Rep Monomer Research Articles

Subunit Communication within Dimeric SF1 DNA Helicases

Monomers of the Superfamily (SF) 1 helicases, E. coli Rep and UvrD, can translocate directionally along single stranded (ss) DNA, but must be activated to function as helicases. In the absence of accessory factors, helicase activity requires Rep and UvrD homo-dimerization. The ssDNA binding sites of SF1 helicases contain a conserved aromatic amino acid (Trp250 in Rep and Trp256 in UvrD) that stacks with the DNA bases. Here we show that mutation of this Trp to Ala eliminates helicase activity in both Rep and UvrD. Rep(W250A) and UvrD(W256A) can still dimerize, bind DNA, and monomers still retain ATP-dependent ssDNA translocase activity, although with ∼10-fold lower rates and lower processivities than wild type monomers. Although neither wtRep monomers nor Rep(W250A) monomers possess helicase activity by themselves, using both ensemble and single molecule methods, we show that helicase activity is achieved upon formation of a Rep(W250A)/wtRep hetero-dimer. An ATPase deficient Rep monomer is unable to activate a wtRep monomer indicating that ATPase activity is needed in both subunits of the Rep hetero-dimer. We find the same results with E. coli UvrD and its equivalent mutant (UvrD(W256A)). Importantly, Rep(W250A) is unable to activate a wtUvrD monomer and UvrD(W256A) is unable to activate a wtRep monomer indicating that specific dimer interactions are required for helicase activity. We also demonstrate subunit communication within the dimer by virtue of Trp fluorescence signals that only are present within the Rep dimer, but not the monomers. These results bear on proposed subunit switching mechanisms for dimeric helicase activity.

Regulation of E. coli Rep helicase activity by PriC

Stalled DNA replication forks can result in incompletely replicated genomes and cell death. DNA replication restart pathways have evolved to deal with repair of stalled forks and E. coli Rep helicase functions in this capacity. Rep and an accessory protein, PriC, assemble at a stalled replication fork to facilitate loading of other replication proteins. A Rep monomer is a rapid and processive single stranded (ss) DNA translocase but needs to be activated to function as a helicase. Activation of Rep in vitro requires self-assembly to form a dimer, removal of its auto-inhibitory 2B sub-domain, or interactions with an accessory protein. Rep helicase activity has been shown to be stimulated by PriC, although the mechanism of activation is not clear. Using stopped flow kinetics, analytical sedimentation and single molecule fluorescence methods, we show that a PriC dimer activates the Rep monomer helicase and can also stimulate the Rep dimer helicase. We show that PriC can self-assemble to form dimers and tetramers and that Rep and PriC interact in the absence of DNA. We further show that PriC serves as a Rep processivity factor, presumably co-translocating with Rep during DNA unwinding. Activation is specific for Rep since PriC does not activate the UvrD helicase. Interaction of PriC with the C-terminal acidic tip of the ssDNA binding protein, SSB, eliminates Rep activation by stabilizing the PriC monomer. This suggests a likely mechanism for Rep activation by PriC at a stalled replication fork.

Processivity of nucleic acid unwinding and translocation by helicases.

Helicases are a class of enzymes that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid (NA) strand separation. Besides the NA unwinding speed, another important factor for the helicase activity is the NA unwinding processivity. Here, we study the NA unwinding processivity with an analytical model that captures the phenomenology of the NA unwinding process. First, we study the processivity of the non-hexameric helicase that can unwind NA efficiently in the form of a monomer and the processivity of the hexameric helicase that can unwind DNA effectively, providing quantitative explanations of the available single-molecule experimental data. Then, we study the processivity of the non-hexameric helicases, in particular UvrD, in the form of a dimer and compare with that in the form of a monomer. The available single-molecule and some biochemical data showing that while UvrD monomer is a highly processive single-stranded DNA translocase it is inactive in DNA unwinding, whereas other biochemical data showing that UvrD is active in both single-stranded DNA translocation and DNA unwinding in the form of a monomer can be explained quantitatively and consistently. In addition, the recent single-molecule data are also explained quantitatively showing that constraining the 2B subdomain in closed conformation by intramolecular cross-linking can convert Rep monomer with a very poor DNA unwinding activity into a superhelicase that can unwind more than thousands of DNA base pairs processively, even against a large opposing force. Proteins 2016; 84:1590-1605. © 2016 Wiley Periodicals, Inc.

