©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Disassembly of the Bacteriophage Mu Transposase for the Initiation of Mu DNA Replication (*)

(Received for publication, April 18, 1995)

Hiroshi Nakai (§) Robert Kruklitis

From the Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D. C. 20007

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Upon catalyzing strand transfer, the Mu transposase (MuA) remains tightly bound to the resulting transposition intermediate, the strand transfer complex (STC), and poses an impediment to host replication proteins. Additional host factors, which can be resolved into two fractions (Mu Replication Factor alpha and beta; MRFalpha and MRFbeta), are required to disassemble the MuA complex and initiate DNA synthesis. MRFalpha modifies the protein content of the STC, removing MuA from the DNA in the process. The MRFbeta promotes initiation of Mu DNA synthesis on the STC altered by the MRFalpha. These host factors cannot promote initiation of Mu DNA synthesis if the STC is damaged by partial proteolysis. Moreover, the mutant protein MuA211 cannot be removed from the STC by MRFalpha, blocking initiation of DNA synthesis. These results indicate that MuA in the STC plays a critical function in beginning a sequence of events leading to the establishment of a Mu replication fork.


INTRODUCTION

Transposition is the process by which movable genetic elements are inserted into new sites of a chromosome. The integration of various transposable elements including retroviral DNA involves similar biochemical steps. A common feature of transposable elements is the function of the transposase they encode (reviewed in (1) ). This enzyme recognizes specific sequences at each end of the transposable element and catalyzes strand transfer, in which the ends of the transposable element are cleaved and joined to target DNA. Reactions that follow strand transfer form the completed transposition product. For some transposable elements, the entire reaction is a conservative process in which the transposing element is cut out of its original site and pasted into the target, forming a simple insert. Other transposable elements recruit replication enzymes to the strand transfer product, duplicating the element in the process of transposition to form a cointegrate.

Bacteriophage Mu employs transposition mechanisms to replicate its DNA during lytic development. We have been interested in the biochemical steps that follow Mu strand transfer in order to understand how a replication fork is established for transposition. For strand transfer, monomers of the Mu transposase (MuA, 663 amino acids) bind to multiple sites at each end of Mu DNA (2) and the transpositional enhancer, which is called the internal activating sequence(3) . Aided by DNA supercoiling and by DNA-binding proteins HU and integration host factor, the Mu ends are brought together in a synaptic complex, in which MuA assembled into a tetramer is tightly bound to each end(4, 5, 6, 7, 8) . This stable synaptic structure is retained as a nick is introduced at each end (cleaved donor complex) and joined to target DNA(4, 8, 9) . The resulting strand transfer product with bound MuA tetramer is an extremely stable nucleoprotein complex (10, 11) that has been termed the type II transpososome or strand transfer complex (STC). (^1)So tightly is MuA bound to the DNA that it blocks DNA replication by Escherichia coli replication proteins which can otherwise replicate the deproteinized intermediate to form a cointegrate(12) .

A system of 8 E. coli replication proteins, which include DNA polymerase III (pol III) holoenzyme, DnaB helicase, and DnaC protein, requires additional host factors to initiate DNA replication on the STC(12) . These additional host factors, which are said to have MuA releasing activity, are necessary to remove the MuA replication block. The MuA in the STC as well as the additional host factors appear to play an active role in the initiation of Mu DNA replication. DNA synthesis promoted by these components strictly requires pol III holoenzyme, DnaB helicase, and DnaC protein(12) , replication proteins required for Mu DNA replication in vivo(13, 14, 15) . For Mu DNA synthesis on the deproteinized intermediate, these replication proteins can be replaced by other host factors.

In this paper we demonstrate that enzyme fractions with MuA releasing activity can be separated into two functionally distinct components. One component modifies the protein content of the STC, removing MuA in the process. The second component allows DNA synthesis to be catalyzed on the altered STC by specific replication proteins. We report that MuA plays a critical function in promoting the modification of the STC, preparing the template for the initiation of DNA synthesis.


EXPERIMENTAL PROCEDURES

Materials

Bacterial Strains, DNA Substrates, and Proteins

E. coli WM433 (dnaA204) was obtained from Dr. W. Messer (Max Planck Institute) via Dr. E. Crooke (Georgetown University). The mini-Mu donor plasmid DNA used in most of the experiments was pGG215 (11) . Where indicated, donor substrate pMK108 (16) was used instead. Phage f1 RFI DNA, phage X174 RFI DNA, and pXP-10 (17) were used as target substrates. The bacterial strain OR1265 bearing the plasmid pRA422(18) , overproducer of MuA211, was obtained from Dr. R. Harshey. MuA211 was purified from this strain to greater than 95% homogeneity by the procedure of Craigie and Mizuuchi(19) . Pol III* (fraction IV, 1.5 10^6 units/mg in the M13Gori replication system) was purified by the procedure of Maki et al.(20) . HU protein was purified to greater than 95% homogeneity from overproducing strain RLM1078 (provided by Dr. R. McMacken, Johns Hopkins University) by the procedure of Dixon et al.(21) with one modification. The first chromatography step was performed with a S-Sepharose (Pharmacia) column rather than a cellulose phosphate column. E. coli DNA ligase and pol I were purchased from New England Biolabs. Purified preparations of MuA, MuB, and all other host proteins were previously described(12) .

