Organized Unidirectional Waves of ATP Hydrolysis within a RecA Filament
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1 Department of Biochemistry, University of Wisconsin, Madison, Wisconsin, United States of America
The RecA protein forms nucleoprotein filaments on DNA, and individual monomers within the filaments hydrolyze ATP. Assembly and disassembly of filaments are both unidirectional, occurring on opposite filament ends, with disassembly requiring ATP hydrolysis. When filaments form on duplex DNA, RecA protein exhibits a functional state comparable to the state observed during active DNA strand exchange. RecA filament state was monitored with a coupled spectrophotometric assay for ATP hydrolysis, with changes fit to a mathematical model for filament disassembly. At 37 °C, monomers within the RecA-double-stranded DNA (dsDNA) filaments hydrolyze ATP with an observed kcat of 20.8 ± 1.5 min1. Under the same conditions, the rate of end-dependent filament disassembly (koff) is 123 ± 16 monomers per minute per filament end. This rate of disassembly requires a tight coupling of the ATP hydrolytic cycles of adjacent RecA monomers. The relationship of kcat to koff infers a filament state in which waves of ATP hydrolysis move unidirectionally through RecA filaments on dsDNA, with successive waves occurring at intervals of approximately six monomers. The waves move nearly synchronously, each one transiting from one monomer to the next every 0.5 s. The results reflect an organization of the ATPase activity that is unique in filamentous systems, and could be linked to a RecA motor function.
Introduction
There are three prominent protein families that both form filaments and hydrolyze nucleoside triphosphates (NTPs). These include the tubulins [1,2,3], the actins [4,5,6], and RecA protein and its homologs [7,8,9,10,11]. Of these, the RecA family is unique in its formation of filaments on a DNA cofactor and in the steady-state hydrolysis of ATP by monomers within an assembled filament [10,11,12]. The function of RecA-mediated ATP hydrolysis has been a source of conjecture and debate for over two decades [10,11,13,14,15,16,17,18,19,20,21].
The bacterial RecA protein promotes the central steps of recombinational DNA repair [12,22]. In vitro, the RecA protein catalyzes a DNA strand exchange reaction that mimics the presumed function of RecA protein in vivo. The RecA protein first forms a filament on single-stranded DNA (ssDNA). The bound ssDNA is then aligned and paired with a homologous double-stranded DNA (dsDNA), forming a short (up to approximately 1 kilobasepair [kbp]) segment of paired DNA and initiating the DNA strand exchange [11,12]. The initial paired DNA can be extended in a reaction that generally requires ATP hydrolysis [10,11,12].
RecA filaments are assembled and disassembled in an end-dependent fashion. Filament assembly proceeds in steps, with a slow nucleation followed by a very rapid 5′ to 3′ extension phase to coat the available DNA [11,12,23,24,25]. The rapid extension phase does not limit overall filament assembly within the pH range normally used for RecA experiments [26,27]. Filament disassembly is also end-dependent and proceeds 5′ to 3′ such that monomers are subtracted from filaments at the end opposite to the end where monomers are added in the extension process (Figure 1) [25,28,29]. In studies carried out to date, we have not detected RecA monomer addition to the disassembling end or RecA monomer subtraction from the assembly (extension) end [25,27,28], although both processes presumably occur at some low rate.
The reaction is limited by a slow nucleation step, followed by rapid extension in the 5′ to 3′ direction. Disassembly is also uniquely 5′ to 3′, proceeding from the end opposite to that where extension occurs. Dissociation of RecA monomers at the disassembling end requires ATP hydrolysis (see text).
The RecA protein also binds to dsDNA, but nucleation onto dsDNA is much slower than onto ssDNA [26,30]. Nucleation directly onto dsDNA is pH-dependent, occurring more rapidly as the pH declines from 7.0 to 6.0 [30,31]. At any pH, filament extension on dsDNA is rapid, with complete filaments incorporating thousands of RecA monomers within a few minutes [25,32,33,34]. Upon binding dsDNA, RecA underwinds the DNA by approximately 40% and extends the helix to about 18 basepairs (bp) per turn relative to the B-form helix [35,36]. A RecA monomer binds three nucleotides (nt) of ssDNA or 3 bp of dsDNA, so one helical turn of the nucleoprotein filament includes approximately six RecA monomers.
RecA protein within a filament hydrolyzes ATP in a reaction that is almost completely DNA-dependent under standard reaction conditions [31]. Under our normal reaction conditions, the intrinsic kcat values for ssDNA and dsDNA-dependent ATP hydrolysis rates are approximately 30 and 20 min1, respectively, as determined in multiple trials over the past two decades [10,11,12]. ATPase rates are independent of pH in the range 6–9 [30,31]. ATP hydrolysis occurs uniformly throughout the filament of RecA-DNA complexes, and there is no detectable change or enhancement at filament ends [37]. In the presence of ATP analogs that are not hydrolyzed, RecA protein will form filaments on DNA and promote substantial DNA pairing and strand exchange [38,39,40]. However, RecA must hydrolyze ATP to bring about net filament disassembly [25,27,28,29,32,33,34], bypass of heterologous inserts during DNA strand exchange [41,42], DNA strand exchange with DNA substrates greater than 3 kbp in length [43], and DNA strand exchange between two duplex DNA molecules [42,44,45]. It is not clear how RecA protein-mediated ATP hydrolysis is coupled to these functions. Intriguingly, the core domain of the RecA protein (residues 34–269) is structurally homologous to several motor proteins, including hexameric helicases [46] and the mitochondrial F1-ATPase [47]. Models for a motor-like coupling of ATP hydrolysis to DNA strand exchange in certain DNA metabolic situations have been proposed [10].