Bacillus stearothermophilus PcrA Monomer Is a Single-stranded DNA Translocase but Not a Processive Helicase in Vitro

Structural studies of the Bacillus stearothermophilus PcrA protein along with biochemical studies of the single-stranded (ss) DNA translocation activity of PcrA monomers have led to the suggestion that a PcrA monomer possesses processive helicase activity in vitro. Yet definitive studies testing whether the PcrA monomer possesses processive helicase activity have not been performed. Here we show, using single turnover kinetic methods, that monomers of PcrA are able to translocate along ssDNA, in the 3' to 5' direction, rapidly and processively, whereas these same monomers display no detectable helicase activity under the same solution conditions in vitro. The PcrA monomer ssDNA translocation activity, although necessary, is not sufficient for processive helicase activity, and thus the translocase and helicase activities of PcrA are separable. These results also suggest that the helicase activity of PcrA needs to be activated either by self-assembly or through interactions with accessory proteins. This same behavior is displayed by both the Escherichia coli Rep and UvrD monomers. Hence, all three of these SF1 enzymes are ssDNA translocases as monomers but do not display processive helicase activity in vitro unless activated. The fact that the translocase and helicase activities are separable suggests that each activity may be used for different functions in vivo.

Repetitive shuttling of a motor protein on DNA

Many helicases modulate recombination, an essential process that needs to be tightly controlled. Mutations in some human disease helicases cause increased recombination, genome instability and cancer. To elucidate the potential mode of action of these enzymes, here we developed a single-molecule fluorescence assay that can visualize DNA binding and translocation of Escherichia coli Rep, a superfamily 1 DNA helicase homologous to Saccharomyces cerevisiae Srs2. Individual Rep monomers were observed to move on single-stranded (ss)DNA in the 3' to 5' direction using ATP hydrolysis. Strikingly, on hitting a blockade, such as duplex DNA or streptavidin, the protein abruptly snapped back close to its initial position, followed by further cycles of translocation and snapback. This repetitive shuttling is likely to be caused by a blockade-induced protein conformational change that enhances DNA affinity for the protein's secondary DNA binding site, thereby resulting in a transient DNA loop. Repetitive shuttling was also observed on ssDNA bounded by a stalled replication fork and an Okazaki fragment analogue, and the presence of Rep delayed formation of a filament of recombination protein RecA on ssDNA. Thus, the binding of a single Rep monomer to a stalled replication fork can lead to repetitive shuttling along the single-stranded region, possibly keeping the DNA clear of toxic recombination intermediates.

Autoinhibition of Escherichia coli Rep monomer helicase activity by its 2B subdomain

DNA helicases catalyze separation of double-helical DNA into its complementary single strands, a process essential for DNA replication, recombination, and repair. The Escherichia coli Rep protein, a superfamily 1 DNA helicase, functions in DNA replication restart and is required for replication of several bacteriophages. Monomers of Rep do not display helicase activity in vitro; in fact, DNA unwinding requires Rep dimerization. Here we show that removal of the 2B subdomain of Rep to form RepDelta2B activates monomer helicase activity, albeit with limited processivity. Although both full length Rep and RepDelta2B monomers can translocate with 3' to 5' directionality along single-stranded DNA, the 2B subdomain inhibits the helicase activity of full length Rep. This suggests an autoregulatory mechanism for Rep helicase, which may apply to other nonhexameric helicases, whereby helicase activity is regulated by the rotational conformational state of the 2B subdomain; formation of a Rep dimer may relieve autoinhibition by altering the 2B subdomain orientation.

DNA-binding Orientation and Domain Conformation of the E. coli Rep Helicase Monomer Bound to a Partial Duplex Junction: Single-molecule Studies of Fluorescently Labeled Enzymes