Reagents and Other Materials

HEPES and polyethylene glycol 8000 were purchased from Fisher. Trizma (Tris base), phosphocreatine, creatine kinase, and ribonucleoside triphosphates were from Sigma. Bovine serum albumin was from Worthington. [alpha-P]dCTP (3000 Ci/mmol) was purchased from DuPont NEN. AMP-PNP and ATPS were from Boehringer Mannheim. Heparin-agarose was a gift from Dr. E. Crooke. Nitrocellulose (BA83, 0.2 µm pore size) was purchased from Schleicher and Schuell. Rabbit antibody against ClpX protein was a gift from Dr. S. Gottesman and Dr. M. Maurizi (National Institutes of Health).

Buffers

Reaction buffer is 25 mM HEPES/KOH (pH 7.5), 12 mM magnesium acetate, 5 mM DTT, 200 mM potassium glutamate, and 50 µg/ml bovine serum albumin. Buffer A is 25 mM HEPES/KOH (pH 7.5), 0.1 mM EDTA, and 0.2 mM DTT. Buffer B is 25 mM HEPES/KOH (pH 7.5), 0.5 mM EDTA, 1 mM DTT, and 10% (v/v) glycerol. Buffer C is 25 mM TrisbulletHCl (pH 7.5), 0.5 mM EDTA, 1 mM DTT, and 10% (v/v) glycerol. Buffer D is 25 mM HEPES/KOH (pH 7.5), 12 mM magnesium acetate, and 200 mM potassium glutamate.

Methods

Reconstitution Assay for Bacteriophage Mu DNA Replication

Mu strand transfer was performed in standard reaction mixtures (12.5-200 µl) that contained reaction buffer, 2 mM ATP, 100 µg/ml creatine kinase, 20 mM creatine phosphate, 32 µg/ml rifampicin, 10 µg/ml pGG215 DNA, 20 µg/ml target DNA, 1.2 µg/ml HU protein, 2.8 µg/ml MuB, and 2.2 µg/ml MuA. Incubation was at 37 °C for 30 min.

Standard reaction mixtures (25 µl) for Mu DNA replication contained DNA from 12.5 µl of the strand transfer reaction, reaction buffer, 100 µg/ml creatine kinase, 20 mM creatine phosphate, 32 µg/ml rifampicin, 2% (w/v) PEG8000, 40 µM NAD, 2 mM ATP, 100 µM each of CTP, GTP, UTP, dATP, dGTP, dTTP, and dCTP (or [alpha-P]dCTP at 1-4 Ci/mmol), 900 ng/ml SSB, 1.3 µg/ml DnaB-DnaC complex, or 2.6 µg/ml DnaB protein and 540 ng/ml DnaC protein, 840 ng/ml DnaG protein, 500 ng/ml pol III*, 160 ng/ml beta-subunit of pol III holoenzyme, 4.5 µg/ml gyrase A subunit, 2.2 µg/ml gyrase B subunit, 4 units/ml DNA ligase, and 0.2 units/ml DNA pol I, 240 units/ml MRFalpha, and 240 units/ml MRFbeta. Incubation was at 37 °C for 30 min. Total nucleotide incorporation was determined by measuring retention of radiolabeled DNA on DE81 filter paper(22) , or DNA products were resolved by electrophoresis on alkaline-agarose gels as described previously(12) . One unit of MRFalpha or MRFbeta activity is the amount required to promote the incorporation of 20 pmol of deoxynucleotides under standard reaction conditions, using 6 units of fraction IIIalpha or fraction IIIbeta (preparation described below) as a source of the complementing MRFalpha or MRFbeta activity.

Preparation of Host Enzyme Fractions with MRFalpha and MRFbeta Activities

Fraction II of E. coli WM433 (9 g wet cells) was prepared by the method of Fuller et al.(23) , adding 0.23 g of ammonium sulfate/ml of lysate supernatant (0.277 g ammonium sulfate/ml was added to prepare fraction II that provided host replication proteins as well as MRFalpha and beta activities to the in vitro reaction). Fraction II (84 mg of protein; 8,000 units of MRFalpha activity; 12,750 units of MRFbeta activity) was dialyzed against buffer A to a conductivity equivalent to buffer A containing 50 mM NaCl. Fraction II was then applied to a heparin-agarose column (3 ml) equilibrated in buffer B containing 50 mM NaCl (flow rate: 9 ml/h). The column was washed with 9 ml of the same buffer and eluted with buffer B containing 0.6 M NaCl. Flow-through fractions containing MRFalpha activity (fraction IIIalpha; 8,280 units; 36 mg of protein; 3.6 ml) and 0.6 M NaCl eluate fractions containing MRFbeta activity (fraction IIIbeta; 12,320 units; 8.1 mg of protein; 2.2 ml) were pooled.