There is now evidence for at least four different functional states of RecA protein, occurring at different reaction stages. These have recently been designated O, Ac, Ao, and P [10,48,49] (Figure 2). The O state is largely inactive and found in the absence of nucleotide cofactors or in the presence of ADP [50]. RecA in the O state can bind to DNA, creating a helical filament with a pitch of 76 [50]. Addition of ATP, ATPγS, or dATP results in a conformation change to an active form that is manifested by extended filaments on DNA with a pitch of 95 [50,51,52,53,54,55,56,57,58]. When RecA filaments form on ssDNA, they are in a structural and functional state designated A. Interconversion between the two different A states, which have somewhat different properties, is mediated largely by the Mg2+ concentration. However, all RecA filaments on ssDNA hydrolyze ATP with a kcat of approximately 30 min1. Addition of a second DNA strand to a RecA filament, as in a filament bound to dsDNA or a filament promoting DNA strand exchange, converts the filament to the P state. The P state is characterized by 30% lower rates of ATP hydrolysis [59,60], higher rates of exchange of RecA monomers into and out of the filament [61,62], and a higher degree of cooperativity in the ATPase function [27,49,61] than the A conformations.
The O state is present in the absence of ATP or ATP analogs, whether the protein is free in solution or bound to DNA. The A state is found on ssDNA in the presence of ATP. Two versions of the A state, Ac and Ao, are found at different Mg2+ concentrations. Addition of a second DNA strand to the RecA filament converts it to the P state.
How can RecA monomers be added to one end of a filament and deleted from the other end in the same test tube The monomer-monomer interfaces are presumably the same at either end (and everywhere else) in the filament. As pointed out by Wegner [63], the dissociation constant, KD, for monomer addition to either filament end cannot be different unless an independent source of chemical energy is provided to affect the binding to one end or the other. As noted above, filament assembly does not require ATP hydrolysis, but filament disassembly does [25,27,28,29,32,33,34]. The hydrolysis of ATP by interior monomers does not generally result in dissociation, and under some conditions ATP hydrolysis can proceed with no evident dissociation of RecA monomers [25,61,64]. A simple model arises: ATP hydrolysis occurs everywhere, resulting in dissociation only for monomers at a disassembling end.
Within a RecA filament, ATP hydrolysis could occur at random, or it could be highly organized as cooperative waves traveling through the filament. There is no convenient method for monitoring the effects of ATP hydrolysis in the middle of a RecA filament. However, important clues can be ascertained by examining one effect of ATP hydrolysis: the 5′ to 3′ end-dependent filament disassembly process. In effect, we cannot see waves of ATP hydrolysis within a filament, but we can monitor one of the waves—the one occurring at the disassembling filament end.
The rate of disassembly can lead directly to an assessment of the degree of coupling of the ATP hydrolytic cycles of adjacent RecA monomers within the filament. For example, since RecA hydrolyzes ATP at a rate of approximately 30 min1 on ssDNA, each RecA monomer is hydrolyzing one ATP molecule every 2 s. If the ATP hydrolytic cycles of adjacent monomers are not coupled in any way, this will be reflected in a predictable rate of filament disassembly. When the end monomer hydrolyzes ATP and dissociates, the next monomer in line could be at any point within its ATP hydrolytic cycle. However, in a large population of filaments, the next monomer will be on average halfway through the cycle, and it will hydrolyze ATP (and dissociate) one second later. Dissociation of one RecA monomer per second will lead to a measured disassembly rate of 60 monomers of RecA per minute per filament end. This is close to the situation observed for RecA monomers formed on ssDNA (functional state A), which disassemble with a rate of 60–70 monomers per minute per filament end [27]. Note that this is the slowest rate of disassembly compatible with the rate of ATP hydrolysis observed under the conditions of this experiment, and requires that every ATP hydrolytic event occurring in the RecA monomer at the disassembly end of the filament results in dissociation of that monomer. If the probability of dissociation upon ATP hydrolysis is less than 100%, the disassembly process would have to be slower than is observed.
A coupling of the ATP hydrolytic cycles of adjacent monomers could in principle lead to greater rates of filament disassembly. We are particularly interested in the status of RecA filaments formed on dsDNA and those promoting DNA strand exchange. As indicated above, these filaments are in the P functional state and exhibit a higher degree of coupling involving the hydrolytic cycles of adjacent monomers than is seen for filaments formed on ssDNA. If ATP hydrolysis is organized into cooperative waves traveling through the filament, then the rate of disassembly from dsDNA will reveal the rate of movement of one of the waves and the interval, i, between successive waves within the entire filament. For example, consider monomers within RecA filaments on dsDNA that are hydrolyzing ATP at approximately 20 min1 (or one ATP every 3 s). If ATP hydrolytic cycles of adjacent monomers are coupled within the filament so that ATP hydrolysis is organized in waves (Figure 3), then a new wave must reach a given monomer every 3 s to account for the observed kcat. If the waves are four monomers apart (i = 4 monomers, Figure 3A), a wave must move from one monomer to the next one in line every 0.75 s. At the disassembling end (the ultimate wave), one monomer would dissociate every 0.75 s, giving a disassembly rate of 80 monomers min1 filament end1. If instead the waves are organized at intervals of six monomers (Figure 3B), then to reach a given monomer every 3 s, a wave would have to move from one monomer to the next every 0.5 s. At the disassembling end, a monomer would dissociate every 0.5 s to yield a disassembly rate of 120 monomers min1 filament end1. We define the rate of end-dependent filament disassembly as koff. Note that the numerical relationship between koff and the kcat for ATP hydrolysis by individual monomers reveals the distance between waves within the filament, i, such that koff/kcat = i. For the examples above, 80/20 = 4, and 120/20 = 6.
