From the Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D. C. 20007
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ABSTRACT |
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The molecular chaperone ClpX of Escherichia coli plays two distinct functions for bacteriophage Mu DNA replication by transposition. As specificity component of a chaperone-linked protease, it recognizes the Mu immunity repressor for degradation by the peptidase component ClpP, thus derepressing Mu transposition functions. After strand exchange has been promoted by MuA transposase, ClpX alone can alter the conformation of the transpososome (the complex of MuA with Mu ends), and the remodeled MuA promotes transition to replisome assembly. Although ClpXP can degrade MuA, the presence of both ClpP and ClpX in the reconstituted transposition system did not destroy MuA essential for initiation of DNA replication by specific host replication enzymes. Levels of ClpXP needed to overcome inhibition by the repressor did not prevent MuA from promoting strand transfer, and ClpP stimulated alteration of the transpososome by ClpX. Apparently intact MuA was still present in the resulting transpososome, promoting initiation of Mu DNA replication by specific replication enzymes. The results indicate that ClpXP can discriminate repressor and MuA in the transpososome as substrates of the protease or the molecular chaperone alone, degrading repressor while remodeling MuA for its next critical function.
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INTRODUCTION |
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Chaperone-linked proteases employ the action of a protease component and a regulatory subunit with ATPase activity. In Escherichia coli, ClpA and ClpX proteins serve as regulatory subunits for the protease subunit ClpP (1-3). These regulatory subunits can by themselves act as molecular chaperones, breaking up aggregates or changing quaternary interactions of specific protein substrates (4, 5). The substrates of the molecular chaperone can become the targets of the chaperone-linked protease when the corresponding protease subunit is present (6). Thus, the molecular chaperones endow the protease with substrate specificity, most likely recognizing the substrate and presenting it to the protease subunit.
ClpP is synthesized as a protein of 207 amino acids (7), of which 14 amino acids at the N terminus are autocatalytically removed to yield the mature enzyme (8). The active protein consists of a tetradecamer, composed of two stacked heptameric rings (9, 10). The structure of ClpP is analogous to the 20 S proteasomes of eukaryotes and Archaebacteria, with multiple active sites residing in the interior of the multimeric rings (10). Presumably, the molecular chaperones unfold the substrate and feed it into the proteolytic chamber of the ClpP tetradecamer, leading to the apparently processive degradation of the substrate (11).
ClpX is known to be involved in two distinct stages of the Mu life cycle. As part of the ClpXP protease, it can promote entry of a lysogen into lytic development by degrading the Mu immunity repressor (12, 13), which serves to shut down Mu transposition functions for the establishment and maintenance of lysogeny. ClpX also promotes initiation of Mu DNA replication, a process that does not require ClpP (14). It activates MuA's function of promoting transition to DNA synthesis after MuA's role in recombination has been completed (15).
During transposition, the MuA transposase binds to the Mu ends (16) and assembles into a tetramer (17), which catalyzes transfer of each Mu end to target DNA (18-20). This forms a branched DNA intermediate that contains a potential replication fork at each Mu end. Replication of Mu DNA by specific host enzymes completes transposition to form the cointegrate product (21-25), and MuA plays a critical role in this process. Upon completing Mu strand transfer, MuA remains very tightly bound to the Mu ends, still holding the Mu ends together in a nucleoprotein complex known as the type II transpososome (or the strand transfer complex 1, STC1)1 (19, 26). Removal of MuA by phenol extraction to create a deproteinized strand transfer product (STP) eliminates the requirement in vitro for specific host replication proteins implicated in Mu replication in vivo (25, 27), and if MuA in STC1 is damaged by partial proteolysis, replication cannot be initiated (25).
ClpX remodels MuA in STC1 to form STC2, destabilizing MuA's tight grip
on DNA without removing it from the nucleoprotein complex and
activating MuA's potential to promote transition to a replisome (15).
Yet unidentified factors (Mu replication factor (MRF) 2)
displace MuA in STC2 to form a new nucleoprotein complex STC3 (15),
which permits initiation of DNA replication only by specific replication proteins including the DNA polymerase (pol) III holoenzyme and constituents of the multiprotein priming apparatus known as the
X174-type primosome (27).