Fraction IIIalpha was precipitated by addition of 0.56 g of ammonium sulfate/ml. The resulting precipitate, which was collected by centrifugation (16,000 g, 4 °C, 40 min), was resuspended with one-eighth volume buffer B containing 0.1 g of ammonium sulfate/ml. The precipitate was again collected by centrifugation (16,000 g, 4 °C, 40 min), dissolved in a minimal amount of buffer B containing 50 mM NaCl, and applied to a Sephacryl S-300 (Pharmacia) gel filtration column (1 30 cm), equilibrated in buffer B containing 50 mM NaCl (flow rate: 0.25 ml/min). Peak fractions containing MRFalpha activity (fraction IV; 2,750 units; 8.8 mg of protein; 1.1 ml), which eluted at an apparent molecular mass of 325 kDa, were pooled, frozen in liquid nitrogen, and stored at -80 °C.

Fraction IIIbeta was dialyzed against buffer C to a conductivity equivalent to buffer C containing 100 mM NaCl. One-fifth of fraction IIIbeta was applied to a Q-Sepharose (Pharmacia) column (0.5 5 cm) equilibrated with buffer C containing 50 mM NaCl (flow rate: 0.25 ml/min). The column was washed with 2 ml of the same buffer and eluted with buffer C containing 0.4 M NaCl. Peak fractions containing MRFbeta activity, present in the 0.4 M NaCl eluate, were pooled, frozen in liquid nitrogen, and stored at -80 °C (fraction IV; 851 units, 0.25 mg of protein; 0.25 ml).

Fraction III containing both MRFalpha and MRFbeta activities was prepared as described previously(12) .

Isolation of Nucleoprotein Complexes by Gel Filtration

The STC was formed in the standard reaction mixtures (62 µl) with the exception that they contained 20 µg/ml pGG215 DNA, 40 µg/ml target DNA, 2.5 µg/ml HU protein, 5.6 µg/ml MuB, and 4.4 µg/ml MuA. The STC was then incubated with MRFalpha or MRFbeta in reaction mixtures (final volume: 92 µl) containing reaction buffer, 100 µg/ml creatine kinase, 20 mM creatine phosphate, 32 µg/ml rifampicin, 2% (w/v) PEG8000, 40 µM NAD, 2 mM ATP, 100 µM each CTP, GTP, UTP, dATP, dGTP, dTTP, dCTP, 11 units/ml DNA ligase, 11 units/ml DNA pol I, and either 540 units/ml MRFalpha (fraction IV) or 540 units/ml MRFbeta (fraction IV). Where indicated, the following proteins were also present at the indicated concentrations: 2.4 µg/ml SSB, 3.5 µg/ml DnaB-DnaC complex, 2.3 µg/ml DnaG primase, 1.4 µg/ml pol III*, 430 ng/ml beta-subunit of pol III holoenzyme, 12 µg/ml gyrase A subunit, and 6.0 µg/ml gyrase B subunit. Incubation was at 37 °C for 30 min. The resulting nucleoprotein complexes were isolated free of nucleotides and unbound proteins by filtration through a Bio-Gel A-5m column (1 ml) equilibrated in buffer D at room temperature. DNA-containing fractions in the void volume (125 µl) were pooled, adjusted to 5 mM DTT and 50 µg/ml bovine serum albumin, and then assayed in the reconstituted Mu DNA replication system. Proteins present in the isolated complexes were resolved on SDS-polyacrylamide (10%) gels by the method of Laemmli(24) . Proteins were transferred to nitrocellulose for detection of MuA using rabbit polyclonal antibody (Bio-Con, Inc.). Donkey anti-rabbit antibody covalently coupled to horseradish peroxidase was used for detection using the enhanced chemiluminescence system (Amersham).

