Kinetic Mechanism of DNA Binding and DNA-Induced Dimerization of the Escherichia coli Rep Helicase † (original) (raw)
Related papers
Biochemistry, 1998
Escherichia coli Rep helicase is a DNA motor protein that unwinds duplex DNA as a dimeric enzyme. Using fluorescence probes positioned asymmetrically within a series of single-stranded (ss) oligodeoxynucleotides, we show that ss-DNA binds with a defined polarity to Rep monomers and to individual subunits of the Rep dimer. Using fluorescence resonance energy transfer and stopped-flow techniques, we have examined the mechanism of ss-oligodeoxynucleotide binding to preformed Rep dimers in which one binding site is occupied by a single-stranded oligodeoxynucleotide, while the other site is free (P 2 S dimer). We show that ss-DNA binding to the P 2 S Rep dimer to form the doubly ligated P 2 S 2 dimer occurs by a multistep process with the initial binding step occurring relatively rapidly with a bimolecular rate constant of k 1) ∼2 × 10 6 M-1 s-1 [20 mM Tris (pH 7.5), 6 mM NaCl, 5 mM MgCl 2 , 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol, 4°C]. A minimal kinetic mechanism is proposed which suggests that the two strands of ss-DNA bound to the Rep homodimer are kinetically distinct even within the P 2 S 2 Rep dimer, indicating that this dimer is functionally asymmetric. The implications of these results for the mechanisms of DNA unwinding and translocation by the functional Rep dimer are discussed.
Allosteric Effects of Nucleotide Cofactors on Escherichia coli Rep Helicase&DNA Binding
Science, 1992
The Escherichia coli Rep helicase unwinds duplex DNA during replication. The functional helicase appears to be a dimer that forms only on binding DNA. Both protomers of the dimer can bind either single-stranded or duplex DNA. Because binding and hydrolysis of adenosine triphosphate (ATP) are essential for helicase function, the energetics of DNA binding and DNA-induced Rep dimerization were studied quantitatively in the presence of the nucleotide cofactors adenosine diphosphate (ADP) and the nonhydrolyzable ATP analog AMPP(NH)P. Large allosteric effects of nucleotide cofactors on DNA binding to Rep were observed. Binding of ADP favored Rep dimers in which both protomers bound singlestranded DNA, whereas binding of AMPP(NH)P favored simultaneous binding of both single-stranded and duplex DNA to the Rep dirner. A rolling model for the active unwinding of duplex DNA by the dirneric Rep helicase is proposed that explains vectorial unwinding and predicts that helicase translocation along DNA is coupled to ATP binding, whereas ATP hydrolysis drives unwinding of multiple DNA base pairs for each catalytic event. Rep-DNA complexes. Dia formed transiently as a result of thermal grammatic fluctuations in the DNA duplex at an untion of the Rep P5.s P5.D p7s7 P2D2 P2SD-The authors are in the Department of Biochemistty and species that Half-s;urated Ilgatlon states Fully saturated ligated states Molecular Biophysics, Washington University School in the presence of Medicine.