However, MuA is also a substrate for the ClpXP protease (28) just as other known substrates of ClpX and ClpA as molecular chaperones are substrates of the corresponding chaperone-linked protease (reviewed in Gottesman et al. (29)). Therefore, can ClpX in the presence of ClpP remodel MuA in STC1 without degrading it? Whether a protein substrate is degraded or remodeled may simply be determined by the availability of ClpP when the substrate is bound by the molecular chaperone. Such a model raises the question of how ClpX could perform the two distinct functions for Mu lytic development, first promoting degradation of repressor by ClpP and then remodeling MuA in STC1 for transition to replisome assembly. The first function requires ClpP but the second function could potentially be disrupted by the engagement of the ClpP proteolytic activity. ClpX would not be expected to act in isolation from ClpP in vivo to perform the second function, raising the question of what effect ClpP has on the DNA replication phase of Mu transposition. Here we report that ClpP does not interfere with MuA-mediated transition to DNA synthesis even when present in saturating amounts and in fact stimulates ClpX-promoted conversion of STC1 to STC2.
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EXPERIMENTAL PROCEDURES |
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Materials
Bacterial Strains, Plasmids, and Proteins--
E.
coli BL21(DE3) (30) and plasmid pET-19b were purchased from
Novagen. Plasmid pWPC9 (8) including the coding sequences for ClpP and
ClpX was obtained from Susan Gottesman (National Institutes of Health).
Plasmid pGG215 (19) bearing a mini-Mu element was used for Mu
transposition in vitro. Phage f1 RFI DNA and X174 RFI DNA
were used as target substrates. Purified preparations of MuA, MuB,
Muc+ (Rep), Mucvir3060 (Vir), and host proteins
required for Mu transposition and replication have been described (15,
31). DNA pol III* was purified from MGC1020 (W3110
malE::Tn10, lexA3, uvrD::kan) obtained
from Dr. Charles McHenry (University of Colorado Health Sciences
Center) as described previously (32). Purification of ClpP is described
below. Restriction enzymes, DNA pol I, DNA pol I large fragment
(Klenow), and E. coli DNA ligase were purchased from New
England BioLabs.
Reagents and Other Materials--
SYBR® Green I was purchased
from Molecular Probes, Inc. HEPES and polyethylene glycol 8000 were
purchased from Fisher. Trizma base, phosphocreatine, creatine kinase,
polyethylenimine,
N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin (Suc-Leu-Tyr-AMC), 7-amido-4-methylcoumarin (AMC),
N-p-tosyl-L-lysine
chloromethyl ketone (TLCK) and ribonucleoside triphosphates were from
Sigma. Bovine serum albumin was from Worthington. Ni-NTA
(nitrilotriacetic acid) agarose was from Qiagen.
[
-32P]dCTP (3000 Ci/mmol) was purchased from NEN Life
Science Products. Glutaraldehyde was from Electron Microscopy Sciences.
Nitrocellulose (BA83, 0.2-µm pore size) was from Schleicher & Schuell. Rabbit antibody against MuA protein was prepared by Biocon,
Inc. The enhanced chemiluminescence system from Amersham was used for
all immunoblots.
Buffers-- Composition of buffers was for buffer A, 25 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1 mM dithiothreitol; buffer B, 20 mM NaPO4 (pH 7.8), 500 mM NaCl; buffer C, 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, 2 mM dithiothreitol, 10% w/v glycerol; cracking buffer, 50 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol, 1% SDS, 0.1% bromphenol blue, 10% glycerol; TAE electrophoresis buffer, 40 mM Tris, 20 mM acetic acid, 2 mM EDTA (pH 8.1); and alkaline electrophoresis buffer, 30 mM NaOH, 2 mM EDTA.
Methods
Fluorogenic Assay for ClpP Activity-- The protease activity of ClpP alone was measured by its ability to cleave the fluorogenic substrate Suc-Leu-Tyr-AMC (33). Suc-Leu-Tyr-AMC (5 mM) was incubated with ClpP in buffer A (25 µl total volume) for 30 min at 37 °C. The reaction was stopped by addition of 475 µl of ice-cold 0.6 M imidazole (pH 7.8) and transferred to a quartz cuvette suitable for analysis in an AMINCO Bowman Series 2 luminescence spectrometer. To determine the amount of AMC generated, the sample was irradiated at 380 nm, and emission at 460 nm was measured and compared with a standard curve for AMC fluorescence. One unit of ClpP activity was defined as 10 pmol of AMC generated under the conditions described here.