The SF1 DNA helicases are multi-domain proteins that can unwind duplex DNA in reactions that are coupled to ATP binding and hydrolysis. Crystal structures of two such helicases, Escherichia coli Rep and Bacillus stearothermophilus PcrA, show that the 2B sub-domain of these proteins can be found in dramatically different orientations (closed versus open) with respect to the remainder of the protein, suggesting that the 2B domain is highly flexible. By systematically using fluorescence resonance energy transfer at the single-molecule level, we have determined both the orientation of an E.coli Rep monomer bound to a 3′-single-stranded-double-stranded (ss/ds) DNA junction in solution, as well as the relative orientation of its 2B sub-domain. To accomplish this, we developed a highly efficient procedure for site-specific fluorescence labeling of Rep and a bio-friendly immobilization scheme, which preserves its activities. Both ensemble and single-molecule experiments were carried out, although the single-molecule experiments proved to be essential here in providing quantitative distance information that could not be obtained by steady-state ensemble measurements. Using distance-constrained triangulation procedures we demonstrate that in solution the 2B sub-domain of a Rep monomer is primarily in the “closed” conformation when bound to a 3′-ss/ds DNA, similar to the orientation observed in the complex of PcrA bound to a 3′-ss/ds DNA. Previous biochemical studies have shown that a Rep monomer bound to such a 3′-ss/ds DNA substrate is unable to unwind the DNA and that a Rep oligomer is required for helicase activity. Therefore, the closed form of Rep bound to a partial duplex DNA appears to be an inhibited form of the enzyme.

Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase.

Helicases are motor proteins that couple conformational changes induced by ATP binding and hydrolysis with unwinding of duplex nucleic acid, and are involved in several human diseases. Some function as hexameric rings, but the functional form of non-hexameric helicases has been debated. Here we use a combination of a surface immobilization scheme and single-molecule fluorescence assays--which do not interfere with biological activity--to probe DNA unwinding by the Escherichia coli Rep helicase. Our studies indicate that a Rep monomer uses ATP hydrolysis to move toward the junction between single-stranded and double-stranded DNA but then displays conformational fluctuations that do not lead to DNA unwinding. DNA unwinding initiates only if a functional helicase is formed via additional protein binding. Partial dissociation of the functional complex during unwinding results in interruptions ('stalls') that lead either to duplex rewinding upon complete dissociation of the complex, or to re-initiation of unwinding upon re-formation of the functional helicase. These results suggest that the low unwinding processivity observed in vitro for Rep is due to the relative instability of the functional complex. We expect that these techniques will be useful for dynamic studies of other helicases and protein-DNA interactions.

E. coli Rep oligomers are required to initiate DNA unwinding in vitro

E. coli Rep protein is a 3′ to 5′ SF1 superfamily DNA helicase which is monomeric in the absence of DNA, but can dimerize upon binding either single-stranded or duplex DNA. A variety of biochemical studies have led to proposals that Rep dimerization is important for its helicase activity; however, recent structural studies of Bacillus stearothermophilus PcrA have led to suggestions that SF1 helicases, such as E. coli Rep and E. coli UvrD, function as monomeric helicases. We have examined the question of whether Rep oligomerization is important for its DNA helicase activity using pre-steady state stopped-flow and chemical quenched-flow kinetic studies of Rep-catalyzed DNA unwinding. The results from four independent experiments demonstrate that Rep oligomerization is required for initiation of DNA helicase activity in vitro. No DNA unwinding is observed when only a Rep monomer is bound to the DNA substrate, even when fluorescent DNA substrates are used that can detect partial unwinding of the first few base-pairs at the ss-ds-DNA junction. In fact, under these conditions, ATP hydrolysis causes dissociation of the Rep monomer from the DNA, rather than DNA unwinding. These studies demonstrate that wild-type Rep monomers are unable to initiate DNA unwinding in vitro, and that oligomerization is required.

Negative control of plasmid pSC101 replication by increased concentrations of both initiator protein and iterons.

Increased intracellular concentrations of the initiator protein Rep (or RepA) interfere with pSC101 DNA replication, and mutated Rep proteins that result in an increase in plasmid copy numbers do not inhibit the replication. A rep mutant (rep(inh)) defective in the inhibitory activity was isolated and found to be a new high copy number mutant. The inhibitory function of Rep was enhanced by the coexistence of directly repeated sequences (DR; iterons) in the replication origin region (ori), but not by the inverted repeat sequences (IR) in ori and the rep promoter. This synergistic effect of Rep and DR sequences for the replication inhibition was dependent on their intracellular concentrations. Considering that DR sequences are the specific binding sites of the Rep monomer form, the Rep monomer-DR complex might be responsible for the inhibition of the plasmid replication. Furthermore, the Rep monomer in the crude cell extracts facilitated dimerization of DR DNA fragments by DNA ligase. Neither synergistic inhibitory function with DR nor Rep mediated dimerization of DR DNA was observed in high copy number mutant Rep proteins. The role of the Rep-iteron complex in the copy number control of pSC101 is discussed.