Journal of Molecular Biology, 1999
Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in reactions that are coupled to ATP binding and hydrolysis. We have investigated the kinetic mechanism of ATP binding and hydrolysis by a proposed intermediate in Rep-catalyzed DNA unwinding, the Rep``P 2 S'' dimer (formed with the single-stranded (ss) oligodeoxynucleotide, (dT) 16), in which only one subunit of a Rep homo-dimer is bound to ssDNA. Pre-steady-state quenched-¯ow studies under both single turnover and multiple turnover conditions as well as¯uorescence stopped-¯ow studies were used (4 C, pH 7.5, 6 mM NaCl, 5 mM MgCl 2 , 10 % (v/v) glycerol). Although steady-state studies indicate that a single ATPase site dominates the kinetics (k cat 17(AE2) s À1 ; K M 3 mM), pre-steady-state studies provide evidence for a two-ATP site mechanism in which both sites of the dimer are catalytically active and communicate allosterically. Single turnover ATPase studies indicate that ATP hydrolysis does not require the simultaneous binding of two ATP molecules, and under these conditions release of product (ADP-P i) is preceded by a slow rate-limiting isomerization ($0.2 s À1). However, product (ADP or P i) release is not rate-limiting under multiple turnover conditions, indicating the involvement of a second ATP site under conditions of excess ATP. Stopped-¯ow¯uorescence studies monitoring ATP-induced changes in Rep's tryptophan¯uorescence displayed biphasic time courses. The binding of the ®rst ATP occurs by a two-step mechanism in which binding (k 1 1.5(AE0.2) Â 10 7 M À1 s À1 , k À1 29(AE2) s À1) is followed by a protein conformational change (k 2 23(AE3) s À1), monitored by an enhancement of Trp¯uorescence. The second Trp¯uorescence quenching phase is associated with binding of a second ATP. The ®rst ATP appears to bind to the DNA-free subunit and hydrolysis induces a global conformational change to form a high energy intermediate state with tightly bound (ADP-P i). Binding of the second ATP then leads to the steady-state ATP cycle. As proposed previously, the role of steady-state ATP hydrolysis by the DNA-bound Rep subunit may be to maintain the DNA-free subunit in an activated state in preparation for binding a second fragment of DNA as needed for translocation and/or DNA unwinding. We propose that the roles of the two ATP sites may alternate upon binding DNA to the second subunit of the Rep dimer during unwinding and translocation using a subunit switching mechanism.
Biochemistry, 1994
The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in a reaction that is coupled to ATP binding and hydrolysis. The Rep protein is a stable monomer in the absence of DNA but dimerizes upon binding either single-stranded or duplex DNA, and the dimer appears to be the functionally active form of the Rep helicase. As a first step toward understanding how ATP binding and hydrolysis are coupled energetically to DNA unwinding, we have investigated the kinetic mechanism of nucleotide binding to the Rep monomer (P) using stopped-flow techniques and the fluorescent ATP analogue, 2'(3')-O-(N-methylanthraniloyl-ATP (mantATP). The fluorescence of mantATP is enhanced upon Rep binding due to energy transfer from tryptophan. The results are consistent with the following two-step mechanism, in which the bimolecular association step is followed by a conformational change in the P-mantATP complex: P-I-mantATP-P-mantATP c* (P-mantATP)*. The following rate and equilibrium constants were determined at 4 "C in 20 mM Tris-HC1 (pH 7.5), 6 mM NaC1, 5 mM MgCl2, and 10% (v/v) glycerol: k+l = (1.1 f 0.2) x lo7 M-' s-l; k-1 = 3.2 (f0.5) s-l; k+2 = 2.9 Koverdl = K1K2 = (2.30 f 0.6) x lo8 M-l. Similar rate and equilibrium constants are obtained with mantATPyS, whereas the apparent rate constant for mantAMPPNP binding is 15-fold lower than for mantATP and equilibrium binding is weaker (Koverdl-lo6 M-l). Rep monomer does bind mantATP in the absence of Mg2+ (Koverdl-5 x los M-l), although the four rate constants in the above reaction increase by at least 8-fold (k-1 and k-2 increase by-100and-lOOO-fold, respectively). The affinities of Mg2+ for P-mantATP and (P-mantATP)* are 10-and 1000-fold higher than those for nucleotidefree Rep monomer, indicating that the second step in the reaction is associated with a marked increase in Mg2+ affinity. The bound Mg2+ in a (P-mantATP)*-Mg2+ complex dissociates at a rate that is comparable to the rate of mantATP release. Single-tumover kinetic studies with the Rep monomer indicate a low, but significant, DNA-independent ATPase activity, with a first-order cleavage rate constant of s-l at 4 "C, which increases with temperature (Eact = 18 f 2 kcal mol-'). These results indicate that Rep is a DNA-stimulated ATPase rather than a DNA-dependent ATPase. The absence of a burst of ADP formation in a multiple ATP tumover experiment suggests that product release is not rate-limiting under these conditions. The approaches described here and in the accompanying paper (Moore & Lohman, 1994) will be useful for subsequent studies of the more complex Rep dimeric species and of other helicases. k+l k+2 k-1 k-2 (f0.5) s-'; k-2 = 0.04 (f0.005) s-'; K1 = k+l/k-1 = (3.4 f 0.8) x lo6 M-'; K2 = k+2/k-2 = 73 (f10); DNA helicases are essential enzymes that function in DNA replication, recombination, and repair to catalyze the unwinding of double-stranded DNA (ds-DNA') to yield the single-stranded DNA (ss-DNA) intermediates that are required during these processes [for reviews, see Matson and +This work was supported in part by grants to T.M.L. from the American Cancer Society (NP-756B) and the NIH (GM 45948) and by a William M. Keck Foundation Fellowship to K.J.M.M.