Overexpression of H-ClpP and Purification by Ni-Column
Chromatography--
Strain BL21(DE3) transformed with plasmid pCPX01
was grown in 10 liters of modified terrific broth (15) in a 14-liter
New Brunswick fermenter (37 °C, A595 = 2.0),
and ClpP overproduction was induced by the addition of isopropyl
-D-thiogalactopyranoside to 0.4 mM. After 30 min, cells were harvested into chilled bottles and collected by
centrifugation at 4 °C (74 g of wet cells). Cells were resuspended
in buffer B to an A595 of 315 and transferred to
Ti45 centrifuge tubes.
Bacteriophage Mu Transposition and Replication by Transposition
in Vitro--
STC1 was prepared as described previously using pGG215
donor substrate and f1 RFI DNA or X174 RFI target DNA substrates
(31). Where indicated, STC1 was deproteinized as described previously to form deproteinized STP (34). Reaction mixtures for Mu replication (50 µl) included STC1 or the deproteinized STP (equivalent of 0.25 µg of donor substrate) and the 8-protein system composed of DNA
gyrase (335 ng), DnaB/DnaC complex (65 ng), DnaG (42 ng), DNA pol III
holoenzyme (58 ng), SSB protein (45 ng), DNA pol I (0.01 unit), and DNA
ligase (0.2 unit) or the 6-protein system, which lacks DNA pol I and
ligase. Where indicated, reactions included 12 units of MRF
(Fraction III) prepared as described previously (31) from E. coli strain AT3327 PriA1::Kan (27), and 0.04 unit each of PriA, PriB, PriC, and DnaT (see Marians (35) for unit
definition). Incubation was at 37 °C for 2-30 min. Reaction conditions and determination of total deoxynucleotide incorporation were as described previously (31).
Gel Disruption Assay to Detect Conversion of STC1 to STC2-- Conversion of STC1 to STC2 was measured by detecting disruption of the synaptic complex of Mu ends by agarose gel electrophoresis. The synaptic complex is unstable in STC2 and is disassembled during electrophoresis (15). Reaction mixtures (25 µl) containing STC1 (equivalent of 0.25 µg of donor substrate, predigested with PstI (300 units/ml at 37 °C for 30 min)), ClpX, and ClpP (concentrations indicated in individual experiments) were incubated at 37 °C for 2-30 min under previously described reaction conditions (15). Reactions were stopped with EDTA (20 mM) and heparin (2 mg/ml), and products were resolved on a 0.6% agarose gel (TAE electrophoresis buffer containing 0.5 µg/ml EtBr). DNA was stained with SYBR® Green I for detection by the Molecular Dynamics Storm 840 system, and bands corresponding to STC1 and the disrupted complex (STC2) were quantitated using ImageQuant software.
Other Methods-- Glutaraldehyde fixation of nucleoprotein complexes, Western blot analysis, and isolation of nucleoprotein complexes by gel filtration were performed as described previously (15). Densitometric analysis of Western blots detected by enhanced chemiluminescence was performed using Scan AnalysisTM software from Biosoft. ClpP protein concentrations were determined by the method of Bradford (36). N-terminal sequencing of purified ClpP protein was performed by Dr. A. Fowler (UCLA Protein Microsequencing Facility).
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RESULTS |
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Overproduction of Histidine-tagged ClpP and Rapid Purification of
the Mature Protein--
In plasmid pCPX01 the clpP and
clpX genes are under the control of the phage T7 10
promoter (Fig. 1A). ClpP
protein is expressed with a polyhistidine tag fused to the N terminus
(h-ClpP) for rapid purification on Ni-NTA agarose columns (see
"Experimental Procedures" and Table
I). Induction of BL21(DE3) harboring
pCPX01 resulted in the overproduction of proteins of molecular mass of 45, 23, and 21 kDa, apparently corresponding to ClpX, h-ClpP, and the
mature form of ClpP (Fig. 1B, lane 2). The
overproduction of mature ClpP suggested that h-ClpP could be processed
to the mature form just as the wild-type precursor ClpP (pre-ClpP) is autocatalytically processed by cleavage of 14 amino acids at the N
terminus (8).