DNA bending of pSC101 ori induced by Rep, a replication initiator protein and IHF.

Purified Rep (or RepA) protein, a replication initiator of plasmid pSC101, is present almost solely in the dimer form, and its binding activity for the directly repeated sequences (iterons) in the replication origin (ori) is very low. When Rep protein was treated with guanidine hydrochloride followed by renaturation, it was shown to bind to the iterons with very high efficiency. A gel shift experiment suggested that guanidine-treated Rep bound to iterons as a monomer form. The Rep monomer bound noncooperatively to the three iterons and induced bending of the DNA helix axis in the same direction (about 100 degrees ). The configuration of the IHF box that is a binding site of another DNA bending protein IHF, the three iterons and an AT-rich region between these sequences was important for efficient bending of the ori region. Furthermore, a mutant Rep protein (Rep(IHF)) which can support the plasmid replication in IHF-deficient host cells was purified, and it was found that affinity of the Rep(IHF) monomer for iterons was similar to that of wild-type Rep and bent DNA only 14 degrees more strongly than did the wild-type Rep. Rep(IHF)-dependent plasmid replication, however, required both enhancer regions, par and IR-1, in addition to "core ori" as a minimal essential ori, whereas only one of these two enhancers was necessary for wild-type Rep-dependent replication. How Rep(IHF) can support plasmid replication in the absence of IHF is discussed.

Kinetic mechanism of DNA binding and DNA-induced dimerization of the Escherichia coli Rep helicase.

The monomeric Escherichia coli Rep protein undergoes a DNA-induced dimerization upon binding either single-stranded (ss) or duplex DNA with the dimer being the active form of the Rep helicase. Using stopped-flow fluorescence, we have determined a minimal kinetic mechanism for this reaction in which Rep monomer (P) binds to ss oligodeoxynucleotides (dN(pN)15) (S) by a two-step mechanism to form PS*, which can then dimerize with P to form P2S as indicated: [reaction in text]. This minimal mechanism is supported by four independent studies in which the kinetics were monitored by changes in fluorescence intensity of three different probes: the intrinsic Rep tryptophan fluorescence, the fluorescence of d(T5(2-AP)T4(2-AP)T5), containing the fluorescent base, 2-aminopurine (2-AP), and dT(pT)15 labeled at its 3'-end with fluorescein (3'-F-dT(pT)15). Simultaneous (global) analysis of the time courses of d(T5(2-AP)T4(2-AP)T5) (100 nM) binding to a range of Rep monomer concentrations (25-400 nM) yields the following rate constants: k1 = (3.3 +/- 0.5) x 10(7) M-1 s-1; k-1 = 1.4 +/- 0.4 s-1; k2 = 2.7 +/- 0.9 s-1; k-2 = 0.21 +/- 0.06 s-1; k3 = (4.5 +/- 0.3) x 10(5) M-1 s-1; k-3 = 0.0027 +/- 0.0008 s-1 [20 mM Tris-HCl, pH 7.5, 6 mM NaCl, 5 mM MgCl2, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol, 4.0 degrees C]. This mechanism provides direct evidence that Rep monomers can bind ss DNA and that ss DNA binding induces a conformational change in the Rep monomer that is probably required for Rep dimerization. This conformational change is likely to be large and global since it is detected by all three fluorescence probes. The apparent bimolecular rate constant for Rep monomer binding to 3'-F-dT(pT)15 [k1(app) = (6.0 +/- 0.7) x 10(7) M-1 s-1] is slightly larger than measured with d(T5(2-AP)T4(2-AP)T5) binding. The apparent rate constant for dissociation of d(T5(2-AP)T4(2-AP)T5) (S) from the half-ligated Rep dimer, P2S, increases with increasing concentration of a nonfluorescent competitor ss DNA (d(T5-AT4AT5)) (C), indicating transient formation of a doubly ligated P2SC intermediate. However, the apparent bimolecular rate constant for binding of C to P2S is extremely slow (> or = 250 M-1 s-1), suggesting the occurrence of a multistep process before dissociation of ss DNA. In the absence of competitor DNA, dissociation of ss DNA from P2S occurs only after slow dissociation of the Rep dimer to form PS* + P. The implications of these results for Rep-catalyzed DNA unwinding are discussed.