(A) The interval between hydrolyzing monomers (i) is set at four monomers. The dark monomers are those at the hydrolytic step of their ATP hydrolytic cycle. Each monomer within the filament (e.g., the one marked with an “X”) is hydrolyzing an ATP every 3 s, so that a new wave must reach it within that time span. If i = 4 monomers, the waves must move every 0.75 s. The last wave, at the disassembling end, results in dissociation.
(B) The same considerations in (A) for i = 4 monomers are illustrated for i = 6 monomers.
The kcat for ATP hydrolysis for RecA monomers on dsDNA is readily measured. An accurate determination of koff, as needed to determine i, is a more substantial challenge that is met in this report.
Results
Experimental Design
A model system has been developed that allows the rate of end-dependent RecA filament disassembly from ssDNA to be determined quantitatively by monitoring the rate of ATP hydrolysis during the disassembly process [10,27]. The system is outlined in Figure 4A. RecA filaments are assembled on a linear ssDNA at a low pH, at which nucleation is fast enough that the DNA is saturated with RecA and there is little net disassembly (disassembling RecA monomers are rapidly replaced). The reaction mixture is then shifted to a higher pH, at which the rate of filament nucleation is slower and net filament disassembly can be observed. The rate of ATP hydrolysis will decline as RecA monomers dissociate from the DNA [27], reaching a nonzero steady-state endpoint (Figure 4B). The steady-state rate reflects the final balance between disassembly and renucleation of RecA protein and filament formation on the ssDNA vacated by the disassembling filament. Since filament extension is very fast relative to disassembly, any nucleation event will result in a filament that extends from the nucleation point to the disassembling end of the first filament, and in effect create a new point for disassembly (Figure 4A). On any of these DNA molecules, there is only one point where net disassembly occurs. Even when the second filament is not perfectly in phase with the first filament, such that some disassembly continues at the junction (Figure 4C), the RecA that dissociates from filament 1 at the junction is rapidly replaced by extension of filament 2 immediately behind it (filament extension is much faster than disassembly). Thus, there is no net change in bound RecA that would be reflected in the observed rates of ATP hydrolysis, except at the end of filament 2. To minimize the rebinding of RecA protein to vacated DNA, ssDNA-binding protein of Escherichia coli (SSB protein) is added at the time of the pH shift [27]. Bound SSB limits the nucleation of RecA filaments on ssDNA [25,65,66,67]. This in turn permits a measurable decline in ATP hydrolysis.
(A) In the model, end-dependent disassembly occurs, with SSB filling in the vacated DNA. At low frequency, additional RecA monomers nucleate filament formation on the vacated DNA, and the new filament (dark ovals) extends until it catches up with the original filament (open ovals). This creates a new disassembling end.
(B) Kinetics of disassembly as monitored by DNA-dependent ATP hydrolysis. This curve is an approximation of curves reported in previous work [27]. The rate of ATP hydrolysis declines as RecA protein dissociates from the ssDNA, until a lower steady-state rate is reached. The steady state reflects a balance between disassembly and new filament formation.
(C) If, upon rebinding, the new filament does match the old one in phase, the junction between the old and new filaments could be a site of RecA monomer exchange with the solution. There will be no net disassembly at this point and no resulting change in the rate of ATP hydrolysis, because any monomers that dissociate will be immediately replaced by extension of the trailing filament.
The process in Figure 4 is modeled by equation 1, where knuc is the rate of renucleation of RecA filaments to vacated DNA during the disassembly process, koff is the end-dependent disassembly rate as noted in the Introduction, ntot is the total number of RecA protein binding sites on the DNA molecule used as substrate, [D-ends] is the concentration of disassembling ends, and kcat is the turnover number for ATP hydrolysis by RecA monomers in the filament.
The derivation of equation 1 is described in detail elsewhere [27]. The assumptions incorporated into the equation and the model of Figure 4 are also detailed elsewhere [27]. In brief, the assumptions are as follows. First, nucleation of filament formation is the rate-limiting step for RecA filament assembly. This assumption is documented in the Introduction of this paper and in previous reports [23,27]. Second, filament extension is faster than filament disassembly under all conditions. The simplest of the arguments [27] underpinning this assertion is that filaments would never form on DNA if RecA were subtracted from the disassembling end faster than it could be added to the extending end. Third, the model assumes that net disassembly is occurring at only one point in each filament. This is addressed in Figure 4C and follows from the fact that filament extension is faster than filament disassembly. There will be no net disassembly except at the 5′ proximal end of the RecA protein tracts on a given linear DNA. Any dissociation in the middle of a filament is rapidly replaced by the growth of trailing filament segments and thus cannot contribute to a net change in ATP hydrolysis. Finally, the assumption is made that the kcat for RecA-mediated ATP hydrolysis does not change in the range used for the pH shifts. This is documented elsewhere [26,27,30].