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ClpXP Protease Relieves Inhibition of Mu Transposition in Vitro by Repressor in the ClpXP-sensitive State-- The Mu immunity repressor inhibits Mu transposition in vitro by competing for MuA binding sites in the operator that enhances transpososome assembly (38, 39). ClpXP protease can degrade the Mu immunity repressor (13), and it potentially plays an important role in inducing Mu DNA replication in lysogens. We determined whether the presence of ClpXP protease allows the reconstituted Mu transposition system to overcome inhibition by the repressor.
Although the wild-type repressor (Rep) is a substrate for the ClpXP protease, it is degraded with a relatively high Km and assumes a ClpXP-resistant state in the presence of DNA (13). A mutant form of the repressor (Vir, repressor encoded by the virulent strain Muvir3060), which has an alteration at the C terminus and is rapidly degraded in vivo by the ClpXP protease (12, 14), is degraded by ClpXP with a 20-fold lower Km and is locked into the high affinity state even when bound to DNA (13). Both Rep and Vir are known to bind operator DNA with nearly equal affinity, Vir having a slightly higher dissociation constant (40). They both inhibited Mu strand transfer in the reconstituted system (Fig. 2, cf. lanes 6 and 10 with lane 2), but only inhibition by Vir could be eliminated by ClpXP (cf. lane 13 with 9). And even though MuA is a ClpXP substrate (28), amounts of ClpXP sufficient to remove Vir inhibition did not destroy MuA needed to catalyze strand transfer (Fig. 2, lanes 5 and 13). MuA is degraded slowly compared with Vir (13), permitting MuA to carry out its transposition function at levels of ClpXP that effectively eliminate the repressor.
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ClpP Stimulates Action of ClpX on MuA Present in STC1-- One method for examining the conversion of STC1 to STC2 is to examine disassembly of the transpososome by agarose gel electrophoresis (15). Treatment of STC1 with ClpX destabilizes tight binding of MuA to the DNA such that it readily disassembles during challenge with heparin and resolution by electrophoresis, and disruption of the synaptic complex of Mu ends results in a change in mobility of the strand transfer product. We used this disassembly assay to compare the kinetics with which ClpX acts on STC1 in the presence or absence of ClpP.
The presence of ClpP increased by 2-3-fold the amount of STC1 disassembled at 30 min by limiting concentrations of ClpX (Fig. 3A). The level of ClpP used was saturating; the presence of 2-fold higher levels of ClpP did not result in any additional stimulation (data not shown). Time course experiments (Fig. 3, B and C) revealed that the extent of STC1 disassembly was approximately proportional to the amount of added ClpX and that ClpP increased the extent of STC1 disassembly at each ClpX concentration. Even in the presence of ClpP, however, an excess of ClpX monomers over STC1 was required for 100% disassembly, with at least 2.5 pmol of ClpX being required to disassemble 0.05 pmol of STC1 (formed from 250 ng of donor substrate in a reaction mixture containing 55 ng of MuA). If sufficient ClpX was present, ClpP was not essential for complete disassembly of STC1 (e.g. see Fig. 5, lane 7) as shown previously (15, 28).
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ClpXP Promotes Transition from STC1 to STC2, Leaving Activated MuA Molecules Bound to the Mu Ends-- The disassembly assay used to detect ClpX action above does not necessarily measure conversion of STC1 to STC2. STC2 includes activated MuA that remains bound to DNA and plays a critical role in the transition to DNA synthesis. The removal of that MuA from the DNA by artificial means such as phenol extraction eliminates the specific requirement in vitro for replication enzymes that are needed in vivo. For example, on the deproteinized STP, potential primers for leading strand synthesis at the Mu left and right ends can be extended by DNA pol I or Klenow fragment, whereas they cannot be extended by pol I in STC1, STC2, or STC3, even when all components of the Mu replication system except pol III holoenzyme are present (15, 27). We determined whether ClpX in the presence of ClpP removes MuA from STC1, leaving the ends of Mu accessible to DNA pol I, or converts STC1 to STC2 in which MuA remains bound to the ends.