Assay to Detect Removal of MuA from the STC

STC was formed in reaction mixtures (30 µl) containing reaction buffer, 170 µg/ml creatine kinase, 33 mM creatine phosphate, 53 µg/ml rifampicin, 3.3% (w/v) PEG8000, 67 µM NAD, 3.3 mM ATP, 167 µM each of CTP, GTP, UTP, dATP, dGTP, dTTP, and dCTP, 17 µg/ml pGG215 DNA, 33 µg/ml phage f1 RFI DNA, 2.4 µg/ml HU protein, 3.8 µg/ml MuB, 7.0 µg/ml MuA or 10 µg/ml MuA211, and 1.8 µg/ml MuA (37 °C for 30 min). Reaction buffer containing host enzymes was then added so that the final mixture (50 µl) contained 12 units/ml DNA pol I, 12 units/ml DNA ligase, and 250 units/ml MRFalpha. Where indicated, the following host factors were also added: 250 units/ml MRFbeta, 4.3 µg/ml SSB, 2.5 µg/ml DnaB-DnaC complex, 1.7 µg/ml DnaG protein, 1.4 µg/ml pol III*, and 320 ng/ml beta-subunit of pol III holoenzyme. Incubation was at 37 °C for 30 min. PstI (10 units) was added, and incubation was continued for 1 h. The reaction was stopped by addition of EDTA to 1.5 mM, heparin to 1 mg/ml, Triton X-100 to 1% (v/v), glycerol to 4% (w/v), and bromcresol green dye marker. To some reaction mixtures, SDS was added to a final concentration of 0.5% and heated at 65 °C for 10 min. The DNA was resolved by electrophoresis on a 0.7% agarose gel (buffer system: 40 mM Tris, 20 mM acetic acid, 2 mM Na(2)EDTA) and was then stained with 0.5 µg/ml ethidium bromide. The gel was then equilibrated in 48 mM Tris, 39 mM glycine, 0.04% SDS over 90 min. Proteins in the agarose gel were transferred to nitrocellulose on a Bio-Rad semi-dry electrophoretic transfer cell (Trans-Blot SD). The blot was probed with polyclonal anti-MuA antibody and stained using goat anti-rabbit antibody covalently coupled to alkaline phosphatase and the substrates nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad). Alternatively, the enhanced chemiluminescence assay described above was used as detection system.

Other Methods

Southern blot analysis of transposition products and preparation of deproteinized strand transfer products were performed as described(25) . Protein concentration was determined by the method of Bradford(26) . Densitometric analysis of autoradiograms and negatives was performed as described previously(12) .


RESULTS

The Potential of the STC to be Replicated Can Be Destroyed by Protease Treatment

Tightly bound MuA in the STC blocks Mu DNA replication catalyzed by a system of 8 purified E. coli replication proteins(12) . The replication proteins consist of DnaB protein, DnaC protein, DnaG primase, DNA pol III holoenzyme, DNA gyrase, single-strand binding protein (SSB), DNA pol I, and DNA ligase (the 8-protein system). Additional host factors from a partially purified cell fraction are able to release the replication block (MuA releasing activity), allowing specific replication proteins to initiate Mu DNA replication. We wished to determine whether proteins present in the STC play any direct role in promoting the completion of transposition. Our previous results had indicated that upon removal of MuA from the STC by phenol extraction, DnaB protein, DnaC protein, and pol III holoenzyme were no longer essential for cointegrate formation and could be replaced by other host factors(12) . The results suggested that MuA may function in the recruitment of specific host factors for the initiation of Mu DNA replication.

After treatment with proteinase K under nondenaturing conditions, the STC could no longer be used as a template for Mu DNA replication even in the presence of MuA releasing activity (Fraction III; Table 1). The proteins in the STC must not have been completely removed by proteinase treatment. Removal of proteins by phenol extraction yields a strand transfer product that can be converted to a cointegrate by the 8-protein system. Protease inhibitor phenylmethylsulfonyl fluoride (PMSF) preserved greater than 50% of the STC replication potential when present during proteinase K treatment (Table 1). As expected, proteinase K treatment of a strand transfer product deproteinized by phenol extraction had no effect on its replication in the 8-protein system with or without MuA releasing activity. Proteolytic damage to proteins involved in strand transfer must therefore be inhibiting replication of the strand transfer product. These results suggest that the damage to proteins in the STC prevented interaction with host factors for initiation of Mu DNA replication. They suggest that proteins in the STC play an important function after strand transfer for the initiation of Mu DNA replication. We previously determined that MuB and HU present in the STC are not required to initiate Mu DNA replication dependent on the 8-protein system and the MuA releasing activity(12) . We therefore focused our investigation on the role of MuA in the initiation of Mu DNA replication.



A Mixed Tetramer of MuA211 and Wild-type MuA Can Catalyze Intermolecular Strand Transfer But the Resulting STC Has Little or No Potential to be Replicated

We suspected that partial digestion of MuA in the STC by proteinase K impaired its ability to interact with host factors necessary to initiate Mu DNA replication. We wished to assemble an STC with a defined alteration in MuA to test this hypothesis.