On dsDNA, the situation becomes more complicated because it is more difficult to define the orientation of the filaments. RecA protein binds to the two strands of a duplex DNA unequally. One strand is bound at the site normally occupied by ssDNA. This strand orients the filament, and it is protected from nuclease digestion better than the second strand of a duplex [33,68]. We will call this first strand the initiating strand [22]. In principle, either strand of a duplex can act as the initiating strand and organize the 5′ to 3′ assembly of RecA filaments. RecA filaments can thus form in two orientations on dsDNA (Figure 5A). If nucleation occurs in the middle of a linear duplex so that the filament extends to one end, and a second nucleation occurs on the same DNA but in the opposite orientation, there could be two filaments on the same DNA, both with disassembling ends (Figure 5A). This in turn would complicate the mathematical modeling of the disassembly reaction. This potential problem can be alleviated by supplying a good nucleation site in the form of a stretch of ssDNA. At pH 8 and above, RecA nucleation onto duplex DNA becomes very slow (measured in hours) [26,30]. If a 5′ single-strand extension is added to one end of the DNA, the nucleation occurs within a few minutes, and RecA readily extends from the single-stranded segment into the adjoining duplex [33]. A 3′ single-strand extension does not work [33], as would be predicted from the polarity of filament assembly from a nucleation site. This effect is seen in the experimental data presented in Figure 5B. RecA binds to a linear duplex of 3,162 bp only after a lag lasting tens of minutes, even at pH 6.6. A linear duplex of similar length, but this time with a 5′ single-strand extension of 30 nt, is bound without a measurable lag at the same pH. This indicates that the filaments are nucleating on the single-strand extension, and virtually all of the resulting filaments in the population will have a unique orientation over the entire length of the DNA that is dictated by the single-stranded tail.
(A) RecA filaments can nucleate on dsDNA so as to have two different orientations. The initiating strand guides the orientation of each filament, with its polarity labeled in each panel. If two nucleations occur on the same filament, there can be two independent disassembly points.
(B) The effect of the 30-nt 5′ extensions on RecA protein filament formation. Reactions contained 4 μM RecA protein, 4 μM DNA, and no SSB and were carried out at pH 6.6. The tailed DNA substrate has 2,900 bp of duplex DNA with the 30-nt 5′ extension on one end. The completely duplex DNA is 3,162 nt in length. The enhanced nucleation afforded by these tails is evident in the rapid ATP hydrolysis seen when the tailed DNA is used as cofactor.
To isolate the disassembly process so that it can be measured accurately, we must limit the rate of RecA rebinding to DNA that has been vacated as the RecA dissociates. Several parameters can be adjusted to accomplish this. The first one is pH. The rate of RecA protein disassembly reaches an apparent maximum at pH 8 and above [33]. The rate of disassembly should reach a maximum when every ATP hydrolytic event at the disassembling end results in dissociation of a RecA monomer. The rate of nucleation on dsDNA declines with increased pH such that working at high pH minimizes RecA rebinding. The second parameter is DNA length, which can affect the consequences of rebinding to duplex DNA. A nucleated filament is rapidly extended to the DNA end. A nucleation event on a long DNA will generate more bound RecA than a nucleation event on a short DNA. Thus, the use of shorter DNAs will limit the effects of each nucleation event. However, the DNA length cannot be reduced too much to suppress the levels of RecA protein rebinding, because a longer DNA (and filament) allows for a more gradual decline in the ATPase reaction that is easier to monitor. Trials using DNAs of several lengths indicated that a linear dsDNA with a length of approximately 3 kbp was optimal for this work [33,69]. These dsDNAs result in bound RecA filaments long enough (about 1,000 monomers) to disassemble over a time period of multiple minutes, while limiting the effects of RecA rebinding. As shown later, we also carried out measurements using dsDNAs of 2 kbp and 4 kbp. A third parameter that can be adjusted is SSB concentration. Adding SSB when the pH shift is carried out inhibits the rebinding of free RecA protein to the ssDNA extension. The concentrations of SSB used in these experiments were determined empirically to provide the maximum possible suppression of RecA renucleation. The effectiveness of this SSB-mediated suppression is illustrated in Figure 6.
(A) Normal RecA filament disassembly protocol. The RecA filaments were formed on the tailed DNA at pH 6.62. The pH was then shifted to 8.0 to allow disassembly to commence. Disassembly curves are shown in the presence and absence of SSB. Reactions contained (after the pH shift) 2 μM RecA protein, 2 μM DNA, and (where indicated) 0.05 μM SSB.
(B) Direct binding of RecA protein to the tailed DNA at pH 8.0. Reactions contained 6 μM RecA protein, 6 μM DNA, and (where indicated) 0.15 μM SSB. When included, SSB was added prior to the RecA protein.