Journal of Molecular Biology, 1996
DNA helicases are motor proteins that unwind duplex DNA during DNA replication, recombination and repair in reactions that are coupled to ATP and Molecular Biophysics binding and hydrolysis. In the process of unwinding duplex DNA Box 8231, Washington processively, DNA helicases must also translocate along the DNA filament. University School of Medicine, 660 S. Euclid Ave. To probe the mechanism of ATP-driven translocation by the dimeric E. coli Rep helicase along single stranded (ss) DNA, we examined the effects of St. Louis, MO 63110, USA ATP on the dissociation kinetics of ssDNA from the Rep dimer. Stopped-flow experiments show that the dissociation rate of a fluorescent ss oligodeoxynucleotide bound to one subunit of the dimeric Rep helicase is stimulated by ssDNA binding to the other subunit, and that the rate of this ssDNA exchange reaction is further stimulated 060-fold upon ATP hydrolysis. This ssDNA exchange process occurs via an intermediate in which ssDNA is transiently bound to both subunits of the Rep dimer. These results suggest a rolling or subunit switching mechanism for processive ATP-driven translocation of the dimeric Rep helicase along ssDNA. Such a mechanism requires the extreme negative cooperativity for DNA binding to the second subunit of the Rep dimer, which insures that the doubly DNA-ligated Rep (P 2 S 2) dimer is formed only transiently and relaxes back to the singly ligated Rep (P 2 S) dimer. The fact that other oligomeric DNA helicases share many functional features with the dimeric Rep helicase suggests that similar mechanisms for translocation and DNA unwinding may apply to other dimeric as well as hexameric DNA helicases.
Biochemistry, 1996
To examine the coupling ofATP hydrolysis to helicase translocation along DNA, we have purified and characterized complexes of the Escherichia coli Rep protein, a dimeric DNA helicase, covalently crosslinked to a singlestranded hexadecameric oligodeoxynucleotide (S). Crosslinked Rep monomers (PS) as well as singly ligated (P2S) and doubly ligated (P2S2) Rep dimers were characterized. The equilibrium and kinetic constants for Rep dimerization as well as the steady-state ATPase activities of both PS and P2S crosslinked complexes were identical to the values determined for un-(vol/vol) glycerol]. Triethyl-ammonium bicarbonate buffer (TEAB, 1 M) was made by bubbling CO2 (g) derived from subliming dry ice into an aqueous solution containing >1 M triethylamine at 0°C for 4 hr or until a pH of 7.5 was achieved; Milli-Q H20 was then added to achieve a final concentration of 1 M. Kinase buffer was 50 mM Tris HCl (pH 7.5 at 25°C), 10 mM MgCl2, and 10 mM 2-mercaptoethanol. Proteins, Enzymes, and Oligodeoxynucleotides. E. coli Rep protein was purified to >99% homogeneity from E. coli MZ-1/ Abbreviations: ssDNA, single-stranded DNA; sulfo-SANPAH, sulfo-N-succinimidyl-6-(4'-azido-2'-nitro-phenylamino)hexanoate.