The MuA211 allele encodes a transposase that is truncated by 47 amino acids at the carboxyl terminus(18) . Deletions in this domain do not affect the catalytic activities necessary for strand transfer; however, they do perturb interaction with the second transposition protein MuB(27, 28) . MuB is an allosteric activator of MuA(27, 29) , promoting intermolecular strand transfer into target DNA to which MuB is bound(30) .

As expected, the mutant transposase MuA211 could not catalyze intermolecular strand transfer in the presence of MuB and target DNA (Fig. 1, lanes 5 and 6). Using the strategy of assembling mixed MuA tetramers(31) , we attempted to form an intermolecular STC with a mixture of MuA211 and wild-type MuA. Levels of MuA that do not produce detectable levels of STC (Fig. 1, lane 3) converted approximately 35% of donor substrates to STC at appropriate concentrations of MuA211 (lane 8). These results are consistent with those of M. Mizuuchi and K. Mizuuchi, (^2)who have determined that a mixed tetramer of at least two wild-type MuA monomers and mutant MuA (missing 48 amino acids at the carboxyl terminus) can interact with MuB to form the intermolecular STC.


Figure 1: MuA211 promotes formation of the STC at limiting concentrations of wild-type MuA. STC products (X174 target DNA) formed with the indicated amounts of MuA and MuA211 (25 µl of reaction mixture) were resolved by alkaline-agarose gel electrophoresis. The amount of STC formed in each reaction was as follows (indicated in equivalents of donor substrates): lanes 1-3 and 5-6, <1 fmol; lane 4, 52 fmol; lane 7, 7 fmol; lane 8, 18 fmol. Lane D contains unreacted donor substrate (pGG215), and lane T contains unreacted target substrate. I, the intermolecular strand transfer product; D(C), covalently closed circular donor DNA; D, linear form of donor DNA; T, target DNA.



However, the STC formed in the presence of MuA211 and MuA was not readily converted to cointegrates. When a crude host enzyme fraction (fraction II) was added to STC formed with pMK108 donor substrate (16) and wild-type MuA, both simple inserts and cointegrates were formed as expected (Fig. 2, lane 4). With a mixture of MuA211 and MuA, approximately 50% the amount of intermolecular STC was formed (quantitated from an autoradiogram shown in Fig. 2, lanes 1 and 3, at a lower exposure), but less than 5% the level of cointegrates was formed from this STC (Fig. 2, lane 2). We were unable to detect any differences in the amount of simple inserts synthesized from STC formed with MuA alone and a mixture of MuA211 and MuA.


Figure 2: The STC formed with a mixture of MuA and MuA211 is not readily converted to cointegrates. STC formed with pMK108 donor DNA, f1 target DNA, and the indicated amounts of MuA and MuA211 (25 µl of reaction mixture for strand transfer) was introduced into the replication system (50 µl of reaction mixture) with fraction II (90 µg) as the source of host enzymes. Products of transposition were resolved by alkaline-agarose gel electrophoresis. A Southern blot of the gel was probed with P-labeled pMK108 DNA. Lane D contains unreacted donor substrate, and lane T contains unreacted target substrate. Arrows indicate the positions of the cointegrate products. D, donor substrate; T, target substrate; I, the intermolecular strand transfer product; SI(C), covalently closed circular simple insert product; SI(L), linear form of the simple insert product (arising from denaturation of DNA); CO(C), covalently closed circular form of the cointegrate; CO(L), linear form of the cointegrate.



Analogous results were obtained when pGG215 was used as donor substrate (data not shown). STC formed from pGG215 donor are predominantly converted to cointegrates, yielding little or no simple insert products (32) . Virtually 100% of the STC formed with MuA was converted to cointegrates in the Mu replication system (the 8-protein system supplemented with MuA releasing activity), whereas less than 15% of the STC formed with MuA211 and MuA was converted to cointegrates. After formation of the STC with wild-type MuA, the presence of MuA211 had no effect on its conversion to a cointegrate, indicating that the MuA211 present in the STC is blocking DNA replication. In addition, no DNA replication could be catalyzed on an intramolecular STC formed with MuA211 alone (data not shown). In the absence of MuB, MuA can catalyze intramolecular strand transfer products, which serve as templates for rolling circle Mu DNA replication(25) . While MuA211 is capable of catalyzing intramolecular strand transfer, no detectable level of Mu DNA replication was catalyzed upon introduction of the intramolecular STC to the replication system (data not shown).

These results suggest that the MuA211 may be defective in interacting with host factors that have MuA releasing activity. This hypothesis is tested in the experiments described below.

Resolution of the MuA Releasing Activity into Two Components

We do not yet know just how many host factors are necessary for MuA releasing activity, which allows initiation of Mu DNA replication on the STC by the 8-protein system. We further fractionated enzyme preparations with MuA releasing activity to determine whether more than a single component is needed for this activity.