Optimizing all of these considerations, the general protocol for these experiments is outlined in Figure 7A. A linear duplex of 2,900 bp, with a 30-nucleotide 5′ single-strand extension, is bound with RecA protein at pH 6.62. The DNA concentration is 12 μM, so that the concentration of RecA binding sites is 2 μM. The RecA protein is added in excess (12 μM) to facilitate saturation of the DNA. After binding is complete, the rate of ATP hydrolysis is measured. The pH is then shifted abruptly to 8 by diluting the solution 2-fold into a solution with another buffer. The RecA and DNA concentrations undergo a 2-fold decrease during the pH shift, but the concentration of ATP and ATP regeneration components are kept constant. The pH shift initiates a net disassembly process that leads to a decline in ATP hydrolysis. Rebinding of RecA protein to the vacated single-strand extension is suppressed by SSB added with the pH shift buffers. The production of ADP is monitored. The ATP regeneration system is set up so as not to limit the observed results. For example, results were the same if the phosphoenolpyruvate (PEP) concentration was reduced from 3 to 1.5 mM. The starting kcat for ATP hydrolysis is reported as the measured rate of ATP hydrolysis(Julia M. Cox, Oleg V. Tso)
The RecA protein forms nucleoprotein filaments on DNA, and individual monomers within the filaments hydrolyze ATP. Assembly and disassembly of filaments are both unidirectional, occurring on opposite filament ends, with disassembly requiring ATP hydrolysis. When filaments form on duplex DNA, RecA protein exhibits a functional state comparable to the state observed during active DNA strand exchange. RecA filament state was monitored with a coupled spectrophotometric assay for ATP hydrolysis, with changes fit to a mathematical model for filament disassembly. At 37 °C, monomers within the RecA-double-stranded DNA (dsDNA) filaments hydrolyze ATP with an observed kcat of 20.8 ± 1.5 min1. Under the same conditions, the rate of end-dependent filament disassembly (koff) is 123 ± 16 monomers per minute per filament end. This rate of disassembly requires a tight coupling of the ATP hydrolytic cycles of adjacent RecA monomers. The relationship of kcat to koff infers a filament state in which waves of ATP hydrolysis move unidirectionally through RecA filaments on dsDNA, with successive waves occurring at intervals of approximately six monomers. The waves move nearly synchronously, each one transiting from one monomer to the next every 0.5 s. The results reflect an organization of the ATPase activity that is unique in filamentous systems, and could be linked to a RecA motor function.
Introduction
There are three prominent protein families that both form filaments and hydrolyze nucleoside triphosphates (NTPs). These include the tubulins [1,2,3], the actins [4,5,6], and RecA protein and its homologs [7,8,9,10,11]. Of these, the RecA family is unique in its formation of filaments on a DNA cofactor and in the steady-state hydrolysis of ATP by monomers within an assembled filament [10,11,12]. The function of RecA-mediated ATP hydrolysis has been a source of conjecture and debate for over two decades [10,11,13,14,15,16,17,18,19,20,21].
The bacterial RecA protein promotes the central steps of recombinational DNA repair [12,22]. In vitro, the RecA protein catalyzes a DNA strand exchange reaction that mimics the presumed function of RecA protein in vivo. The RecA protein first forms a filament on single-stranded DNA (ssDNA). The bound ssDNA is then aligned and paired with a homologous double-stranded DNA (dsDNA), forming a short (up to approximately 1 kilobasepair [kbp]) segment of paired DNA and initiating the DNA strand exchange [11,12]. The initial paired DNA can be extended in a reaction that generally requires ATP hydrolysis [10,11,12].
RecA filaments are assembled and disassembled in an end-dependent fashion. Filament assembly proceeds in steps, with a slow nucleation followed by a very rapid 5′ to 3′ extension phase to coat the available DNA [11,12,23,24,25]. The rapid extension phase does not limit overall filament assembly within the pH range normally used for RecA experiments [26,27]. Filament disassembly is also end-dependent and proceeds 5′ to 3′ such that monomers are subtracted from filaments at the end opposite to the end where monomers are added in the extension process (Figure 1) [25,28,29]. In studies carried out to date, we have not detected RecA monomer addition to the disassembling end or RecA monomer subtraction from the assembly (extension) end [25,27,28], although both processes presumably occur at some low rate.
The reaction is limited by a slow nucleation step, followed by rapid extension in the 5′ to 3′ direction. Disassembly is also uniquely 5′ to 3′, proceeding from the end opposite to that where extension occurs. Dissociation of RecA monomers at the disassembling end requires ATP hydrolysis (see text).
The RecA protein also binds to dsDNA, but nucleation onto dsDNA is much slower than onto ssDNA [26,30]. Nucleation directly onto dsDNA is pH-dependent, occurring more rapidly as the pH declines from 7.0 to 6.0 [30,31]. At any pH, filament extension on dsDNA is rapid, with complete filaments incorporating thousands of RecA monomers within a few minutes [25,32,33,34]. Upon binding dsDNA, RecA underwinds the DNA by approximately 40% and extends the helix to about 18 basepairs (bp) per turn relative to the B-form helix [35,36]. A RecA monomer binds three nucleotides (nt) of ssDNA or 3 bp of dsDNA, so one helical turn of the nucleoprotein filament includes approximately six RecA monomers.
RecA protein within a filament hydrolyzes ATP in a reaction that is almost completely DNA-dependent under standard reaction conditions [31]. Under our normal reaction conditions, the intrinsic kcat values for ssDNA and dsDNA-dependent ATP hydrolysis rates are approximately 30 and 20 min1, respectively, as determined in multiple trials over the past two decades [10,11,12]. ATPase rates are independent of pH in the range 6–9 [30,31]. ATP hydrolysis occurs uniformly throughout the filament of RecA-DNA complexes, and there is no detectable change or enhancement at filament ends [37]. In the presence of ATP analogs that are not hydrolyzed, RecA protein will form filaments on DNA and promote substantial DNA pairing and strand exchange [38,39,40]. However, RecA must hydrolyze ATP to bring about net filament disassembly [25,27,28,29,32,33,34], bypass of heterologous inserts during DNA strand exchange [41,42], DNA strand exchange with DNA substrates greater than 3 kbp in length [43], and DNA strand exchange between two duplex DNA molecules [42,44,45]. It is not clear how RecA protein-mediated ATP hydrolysis is coupled to these functions. Intriguingly, the core domain of the RecA protein (residues 34–269) is structurally homologous to several motor proteins, including hexameric helicases [46] and the mitochondrial F1-ATPase [47]. Models for a motor-like coupling of ATP hydrolysis to DNA strand exchange in certain DNA metabolic situations have been proposed [10].