ATPase activity of Escherichia coli Rep helicase is dramatically dependent on DNA ligation and protein oligomeric states.

The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA using the energy derived from ATP binding and hydrolysis. Rep functions as a dimer but assembles to its active dimeric form only on binding DNA. Each promoter of a dimer contains a DNA binding site that can bind either single-stranded (S) or duplex (D) DNA. The dimer can bind up to two oligodeoxynucleotides in five DNA-ligation states: two half-ligated states, P2S and P2D, and three fully-ligated states, P2S2, P2D2, and P2SD. We have previously shown that the relative stabilities of these ligation states are allosterically regulated by the binding and hydrolysis of ATP and have proposed an "active rolling" model for DNA unwinding where the enzyme cycles through a series of these ligation states in a process that is coupled to the catalytic cycle of ATP hydrolysis [Wong, I., & Lohman, T.M., (1992), Science 256, 350-355]. THe basal ATPase activity of Rep protein is stimulated by ss DNA binding and by protein dimerization. We have measured the steady-state ATPase activities of Rep bound to dT(pT)15 in each distinct ss DNA ligation state (PS, P2S, and P2S2) to compare with our previous measurements with unligated Rep monomer (P) [Moore, K.J.M., & Lohman, T.M. (1994) Biochemistry 33, 14550]. We find the ATPase activity of Rep is influenced dramatically by both dimerization and ss DNA ligation state, with the following kcat values for ATP hydrolysis increasing by over 4 orders of magnitude: 2.1 x 10(-3) s(-1) for P, 2.17 +/- 0.04 s(-1) for PS, 16.5 +/- 0.2 s(-1) for P2S, and 71 +/- 2.5 s(-1) for P2S2 (20 mM Tris-HCl, pH 7.5, 6mM NaCl, 5 mM MgCl2, 10% glycerol, 4 degrees C). The apparent KM's for ATP hydrolysis are 2.05 +/- 0.1 microM for PS and 2.7 +/- 0.2 microM for P2S. These widely different ATPase activities reflect the allosteric effects of DNA ligation and demonstrate that cooperative communication occurs between the ATP and DNA site of both subunits of the Rep dimer. These results further emphasize the need to explicitly consider the population distribution of oligomerization and DNA ligation states of the helicase when attempting to infer information about elementary processes such as helicase translocation based solely on macroscopic steady-state ATPase measurements.

Kinetic mechanism of adenine nucleotide binding to and hydrolysis by the Escherichia coli Rep monomer. 1. Use of fluorescent nucleotide analogs

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 + mantATP [formula: see text] P-mantATP [formula: see text] (P-mantATP). The following rate and equilibrium constants were determined at 4 degrees C in 20 mM Tris.HCl (pH 7.5), 6 mM NaCl, 5 mM MgCl2, and 10% (v/v) glycerol: k+1 = (1.1 +/- 0.2) x 10(7) M-1 s-1; k-1 = 3.2 (+/- 0.5) s-1; k+2 = 2.9 (+/- 0.5) s-1; k-2 = 0.04 (+/- 0.005) s-1; K1 = k+1/k-1 = (3.4 +/- 0.8) x 10(6) M-1; K2 = k+2/k-2 = 73 (+/- 10); Koverall = K1K2 = (2.30 +/- 0.6) x 10(8) M-1. Similar rate and equilibrium constants are obtained with mantATP gamma S, whereas the apparent rate constant for mantAMPPNP binding is 15-fold lower than for mantATP and equilibrium binding is weaker (Koverall approximately 10(6) M-1). Rep monomer does bind mantATP in the absence of Mg2+ (Koverall approximately 5 x 10(5) M-1), although the four rate constants in the above reaction increase by at least 8-fold (k-1 and k-2 increase by approximately 100- and approximately 1000-fold, respectively). The affinities of Mg2+ for P-mantATP and (P-mantATP)* are 10- and 1000-fold higher than those for nucleotide-free 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetic mechanism of adenine nucleotide binding to and hydrolysis by the Escherichia coli Rep monomer. 2. Application of a kinetic competition approach

