5'-triphosphate (NTP) hydrolysis to perform the in the DNA unwinding reaction. mechanical work of disrupting the base pairs between comple- mentary strands of duplex DNA to form single-stranded DNA MATERIALS AND METHODS (ssDNA) intermediates (1, 2). These molecular motors are es- Reagents and Buffers. [a-32P]ATP (3000 Ci/mmol; 1 Ci = 37 sential components of most DNA metabolic and processing GBq) was obtained from Amersham. Spectrophotometric grade machineries in all organisms (2), and mutations in helicases glycerol, HPLC grade methanol, and 99% triethylamine were involved in DNA repair processes have been linked to a number obtained from Aldrich. Sulfo-N-succinimidyl-6-(4'-azido-2'- of human skin cancers (3-5). Despite the importance of these nitrophenylamino)hexanoate (sulfo-SANPAH) was obtained enzymes and the large number of DNA helicases that have been from Pierce. Amino-modifier-C2-dT phosphoramidite was ob- identified, mechanistic studies on these enzymes have only begun tained from Glen Research (Sterling, VA). All solutions were recently (for reviews, see refs. 1, 6, and 7). made with reagent grade chemicals, except as noted above, using DNA helicases must translocate along DNA to unwind DNA Milli-Q H20-i.e., distilled H20 that was de-ionized using a processively and thus have features in common with motor Milli-Q Water Purification System (Millipore). All binding ex- proteins such as cytoplasmic kinesin (8-10). DNA helicases periments and ATPase assays were carried out in BBM buffer [20 appear generally to be oligomeric, primarily dimeric or hexameric mM Tris HCl, pH 7.5 at 4°C/6 mM NaCl/5 mM MgCl2/10% (1, 6, 7); for example, the Escherichia coli Rep helicase functions (vol/vol) glycerol]. Triethyl-ammonium bicarbonate buffer as a dimer (11), with both subunits able to bind DNA and ATP (TEAB, 1 M) was made by bubbling CO2 (g) derived from (10, 12, 13). Of particular mechanistic interest is how helicases subliming dry ice into an aqueous solution containing >1 M translocate along DNA and how this is coupled to NTP hydro- triethylamine at 0°C for 4 hr or until a pH of 7.5 was achieved; lysis. In this regard, most helicases display a macroscopic "polar- Milli-Q H20 was then added to achieve a final concentration of ity" in DNA unwinding assays in vitro-i.e., unwinding by most 1 M. Kinase buffer was 50 mM Tris HCl (pH 7.5 at 25°C), 10 mM helicases is stimulated if the DNA duplex possesses a 5' ssDNA MgCl2, and 10 mM 2-mercaptoethanol. "tail" flanking the duplex (a 5' ->3' helicase) or a 3' ssDNA tail Proteins, Enzymes, and Oligodeoxynucleotides. E. coli Rep (a 3' ->5' helicase). This observation has led to the suggestion protein was purified to >99% homogeneity from E. coli MZ-1/ that helicases couple the energy derived from ATP hydrolysis to Abbreviations: ssDNA, single-stranded DNA; sulfo-SANPAH, sulfo- The publication costs of this article were defrayed in part by page charge N-succinimidyl-6-(4'-azido-2'-nitro-phenylamino)hexanoate. payment. This article must therefore be hereby marked "advertisement" in tTo whom reprint requests should be addressed. e-mail: accordance with 18 U.S.C. §1734 solely to indicate this fact. [email protected]. 10051 Downloaded by guest on September 27, 2021 10052 Biochemistry: Wong and Lohman Proc. Natl. Acad. Sci. USA 93 (1996) pRepO (15) as described (16). Rep concentration was deter- sample to 25 ml and the sample was dialyzed for 6 h against mined spectrophotometrically using s20 = 7.68 x 104 M-l cm-I 1 liter of the same buffer containing 100 mM NaCl. The for Rep monomer (14). An ATPase-deficient mutant of Rep, dialysate was reloaded onto the Macro-Prep Hi-Q column K281, in which Lys-28 was replaced with Ile, was constructed and that had been reequilibrated with 100 mM NaCl. The column purified to >99% purity (unpublished data). T4 polynucleotide was rinsed with 10 ml buffer containing 100 mM NaCl and kinase was purchased from United States Biochemical. Oligode- eluted with 1 M NaCl as before. The fractions containing oxynucleotides dT16, d(T3C2T12), d(T7C2T8), d(T12C2T3), where protein and DNA were pooled and dialyzed against 5x C2 denotes amino-modifier-C2-dT, were synthesized using an binding buffer. Due to the thermodynamic stability of P2S Applied Biosystems model PCR-mate 391 DNA synthesizer and and the negative cooperativity of binding DNA to P2S to were purified to >99% homogeneity as described (17) and form P2S2, the purified crosslinked mixture contained a signif- dialyzed (Spectra/Por 7 MWCO 1000) into Milli-Q H20 for icant fraction of uncrosslinked Rep subunits in the form of P2S. storage. Oligodeoxynucleotide concentrations were determined However, all uncrosslinked DNA has been removed. spectrophotometrically in 10 mM Tris-HCl (pH 7.5), 1 mM ATPase Assay. ATPase activity was determined at 4°C by EDTA, and 150 mM NaCl at 25°C using 8260 = 1.29x105 measuring the initial rate of conversion ofATP to ADP using M-l cm-1 per molecule. [a-32P]ATP (Amersham) in BBM buffer. Reactions were Synthesis and Purification ofd(T7S2T8). Solid sulfo-SANPAH typically initiated by addition of 10 ,ul of 10 mM ATP to 90 was added to d(T7C2T8) (350 ,uM) in 0.5 M Na-carbonate ,ul protein. At regular time intervals, 10 ,lI aliquots were (pH 9.0) buffer to a final concentration of 25 mM and removed and quenched into 10 ,ul 0.5 M EDTA (pH 8.5). allowed to react in the dark at 25°C for .4 h. Three volumes Intervals between time points ranged from S to 20 s depend- of ice-cold ethanol was added and the sample chilled at ing on the anticipated rate of hydrolysis. Eight to 10 time -20°C for 1 h to precipitate the DNA. The large bright points were taken per assay, but only the linear portion of orange pellet was collected by centrifugation at 4°C and each time course, <60% product formation, was used to rinsed with 1 volume of 1,4-dioxane, followed by 1 volume of determine the initial velocity. The extent of ADP formation 100% ethanol, then dried and resuspended in Milli-Q H20 was monitored by spotting 1 ,lI onto polyethyleneimine- at 55°C for 5 min. The sample was chilled on ice for 30 min cellulose TLC plates (Merck).