There is now evidence for at least four different functional states of RecA protein, occurring at different reaction stages. These have recently been designated O, Ac, Ao, and P [10,48,49] (Figure 2). The O state is largely inactive and found in the absence of nucleotide cofactors or in the presence of ADP [50]. RecA in the O state can bind to DNA, creating a helical filament with a pitch of 76 [50]. Addition of ATP, ATPγS, or dATP results in a conformation change to an active form that is manifested by extended filaments on DNA with a pitch of 95 [50,51,52,53,54,55,56,57,58]. When RecA filaments form on ssDNA, they are in a structural and functional state designated A. Interconversion between the two different A states, which have somewhat different properties, is mediated largely by the Mg2+ concentration. However, all RecA filaments on ssDNA hydrolyze ATP with a kcat of approximately 30 min1. Addition of a second DNA strand to a RecA filament, as in a filament bound to dsDNA or a filament promoting DNA strand exchange, converts the filament to the P state. The P state is characterized by 30% lower rates of ATP hydrolysis [59,60], higher rates of exchange of RecA monomers into and out of the filament [61,62], and a higher degree of cooperativity in the ATPase function [27,49,61] than the A conformations.
The O state is present in the absence of ATP or ATP analogs, whether the protein is free in solution or bound to DNA. The A state is found on ssDNA in the presence of ATP. Two versions of the A state, Ac and Ao, are found at different Mg2+ concentrations. Addition of a second DNA strand to the RecA filament converts it to the P state.
How can RecA monomers be added to one end of a filament and deleted from the other end in the same test tube The monomer-monomer interfaces are presumably the same at either end (and everywhere else) in the filament. As pointed out by Wegner [63], the dissociation constant, KD, for monomer addition to either filament end cannot be different unless an independent source of chemical energy is provided to affect the binding to one end or the other. As noted above, filament assembly does not require ATP hydrolysis, but filament disassembly does [25,27,28,29,32,33,34]. The hydrolysis of ATP by interior monomers does not generally result in dissociation, and under some conditions ATP hydrolysis can proceed with no evident dissociation of RecA monomers [25,61,64]. A simple model arises: ATP hydrolysis occurs everywhere, resulting in dissociation only for monomers at a disassembling end.
Within a RecA filament, ATP hydrolysis could occur at random, or it could be highly organized as cooperative waves traveling through the filament. There is no convenient method for monitoring the effects of ATP hydrolysis in the middle of a RecA filament. However, important clues can be ascertained by examining one effect of ATP hydrolysis: the 5′ to 3′ end-dependent filament disassembly process. In effect, we cannot see waves of ATP hydrolysis within a filament, but we can monitor one of the waves—the one occurring at the disassembling filament end.
The rate of disassembly can lead directly to an assessment of the degree of coupling of the ATP hydrolytic cycles of adjacent RecA monomers within the filament. For example, since RecA hydrolyzes ATP at a rate of approximately 30 min1 on ssDNA, each RecA monomer is hydrolyzing one ATP molecule every 2 s. If the ATP hydrolytic cycles of adjacent monomers are not coupled in any way, this will be reflected in a predictable rate of filament disassembly. When the end monomer hydrolyzes ATP and dissociates, the next monomer in line could be at any point within its ATP hydrolytic cycle. However, in a large population of filaments, the next monomer will be on average halfway through the cycle, and it will hydrolyze ATP (and dissociate) one second later. Dissociation of one RecA monomer per second will lead to a measured disassembly rate of 60 monomers of RecA per minute per filament end. This is close to the situation observed for RecA monomers formed on ssDNA (functional state A), which disassemble with a rate of 60–70 monomers per minute per filament end [27]. Note that this is the slowest rate of disassembly compatible with the rate of ATP hydrolysis observed under the conditions of this experiment, and requires that every ATP hydrolytic event occurring in the RecA monomer at the disassembly end of the filament results in dissociation of that monomer. If the probability of dissociation upon ATP hydrolysis is less than 100%, the disassembly process would have to be slower than is observed.
A coupling of the ATP hydrolytic cycles of adjacent monomers could in principle lead to greater rates of filament disassembly. We are particularly interested in the status of RecA filaments formed on dsDNA and those promoting DNA strand exchange. As indicated above, these filaments are in the P functional state and exhibit a higher degree of coupling involving the hydrolytic cycles of adjacent monomers than is seen for filaments formed on ssDNA. If ATP hydrolysis is organized into cooperative waves traveling through the filament, then the rate of disassembly from dsDNA will reveal the rate of movement of one of the waves and the interval, i, between successive waves within the entire filament. For example, consider monomers within RecA filaments on dsDNA that are hydrolyzing ATP at approximately 20 min1 (or one ATP every 3 s). If ATP hydrolytic cycles of adjacent monomers are coupled within the filament so that ATP hydrolysis is organized in waves (Figure 3), then a new wave must reach a given monomer every 3 s to account for the observed kcat. If the waves are four monomers apart (i = 4 monomers, Figure 3A), a wave must move from one monomer to the next one in line every 0.75 s. At the disassembling end (the ultimate wave), one monomer would dissociate every 0.75 s, giving a disassembly rate of 80 monomers min1 filament end1. If instead the waves are organized at intervals of six monomers (Figure 3B), then to reach a given monomer every 3 s, a wave would have to move from one monomer to the next every 0.5 s. At the disassembling end, a monomer would dissociate every 0.5 s to yield a disassembly rate of 120 monomers min1 filament end1. We define the rate of end-dependent filament disassembly as koff. Note that the numerical relationship between koff and the kcat for ATP hydrolysis by individual monomers reveals the distance between waves within the filament, i, such that koff/kcat = i. For the examples above, 80/20 = 4, and 120/20 = 6.