A host enzyme fraction (fraction II) containing MuA releasing activity was resolved by heparin-agarose chromatography into two enzyme fractions, fraction IIIalpha and fraction IIIbeta. Neither of these two fractions alone had MuA releasing activity (i.e. the ability to promote Mu DNA replication on the STC by the 8-protein system; Fig. 3). The activity could be reconstituted by mixing the two enzyme fractions, which will be referred to as Mu replication factors alpha and beta (MRFalpha and MRFbeta; Fig. 3). This reconstitution assay was used to further fractionate MRFalpha by gel filtration chromatography (MRFalpha, fraction IV) and MRFbeta by Q-Sepharose chromatography (MRFbeta, fraction IV). Supplementation of the 8-protein system with both these enzyme fractions was necessary for Mu DNA replication and cointegrate formation that was dependent on DnaB protein, DnaC protein, and pol III holoenzyme (Table 2).


Figure 3: Factors present in fractions IIIalpha and IIIbeta are required to catalyze Mu DNA synthesis on the STC with the 8-protein system. Mu DNA synthesis was catalyzed in the presence of the 8-protein system and varying amounts of fraction II (closed circles), fraction IIIalpha with (closed squares) or without (open squares) 6 units of fraction IIIbeta, and fraction IIIbeta with (closed triangles) or without (open triangles) 6 units of fraction IIIalpha (standard 25-µl reaction mixture).





The essential host factors in each enzyme fraction have not yet been purified to homogeneity; therefore, the actual number of separable factors in each enzyme fraction is not known. Nevertheless, as we demonstrate below, distinct functions are associated with each enzyme fraction.

MRFalpha Removes MuA from the STC but Cannot Remove a Mixed Tetramer of MuA211 and Wild-type MuA

We tested the enzyme fractions containing MRFalpha and MRFbeta to determine whether they could remove MuA from the STC. For this analysis, STC was formed using pGG215 as donor substrate and bacteriophage f1 RFI DNA as target substrate. The STC was cleaved with PstI and resolved by electrophoresis on a neutral agarose gel (Fig. 4, lane 1). PstI cleaves the donor substrate twice, once within the mini-Mu element and once within the vector sequence, whereas it does not cleave target DNA. The STC digested with PstI resolves as a single tight band.


Figure 4: Removal of MuA from the STC in the presence of MRFalpha. The STC was incubated with E. coli replication proteins supplemented with MRFalpha or MRFbeta as indicated (see ``Experimental Procedures''), and the reaction was stopped by heating at 65 °C. The DNA was digested with PstI and resolved by agarose gel electrophoresis. A, the agarose gel stained with ethidium bromide; B, the immunoblot probed with anti-MuA antibody and stained with the alkaline phosphatase detection system. Lane D(P) contains unreacted donor DNA digested with PstI, and lane D contains undigested donor DNA. Lane T contains unreacted target substrate. Lane 1 contains STC that has not been subjected to heating at 65 °C. Heating at 65 °C caused some STC molecules to aggregate, forming a ladder of oligomers (lane 2). CDC, position of the structure formed by cleaving once within the mini-Mu element and once within the vector of the cleaved donor complex.



Treatment of STC with the E. coli replication proteins or the replication proteins supplemented with MRFbeta did not remove MuA. The immunoblot of the gel probed with polyclonal anti-MuA antibody indicates that MuA was still associated with DNA (Fig. 4B, lanes 3 and 5). On the other hand, MuA was removed from the DNA in the presence of MRFalpha and the replication proteins. The resulting DNA product migrated faster (Fig. 4A, lane 4), and no detectable MuA was associated with the DNA (Fig. 4B, lane 4). No alteration of this strand transfer product could be detected when it was deproteinized by SDS treatment and resolved by alkaline-agarose gel electrophoresis (data not shown).

The removal of MuA by MRFalpha did not require the addition of the E. coli replication proteins necessary to catalyze Mu DNA synthesis (Fig. 5, lane 6). Treatment with MRFalpha (fraction IV) caused virtually 100% of the strand transfer product (digested with PstI) to migrate faster as a single band (Fig. 5A, cf. lanes 6 and 5). No detectable MuA was associated with this DNA band (Fig. 5B, lane 6).


Figure 5: The stable MuA nucleoprotein complex formed with MuA211 cannot be disrupted by MRFalpha. The STC formed with MuA or a mixture of MuA211 and MuA (30-µl reaction mixture) was subjected to treatment with MRFalpha. The DNA was digested with PstI and resolved by agarose gel electrophoresis as described under ``Experimental Procedures.'' The ethidium-stained gel (A) and immunoblot (B) probed with anti-MuA antibody (enhanced chemiluminescence detection system) are shown. The arrow indicates the position of the DNA after treatment of the STC with SDS. D(P) indicates the position of the two bands that would arise from digestion of the unreacted donor substrate with PstI. T, target substrate; CDC, cleaved donor complex.