Cell, 1997
Crystal structures of binary and ternary complexes of (Amaratunga and Lohman, 1993; Bjornson et al., 1994; the E. coli Rep helicase bound to single-stranded (ss) Lohman and Bjornson, 1996). Mutations in the rep gene DNA or ssDNA and ADP were determined to a resolureduce the rate of movement of the E. coli replication tion of 3.0 Å and 3.2 Å , respectively. The asymmetric fork (Lane and Denhardt, 1975a, 1975b). The Rep protein unit in the crystals contains two Rep monomers difis a stable monomer in the absence of DNA (Lohman fering from each other by a large reorientation of one et al., 1989), and individual monomers can bind one of the domains, corresponding to a swiveling of 130؇ nucleotide (Moore and Lohman, 1994a, 1994b) and eiabout a hinge region. Such domain movements are ther ssDNA or dsDNA, competitively (Wong and Lohsufficiently large to suggest that these may be coupled man, 1992; Wong et al., 1992). However, Rep dimers are to translocation of the Rep dimer along DNA. The induced upon binding DNA, and the active form of the ssDNA binding site involves the helicase motifs Ia, III, helicase is a dimer (Chao and Lohman, 1991; Wong and and V, whereas the ADP binding site involves helicase Lohman, 1992; Wong et al., 1992, 1996; Amaratunga and motifs I and IV. Residues in motifs II and VI may func-Lohman, 1993; Bjornson et al., 1996a). Both subunits tion to transduce the allosteric effects of nucleotides of the Rep dimer can bind either ss or duplex DNA on DNA binding. These structures represent the first competitively, and there is a strong negative cooperativview of a DNA helicase bound to DNA. ity for binding DNA to the second site of the dimer (Wong et al., 1992; Bjornson et al., 1996b). Nucleotide cofactors *To whom correspondence should be addressed.
Escherichia coli Rep helicase unwinds DNA by an active mechanism
Biochemistry, 1993
DNA helicases unwind duplex DNA to form the single-stranded (ss) DNA intermediates required for replication, recombination, and repair in reactions that require nucleoside 5'-triphosphate hydrolysis. Helicases generally require a ss-DNA flanking the duplex in order to initiate unwinding in uitro; however, the precise function of the ss-DNA is not understood. If a helicase unwinds DNA by a "passive" mechanism, it would bind to and translocate unidirectionally along the ss-DNA and facilitate duplex unwinding by translocating onto the ss-DNA that is formed transiently by thermal fluctuations in the duplex. We have examined the kinetics of DNA unwinding by Escherichia coli Rep protein (a 3' to 5' helicase) by rapid quench-flow methods using a series of novel, nonnatural DNA substrates possessing 3' flanking ss-DNA within which is embedded either a segment of ss-DNA possessing reversed backbone polarity or a rron-DNA [poly(ethylene glycol)] spacer, either of which should block unwinding by a passive helicase. The E. coli Rep helicase effectively unwinds these DNA substrates, ruling out a passive mechanism of unwinding. Instead, the results are consistent with an "active" rolling mechanism during which Rep binds to ss-DNA and duplex DNA simultaneously.
Journal of Biological Chemistry, 2010
a The size of the ss-3Ј-tail is indicated in parentheses. b Bases corresponding to non-complementary ss-3Ј-tails are underlined. c I 22*Al(20) , I 22*Al(30) , Q 45*Al(20) , and Q 45*Al(30) are the equivalent oligonucleotides with a longer ss-3Ј-tail: 20 nucleotides (AGTTAGGGTTTTTTTTTTTA-3Ј) for I 22*Al(20) and Q 45*Al(20) and 30 nucleotides (AGTTAGGGTTTTTTTTTTTAAGTTAGGGTA-3Ј) for I 22*Al(30) and Q 45*Al(30) . d J 22*Te(20) , J 22*Te(30) , R 45*Te(20) , and R 45*Te(30) are the equivalent oligonucleotides with a longer ss-3Ј-tail: 20 nucleotides (GATTGTTATTTTTTTTTTTA-3Ј) for J 22*Te(20) and R 45*Te(20) and 30 nucleotides (GATTGTTATTTTTTTTTTTAGATTGTTATA-3Ј) for J 22*Te(30) and R 45*Te(30) .