(A) The interval between hydrolyzing monomers (i) is set at four monomers. The dark monomers are those at the hydrolytic step of their ATP hydrolytic cycle. Each monomer within the filament (e.g., the one marked with an “X”) is hydrolyzing an ATP every 3 s, so that a new wave must reach it within that time span. If i = 4 monomers, the waves must move every 0.75 s. The last wave, at the disassembling end, results in dissociation.
(B) The same considerations in (A) for i = 4 monomers are illustrated for i = 6 monomers.
The kcat for ATP hydrolysis for RecA monomers on dsDNA is readily measured. An accurate determination of koff, as needed to determine i, is a more substantial challenge that is met in this report.
Results
Experimental Design
A model system has been developed that allows the rate of end-dependent RecA filament disassembly from ssDNA to be determined quantitatively by monitoring the rate of ATP hydrolysis during the disassembly process [10,27]. The system is outlined in Figure 4A. RecA filaments are assembled on a linear ssDNA at a low pH, at which nucleation is fast enough that the DNA is saturated with RecA and there is little net disassembly (disassembling RecA monomers are rapidly replaced). The reaction mixture is then shifted to a higher pH, at which the rate of filament nucleation is slower and net filament disassembly can be observed. The rate of ATP hydrolysis will decline as RecA monomers dissociate from the DNA [27], reaching a nonzero steady-state endpoint (Figure 4B). The steady-state rate reflects the final balance between disassembly and renucleation of RecA protein and filament formation on the ssDNA vacated by the disassembling filament. Since filament extension is very fast relative to disassembly, any nucleation event will result in a filament that extends from the nucleation point to the disassembling end of the first filament, and in effect create a new point for disassembly (Figure 4A). On any of these DNA molecules, there is only one point where net disassembly occurs. Even when the second filament is not perfectly in phase with the first filament, such that some disassembly continues at the junction (Figure 4C), the RecA that dissociates from filament 1 at the junction is rapidly replaced by extension of filament 2 immediately behind it (filament extension is much faster than disassembly). Thus, there is no net change in bound RecA that would be reflected in the observed rates of ATP hydrolysis, except at the end of filament 2. To minimize the rebinding of RecA protein to vacated DNA, ssDNA-binding protein of Escherichia coli (SSB protein) is added at the time of the pH shift [27]. Bound SSB limits the nucleation of RecA filaments on ssDNA [25,65,66,67]. This in turn permits a measurable decline in ATP hydrolysis.
(A) In the model, end-dependent disassembly occurs, with SSB filling in the vacated DNA. At low frequency, additional RecA monomers nucleate filament formation on the vacated DNA, and the new filament (dark ovals) extends until it catches up with the original filament (open ovals). This creates a new disassembling end.
(B) Kinetics of disassembly as monitored by DNA-dependent ATP hydrolysis. This curve is an approximation of curves reported in previous work [27]. The rate of ATP hydrolysis declines as RecA protein dissociates from the ssDNA, until a lower steady-state rate is reached. The steady state reflects a balance between disassembly and new filament formation.
(C) If, upon rebinding, the new filament does match the old one in phase, the junction between the old and new filaments could be a site of RecA monomer exchange with the solution. There will be no net disassembly at this point and no resulting change in the rate of ATP hydrolysis, because any monomers that dissociate will be immediately replaced by extension of the trailing filament.
The process in Figure 4 is modeled by equation 1, where knuc is the rate of renucleation of RecA filaments to vacated DNA during the disassembly process, koff is the end-dependent disassembly rate as noted in the Introduction, ntot is the total number of RecA protein binding sites on the DNA molecule used as substrate, [D-ends] is the concentration of disassembling ends, and kcat is the turnover number for ATP hydrolysis by RecA monomers in the filament.
The derivation of equation 1 is described in detail elsewhere [27]. The assumptions incorporated into the equation and the model of Figure 4 are also detailed elsewhere [27]. In brief, the assumptions are as follows. First, nucleation of filament formation is the rate-limiting step for RecA filament assembly. This assumption is documented in the Introduction of this paper and in previous reports [23,27]. Second, filament extension is faster than filament disassembly under all conditions. The simplest of the arguments [27] underpinning this assertion is that filaments would never form on DNA if RecA were subtracted from the disassembling end faster than it could be added to the extending end. Third, the model assumes that net disassembly is occurring at only one point in each filament. This is addressed in Figure 4C and follows from the fact that filament extension is faster than filament disassembly. There will be no net disassembly except at the 5′ proximal end of the RecA protein tracts on a given linear DNA. Any dissociation in the middle of a filament is rapidly replaced by the growth of trailing filament segments and thus cannot contribute to a net change in ATP hydrolysis. Finally, the assumption is made that the kcat for RecA-mediated ATP hydrolysis does not change in the range used for the pH shifts. This is documented elsewhere [26,27,30].