Removal of MuA from the STC required ATP (data not shown). The presence of 2 mM ATP as sole nucleotide cofactor was sufficient to support MuA removal. Removal of MuA was not detected if nucleoside triphosphates were absent or if ATP was replaced with nonhydrolyzable analogs AMP-PNP or ATPS (present at 2 mM).

These experiments identified a potential host function that is needed for Mu DNA replication and that most likely involves an interaction between the host factors and MuA. When the STC was formed with a mixture of MuA211 and MuA, less than 2% of the nucleoprotein complex was converted to the faster migrating species by MRFalpha (Fig. 5A, lane 3), and MuA remained bound to the STC (Fig. 5B, lane 3). Because the mobility of the STC containing MuA211 is unaltered by treatment with MRFalpha, the synaptic complex of the Mu ends must have been maintained. Disruption of the synaptic complex with SDS caused the DNA to migrate faster (Fig. 5, lanes 1 and 4). These results suggest that the inefficient replication of the STC formed in the presence of MuA211 and MuA is due to the failure of MRFalpha to remove the mixed MuA tetramer from DNA. In the following experiments we demonstrate that the removal of MuA by MRFalpha is an important step for the initiation of Mu DNA replication.

The STC Altered by MRFalpha Requires MRFbeta, DnaB Helicase, DnaC Protein, and Pol III Holoenzyme for Its Replication

Conceivably, MuA in STC could be removed by a number of mechanisms. For example, we might speculate that a nonspecific protease present in the MRFalpha fraction destroyed MuA, and such a process may not be relevant to the mechanism of Mu DNA replication. Thus we investigated whether the removal of MuA by MRFalpha is an important step in the establishment of a replication fork with MRFbeta and the host replication proteins.

An important feature of Mu DNA replication on the STC is the requirement for DnaB, DnaC, pol III holoenzyme, and MuA releasing activity(12) . On the strand transfer product deproteinized by phenol extraction, the DnaB-DnaC complex or pol III holoenzyme is not required if SSB or pol I, respectively, are present at appropriately high concentrations. This relaxed host enzyme requirement was observed whether the MuA releasing activity was present or not. Thus, if the removal of MuA from the STC by MRFalpha were a relevant step for initiation of Mu DNA synthesis, then MRFbeta and the specific replication enzymes should be required for replication of the altered STC.

The STC that was treated with MRFalpha and isolated by gel filtration to remove unbound proteins was unable to replicate in the 8-protein system unless it was supplemented with the MRFbeta (Table 3). MRFalpha was not necessary to replicate the altered STC (Table 3); the presence of MRFalpha had no measurable effect on its replication (data not shown). In addition, no DNA replication was catalyzed if DnaB protein, DnaC protein, or pol III holoenzyme was omitted (Table 3). However, if the isolated DNA was deproteinized by phenol extraction, the strand transfer product could be replicated in the absence of DnaB protein, DnaC protein, pol III holoenzyme, or MRFbeta. These results indicate that the STC altered by MRFalpha is not identical to the deproteinized strand transfer product. In fact, treatment of the deproteinized strand transfer product with MRFalpha was not sufficient to form the altered STC, which requires MRFbeta for its replication. The deproteinized strand transfer product treated with MRFalpha could be replicated in the absence of MRFbeta (Table 4). Even in the presence of MRFbeta, the replication of the treated deproteinized strand transfer product was not dependent on the presence of DnaB protein, DnaC protein, and pol III holoenzyme. The results indicate that the alteration of the STC by MRFalpha is an important step in initiating DNA replication by MRFbeta, DnaB protein, DnaC protein, and pol III holoenzyme.





On the other hand, treatment of the STC with MRFbeta even in the presence of the 8-protein system did not form an altered STC that could be replicated in the absence of MRFbeta (Table 5). The STC that had been incubated with MRFbeta and then isolated by gel filtration could only be replicated in the 8-protein system supplemented with both MRFalpha and MRFbeta.



Our results suggest that the STC altered by MRFalpha has bound protein that inhibits the initiation of Mu DNA replication, a blockade that may be removed by MRFbeta. Upon treatment of STC isolated by gel filtration with MRFalpha, there was no major degradation of MuA (Fig. 6, lane 2). However, no detectable amount of MuA was associated with the altered STC isolated a second time by gel filtration (Fig. 6, lane 4). These results indicate that MuA's function is complete upon formation of the altered STC.