The Escherichia coli Rep protein is a DNA helicase that functions as a homodimer to catalyze the unwinding of duplex DNA during DNA replication in a reaction that is coupled to the binding and hydrolysis of ATP. As a first step toward a molecular understanding of the interactions of Rep with adenine nucleotides, we have investigated the kinetic mechanism of adenine nucleotide binding to the Rep monomer, which is the state of the protein in the absence of DNA. Although ATP binding to Rep does not significantly change the intrinsic tryptophan fluorescence, the binding of the fluorescent nucleotide analogue, 2'(3')-O-(N-methylanthraniloyl)-ATP (mantATP) is associated with a large increase in mant nucleotide fluorescence intensity [lambda ex = 290 nm, lambda em > 420 nm; Moore, K. J. M., & Lohman, T. M. (1994) Biochemistry (preceding article in this issue)]. We have used the fluorescence signal from mantATP binding to monitor the kinetics of nonfluorescent nucleotide binding to Rep by a kinetic competition approach. The simultaneous and parallel binding of a mixture of mantATP and ATP to the Rep monomer is associated with a complex triphasic fluorescence transient during the approach to equilibrium. Global analysis of the fluorescence transients over a range of [ATP] by numerical integration techniques was used to define the kinetic mechanism of ATP binding and to determine the elementary rate constants. Using this approach, the kinetic rate constants for ADP, ATP gamma S, AMPPNP, AMP, adenosine, and inorganic phosphate were also determined at 4 degrees C in 20 mM Tris.HCl (pH 7.5), 6 mM NaCl, 10% (v/v) glycerol, and 5 mM MgCl2. The kinetics of adenine nucleotide binding to the Rep monomer are similar to those observed with the mant nucleotides under identical experimental conditions (Moore & Lohman, 1994). The kinetic competition data are consistent with the following two-step mechanism for the binding of ATP, ADP, and ATP gamma S, where P is the Rep monomer and A is the adenine nucleotide: P+A [formula: see text] P-A [formula: see text] (P-A). In the presence of 5 mM MgCl2, the values of K+1 (approximately 10(7) M-1 s-1) and k+2 (approximately 10 s-1) are comparable for each nucleotide, whereas k+2 > k-1 for ATP and ATP gamma S while for ADP k+2 << k-1; hence, differences in the overall equilibrium binding affinities of these nucleotides are primarily due to changes in k-1.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA-induced dimerization of the Escherichia coli rep helicase. Allosteric effects of single-stranded and duplex DNA.

The Escherichia coli Rep helicase is a stable monomer (Mr = 72,802) in the absence of DNA; however, binding of single-stranded (ss) or duplex (ds) DNA induces Rep monomers to dimerize. Furthermore, a chemically cross-linked Rep dimer retains both its DNA-dependent ATPase and helicase activities, suggesting that the functionally active Rep helicase is a dimer (Chao, K., and Lohman, T. M. (1991) J. Mol. Biol. 221, 1165-1181). Using a modified "double-filter" nitrocellulose filter binding assay, we have examined quantitatively the equilibrium binding of Rep to a series of ss-oligodeoxynucleotides, d(pN)n (8 less than or equal to n less than or equal to 20) and two 16-base pair duplex oligodeoxynucleotides, which are short enough so that only a single Rep monomer can bind to each oligonucleotide. This strategy has enabled us to examine the linkage between DNA binding and dimerization. We also present a statistical thermodynamic model to describe the DNA-induced Rep dimerization in the presence of ss- and/or ds-oligodeoxynucleotides. We observe quantitative agreement between this model and the experimental binding isotherms and have analyzed these isotherms to obtain the seven independent interaction constants that describe Rep-DNA binding and Rep dimerization. We find that Rep monomers (P) can bind either ss-DNA (S) or ds-DNA (D) to form PS or PD, respectively, which can then dimerize to form P2S or P2D. Furthermore, both protomers of the DNA-induced Rep dimer can bind DNA to form either P2S2, P2D2 or the mixed dimer species P2SD and ss- and ds-DNA compete for the same sites on the Rep protein. When bound to DNA, the Rep dimerization constants are approximately 1-2 x 10(8) M-1 (6 mM NaCl, pH 7.5, 4 degrees C), which are greater than the dimerization constant for free Rep monomers by at least 10(4)-fold. The Rep-ss-DNA interaction constants are independent of base composition and sequence, consistent with its role as a nonspecific DNA-binding protein. Allosteric effects are associated with ss- and ds-DNA binding to the half-saturated Rep dimers, i.e. the affinity of either ss- or ds-DNA to the free promoter of a half-saturated Rep dimer is clearly influenced by the conformation of DNA bound to the first protomer. These allosteric effects further support the proposal that the Rep dimer is functionally important and that the Rep-DNA species P2S2 and P2SD may serve as useful models for intermediates that occur during DNA unwinding.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA-induced dimerization of the Escherichia coli Rep helicase