On dsDNA, the situation becomes more complicated because it is more difficult to define the orientation of the filaments. RecA protein binds to the two strands of a duplex DNA unequally. One strand is bound at the site normally occupied by ssDNA. This strand orients the filament, and it is protected from nuclease digestion better than the second strand of a duplex [33,68]. We will call this first strand the initiating strand [22]. In principle, either strand of a duplex can act as the initiating strand and organize the 5′ to 3′ assembly of RecA filaments. RecA filaments can thus form in two orientations on dsDNA (Figure 5A). If nucleation occurs in the middle of a linear duplex so that the filament extends to one end, and a second nucleation occurs on the same DNA but in the opposite orientation, there could be two filaments on the same DNA, both with disassembling ends (Figure 5A). This in turn would complicate the mathematical modeling of the disassembly reaction. This potential problem can be alleviated by supplying a good nucleation site in the form of a stretch of ssDNA. At pH 8 and above, RecA nucleation onto duplex DNA becomes very slow (measured in hours) [26,30]. If a 5′ single-strand extension is added to one end of the DNA, the nucleation occurs within a few minutes, and RecA readily extends from the single-stranded segment into the adjoining duplex [33]. A 3′ single-strand extension does not work [33], as would be predicted from the polarity of filament assembly from a nucleation site. This effect is seen in the experimental data presented in Figure 5B. RecA binds to a linear duplex of 3,162 bp only after a lag lasting tens of minutes, even at pH 6.6. A linear duplex of similar length, but this time with a 5′ single-strand extension of 30 nt, is bound without a measurable lag at the same pH. This indicates that the filaments are nucleating on the single-strand extension, and virtually all of the resulting filaments in the population will have a unique orientation over the entire length of the DNA that is dictated by the single-stranded tail.
(A) RecA filaments can nucleate on dsDNA so as to have two different orientations. The initiating strand guides the orientation of each filament, with its polarity labeled in each panel. If two nucleations occur on the same filament, there can be two independent disassembly points.
(B) The effect of the 30-nt 5′ extensions on RecA protein filament formation. Reactions contained 4 μM RecA protein, 4 μM DNA, and no SSB and were carried out at pH 6.6. The tailed DNA substrate has 2,900 bp of duplex DNA with the 30-nt 5′ extension on one end. The completely duplex DNA is 3,162 nt in length. The enhanced nucleation afforded by these tails is evident in the rapid ATP hydrolysis seen when the tailed DNA is used as cofactor.
To isolate the disassembly process so that it can be measured accurately, we must limit the rate of RecA rebinding to DNA that has been vacated as the RecA dissociates. Several parameters can be adjusted to accomplish this. The first one is pH. The rate of RecA protein disassembly reaches an apparent maximum at pH 8 and above [33]. The rate of disassembly should reach a maximum when every ATP hydrolytic event at the disassembling end results in dissociation of a RecA monomer. The rate of nucleation on dsDNA declines with increased pH such that working at high pH minimizes RecA rebinding. The second parameter is DNA length, which can affect the consequences of rebinding to duplex DNA. A nucleated filament is rapidly extended to the DNA end. A nucleation event on a long DNA will generate more bound RecA than a nucleation event on a short DNA. Thus, the use of shorter DNAs will limit the effects of each nucleation event. However, the DNA length cannot be reduced too much to suppress the levels of RecA protein rebinding, because a longer DNA (and filament) allows for a more gradual decline in the ATPase reaction that is easier to monitor. Trials using DNAs of several lengths indicated that a linear dsDNA with a length of approximately 3 kbp was optimal for this work [33,69]. These dsDNAs result in bound RecA filaments long enough (about 1,000 monomers) to disassemble over a time period of multiple minutes, while limiting the effects of RecA rebinding. As shown later, we also carried out measurements using dsDNAs of 2 kbp and 4 kbp. A third parameter that can be adjusted is SSB concentration. Adding SSB when the pH shift is carried out inhibits the rebinding of free RecA protein to the ssDNA extension. The concentrations of SSB used in these experiments were determined empirically to provide the maximum possible suppression of RecA renucleation. The effectiveness of this SSB-mediated suppression is illustrated in Figure 6.
(A) Normal RecA filament disassembly protocol. The RecA filaments were formed on the tailed DNA at pH 6.62. The pH was then shifted to 8.0 to allow disassembly to commence. Disassembly curves are shown in the presence and absence of SSB. Reactions contained (after the pH shift) 2 μM RecA protein, 2 μM DNA, and (where indicated) 0.05 μM SSB.
(B) Direct binding of RecA protein to the tailed DNA at pH 8.0. Reactions contained 6 μM RecA protein, 6 μM DNA, and (where indicated) 0.15 μM SSB. When included, SSB was added prior to the RecA protein.
Optimizing all of these considerations, the general protocol for these experiments is outlined in Figure 7A. A linear duplex of 2,900 bp, with a 30-nucleotide 5′ single-strand extension, is bound with RecA protein at pH 6.62. The DNA concentration is 12 μM, so that the concentration of RecA binding sites is 2 μM. The RecA protein is added in excess (12 μM) to facilitate saturation of the DNA. After binding is complete, the rate of ATP hydrolysis is measured. The pH is then shifted abruptly to 8 by diluting the solution 2-fold into a solution with another buffer. The RecA and DNA concentrations undergo a 2-fold decrease during the pH shift, but the concentration of ATP and ATP regeneration components are kept constant. The pH shift initiates a net disassembly process that leads to a decline in ATP hydrolysis. Rebinding of RecA protein to the vacated single-strand extension is suppressed by SSB added with the pH shift buffers. The production of ADP is monitored. The ATP regeneration system is set up so as not to limit the observed results. For example, results were the same if the phosphoenolpyruvate (PEP) concentration was reduced from 3 to 1.5 mM. The starting kcat for ATP hydrolysis is reported as the measured rate of ATP hydrolysis(Julia M. Cox, Oleg V. Tso)