Upon treatment of STC1 with ClpXP, the leading strand primers at the Mu left and right ends could not be appreciably extended by the Klenow fragment (Fig. 4B, lanes 5-7). This was true even after incubation for 60 min, twice the time required for 100% of STC1 to be disassembled by the gel electrophoresis assay (Fig. 3B). Identical results were obtained with STC1 treated with ClpX alone (Fig. 4B, lanes 2-4). Although a low level of nucleotide incorporation was detected after prolonged incubation of ClpXP-treated STC with Klenow, this was not associated with any measurable extension of the leading strand primers. Thus, only a limited amount of leading strand synthesis if any could have been catalyzed, and most of the incorporation was due to nonspecific DNA synthesis such as the alternating action of 3
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Specific Host Proteins Are Required to Initiate Mu DNA Replication
on ClpXP-treated STC--
The most important function of MuA bound to
STC2 is to allow initiation of Mu DNA replication only by a specific
group of host proteins, which include DnaB helicase, DnaC protein (15), and X174-type primosome proteins PriA and DnaT (27). Proteolytic damage to MuA in STC1 can block Mu replication even by these specific proteins (31). However, ClpXP-treated STC allowed Mu DNA synthesis to
proceed with kinetics comparable to that on STC treated with ClpX alone
(Fig. 7A). We have previously
demonstrated that the primers at the ends of Mu in STC are not extended
when all components required for replication except PriA or DnaBC are
present (27). Likewise, on both ClpX-treated and ClpXP-treated STC,
initiation of Mu DNA replication required MRF
, DnaB, DnaC, and PriA
(Fig. 7B, cf. lanes 3 and 4 with
2, and lanes 8 and 9 with
7). Mu DNA synthesis was also dependent upon DnaT, the low
levels of Mu DNA synthesis catalyzed without added DnaT (Fig.
7B, lanes 5 and 10) most likely being
due to DnaT present in the MRF
fractions (27). On the deproteinized
STP, limited extension of the leading strand primers at the left and
right ends was catalyzed in the absence of these factors (Fig.
7B, lane 11), whereas no limited synthesis was
catalyzed on ClpX-treated and ClpXP-treated STC.
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DISCUSSION |
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ClpX plays at least two critical functions in the phage Mu life cycle. Together with protease component ClpP, it can catalyze degradation of the Mu immunity repressor to induce lytic development (12-14). ClpX is also needed for Mu DNA replication (14), activating MuA in the transpososome so that MuA can promote transition to DNA synthesis (15). The latter function does not require ClpP and in theory could be disrupted by its presence. If the presence of ClpP is sufficient to promote degradation of substrates bound by ClpX, MuA in the transpososome would be degraded and would no longer be available to regulate access of host enzymes to the potential Mu replication forks. The results of this work indicate that the ClpX function of activating MuA can be carried out in the presence of the ClpP protein despite the fact that MuA can be a protease substrate (28, 41). ClpP stimulates ClpX without degrading crucial MuA needed for STC2 function. Thus, ClpX in the presence of ClpP can simultaneously function as a molecular chaperone as well as a specificity component for the protease.
The mechanism by which substrates are specifically recognized by ClpX
and ClpP is not fully understood. Substrates that can be remodeled by
molecular chaperone ClpA or ClpX alone can also be degraded when ClpP
is also present. For example, aggregates of the phage O protein,
which is the initiator for
DNA replication, can be disaggregated by
ClpX alone (5), and
O is degraded by the ClpXP protease (3). MuA is
also an example of a protein that is both a substrate of the ClpX
molecular chaperone and a substrate of the ClpXP protease (28).
Wawrzynow et al. (42) have hypothesized that determinants on
the substrate specify whether ClpX will release the substrate to
promote protein folding or present the substrate to ClpP for
degradation. Determinants necessary for degradation are present in the
C-terminal regions of the ClpXP substrates MuA (28, 41) and Mu Vir
repressor (43). The last 10 amino acids at the C terminus of MuA, when
attached to the C terminus of the phage P22 Arc protein, are sufficient
to convert Arc into a ClpXP substrate (41). However, when 7 amino acids from the C terminus of Vir3061 are fused to F plasmid proteins CcdA and
CcdB, only CcdA becomes a ClpXP substrate (43). This leaves open the
possibility that additional determinants present in Arc and CcdA but
not in CcdB are also needed to promote degradation of the substrate by
the protease.