The Escherichia coli Rep protein is a DNA helicase that is involved in DNA replication. We have examined the effects of DNA binding on the assembly state of the Rep protein using small-zone gel permeation chromatography and chemical crosslinking of the protein. Complexes of Rep protein were formed with short single-stranded and duplex hairpin oligodeoxynucleotides with lengths such that only a single Rep monomer could bind per oligodeoxynucleotide (i.e. 2 Rep monomers could not bind contiguously on the oligodeoxynucleotides). In the absence of DNA, Rep protein is monomeric (Mr 72,800) up to concentrations of at least 8 μm (monomer), even in the presence of its nucleotide cofactors (ATP, ADP, ATP-γ-S). However, the binding of Rep monomers to single-stranded (ss) oligodeoxynucleotides, d(pN)n (12 ≤ n ≤ 20), induces the Rep monomers to oligomerize. Upon treatment of the Rep-ss oligodeoxynucleotide complexes with the protein crosslinking reagent dimethyl-suberimidate (DMS) and subsequent removal of the DNA, crosslinked Rep dimers are observed, independent of oligodeoxynucleotide length (n ≤ 20). Furthermore, short duplex oligodeoxynucleotides also induce the Rep monomers to dimerize. Formation of the Rep dimers results from an actual DNA-induced dimerization, rather than the adventitious crosslinking of Rep monomers bound contiguously to a single oligodeoxynucleotide. The purified DMS-crosslinked Rep dimer shows increased affinity for DNA and retains DNA-dependent ATPase and DNA helicase activities, as shown by its ability to unwind M13 RF DNA in the presence of the bacteriophage f1 gene II protein. On the basis of these observations and since the dimer is the major species when Rep is bound to DNA, we suggest that a DNA-induced Rep dimer is the functionally active form of the Rep helicase.

DNA-induced dimerization of the Escherichia coli Rep helicase

The Escherichia coli Rep protein is a DNA helicase that is involved in DNA replication. We have examined the effects of DNA binding on the assembly state of the Rep protein using small-zone gel permeation chromatography and chemical crosslinking of the protein. Complexes of Rep protein were formed with short single-stranded and duplex hairpin oligodeoxynucleotides with lengths such that only a single Rep monomer could bind per oligodeoxynucleotide (i.e. 2 Rep monomers could not bind contiguously on the oligodeoxynucleotides). In the absence of DNA, Rep protein is monomeric ( M r 72,800) up to concentrations of at least 8 μ m (monomer), even in the presence of its nucleotide cofactors (ATP, ADP, ATP-γ-S). However, the binding of Rep monomers to single-stranded (ss) oligodeoxynucleotides, d(pN) n (12 ≤ n ≤ 20), induces the Rep monomers to oligomerize. Upon treatment of the Rep-ss oligodeoxynucleotide complexes with the protein crosslinking reagent dimethyl-suberimidate (DMS) and subsequent removal of the DNA, crosslinked Rep dimers are observed, independent of oligodeoxynucleotide length ( n ≤ 20). Furthermore, short duplex oligodeoxynucleotides also induce the Rep monomers to dimerize. Formation of the Rep dimers results from an actual DNA-induced dimerization, rather than the adventitious crosslinking of Rep monomers bound contiguously to a single oligodeoxynucleotide. The purified DMS-crosslinked Rep dimer shows increased affinity for DNA and retains DNA-dependent ATPase and DNA helicase activities, as shown by its ability to unwind M13 RF DNA in the presence of the bacteriophage f1 gene II protein. On the basis of these observations and since the dimer is the major species when Rep is bound to DNA, we suggest that a DNA-induced Rep dimer is the functionally active form of the Rep helicase.