In the in vitro transposition system, ClpXP degraded Vir to remove the transposition block while leaving MuA in STC2 intact. Although ClpXP can degrade monomeric MuA in solution (41), MuA needed for STC2 function was not degraded even after treating the transpososome with excess ClpXP for an extended period (60 min). This indicates that ClpP will not necessarily degrade all molecules recognized by ClpX and that some property of the ClpX substrate determines whether it is presented to ClpP for degradation. Once ClpX binds to substrate recognition sites on MuA or repressor, other signals on these proteins may determine whether they are degraded. The accessibility of such signals that promote substrate degradation may be determined by the protein's tertiary or quaternary structure or its interactions with other macromolecules (44). DNA binding and quaternary interactions of MuA in the transpososome could shield determinants that promote its presentation to ClpP. ClpXP can discriminate Vir bound to operator and MuA in STC1 as substrates for degradation and for remodeling, respectively, and this plays an important function in determining the course of Mu lytic development.
ClpP only stimulates ClpX function by 2-3-fold for the transition of STC1 to STC2, ClpX being able to perform this function alone when present at sufficiently high levels, and this is consistent with the finding that ClpP is not required for Mu DNA replication in vivo (14). The stimulation of ClpX activity suggests that ClpP is interacting with ClpX; however, how ClpP interacts with ClpX to stimulate chaperone activity is not yet clear. Our results indicate that a molar excess of ClpX monomers over STC1 was required for complete conversion of STC1 to STC2 and that the extent of STC2 formation was proportional to the ClpX concentration. The molar excess of ClpX used in the STC1 disassembly assay is typical of amounts used in previous work (15, 28, 41), and it is possible that ClpX is not turning over catalytically in this reaction. Through its interaction with ClpX, ClpP may be increasing the effective concentration of active ClpX in the reaction mixture. ClpP could allosterically activate the ClpX molecular chaperone or stabilize an active multimeric configuration of ClpX.
Alternatively, the ClpP proteolytic function could facilitate transition to STC2. STC2 formed in the presence of ClpXP was functionally identical to that formed with ClpX alone, but we consistently noticed some decrease in total MuA upon treatment of STC1 with ClpXP. Transpososomes are made up of a core tetramer of MuA plus additional, more loosely bound MuA protomers that play an auxiliary function (45). It is possible that these auxiliary MuA protomers are degraded during ClpXP treatment, thereby facilitating STC1 to STC2 transition. The oligomeric structure of MuA must nevertheless be retained in the resulting STC2 for Mu ends to be held together in a synaptic complex. When ClpX promotes transition of STC1 to STC2, the oligomeric structure of MuA is preserved (15).
The versatility of ClpXP to act either as molecular chaperone or protease may play a key role in determining the course of biochemical reactions such as MuA-catalyzed transposition. While the decision not to degrade MuA in STC1 and to promote transition to STC2 would assure Mu DNA replication, degradation of MuA under certain cellular conditions could permit other pathways for processing the strand transfer intermediate to yield the final recombination product. Thus, the choice of acting as protease or chaperone could influence key decision points in biochemical pathways.
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ACKNOWLEDGEMENTS |
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We thank our collaborators Elliott Crooke, Kirsten Skarstad, and Nick Dixon, with whom we maintain our supplies of E. coli replication proteins. We thank Susan Gottesman for providing pWPC9. Finally, we thank Sam Rabkin for critically reading this manuscript.
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FOOTNOTES |
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* This investigation was supported by National Institutes of Health Grant R01 GM49649 (to H. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Rd., NW, Washington, DC 20007. Tel.: 202-687-1442; Fax:
202-687-7186; E-mail: nakai{at}bc.georgetown.edu.
1
The abbreviations used are: STC, strand transfer
complex; STP, strand transfer product; RFI, closed circular duplex DNA;
MRF, Mu replication factor; pol, polymerase; Rep, Muc+
repressor protein; Vir, Mucvir3060 repressor protein;
h-ClpP, histidine-tagged ClpP protein; pre-ClpP, ClpP protein with
precursor peptide intact; SSB, single-strand binding protein;
Suc-Leu-Tyr-AMC, N-succinyl-leucine-tyrosine-7-amido-4-methylcoumarin; AMC,
7-amido-4-methylcoumarin; NTA, nitrilotriacetic acid; TLCK,
N-p-tosyl-L-lysine
chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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