Figure 6: MuA is not degraded as it is removed from the STC by MRFalpha. The STC was challenged with 2 mg/ml heparin (0 °C for 15 min) to adsorb unbound MuA and filtered through a Bio-Gel A-5m column. The STC (equivalent of 0.5 µg of donor substrate), purified free of unbound proteins, was treated as indicated with 20 units of MRFalpha (fraction IV) in standard reaction mixtures (125 µl). One-half of the reaction mixture was subjected to a second gel filtration step to isolate the altered STC. Proteins present in samples before and after the second gel filtration step were resolved by SDS-polyacrylamide gel electrophoresis. Immunoblots were probed with anti-MuA antibody (enhanced chemiluminescence detection system).




DISCUSSION

Fundamental Steps Involved in the Initiation of Mu DNA Replication

In this paper we have demonstrated that the removal of MuA from the STC is an important step in initiation of Mu DNA replication and that this step may precede the assembly of the replisome at the Mu fork. At least two distinguishable factors (MRFalpha and MRFbeta) are necessary to catalyze Mu DNA replication on the STC using a system of 8 host replication proteins. MRFalpha removes MuA from the STC without major proteolytic degradation of MuA. However, the altered STC isolated free of MuA by gel filtration will still not allow DNA replication to be catalyzed by the replication proteins. MRFbeta is necessary to initiate Mu DNA replication that specifically requires DnaB and DnaC proteins and pol III holoenzyme. If instead, the STC altered by MRFalpha is deproteinized by phenol extraction, Mu DNA replication can be catalyzed in the absence of the MRFbeta; however, replication of the deproteinized intermediate does not specifically require DnaB and DnaC proteins and pol III holoenzyme. The requirement for these replication enzymes cannot be restored simply by adding MRFalpha and MRFbeta to the deproteinized intermediate. These results suggest that a host factor is bound to the Mu fork as MuA is released from the STC, preparing the template for assembly of the replisome.

These results indicate that MuA plays an important function in the initiation of DNA replication after it has completed strand transfer. Although it does not appear to play a direct role in the assembly of the replisome, MuA may promote the binding of a crucial host factor that is required for assembly of specific replication enzymes at the Mu fork possibly with the aid of the MRFbeta.

MuA Contains a Domain Required for its Removal from DNA during Replicative Transposition

The hypothesis that MuA plays a critical function after strand transfer is supported by our experimental evidence that functional MuA is required in the STC for the initiation of DNA replication. Mu DNA replication cannot be initiated in the presence of MRFalpha and MRFbeta when MuA in the STC is damaged by proteinase K. Moreover, MuA truncated by 47 amino acids at the carboxyl terminus cannot be removed from the STC by MRFalpha, thus blocking initiation of DNA replication. The carboxyl terminus of MuA has been determined to be a domain required for interaction with MuB (27, 28) . Our results indicate that this domain is also required for the release of MuA for the initiation of Mu DNA replication. This domain may be involved in interaction with the host factor that catalyzes removal of MuA from the DNA. It is also possible that the loss of this domain further stabilizes the MuA nucleoprotein complex so that it cannot be disrupted by the host factor.

Nonetheless, these functions of MuA, interaction with MuB and MRFalpha, are clearly distinguishable. A mixed tetramer containing at least two monomers of wild-type MuA is capable of interacting with MuB to form the intermolecular STC. (^3)In the experiments presented in this paper, we used a 5-6-fold higher concentration of MuA211 with respect to MuA, thus favoring formation of a STC that includes MuA211. Clearly, a MuA tetramer assembled under these conditions can interact with MuB to form the intermolecular STC but cannot be disassembled by MRFalpha for the initiation of Mu DNA replication.

Possible Function of MuA in the STC for Initiation of Mu DNA Replication

For replication, Mu must efficiently recruit the host replication apparatus to amplify its genome 100-fold or more during the course of lytic development, which is less than 1 h in duration. During strand transfer, a template for Mu DNA replication is created. An important question about the mechanism of Mu DNA replication has been how replication enzymes would be assembled to create the Mu replication fork. A hypothetical factor that mediates the assembly of the replisome on the strand transfer product would help explain how Mu effectively directs many rounds of transposition with replication enzymes present at very low concentrations such as the pol III holoenzyme.

Our previous results had indicated that the MuA remaining tightly bound to the strand transfer product and the host factors with MuA releasing activity were important components for reconstituting Mu DNA replication that requires DnaB protein, DnaC protein, and pol III holoenzyme(12) , enzymes known to be required for Mu DNA replication in vivo(13, 14, 15) . These results had suggested that MuA could play some role in mediating the assembly of the replication enzymes, blocking the action of inappropriate enzymes that might interfere with the catalysis of Mu DNA replication.

The results presented in this paper further clarify the function of MuA in the initiation of DNA replication and the roles of the host factors needed along with MuA to initiate DNA replication. Clearly, the function of MuA can be completed by the time replication proteins assemble at the fork. The results suggest that MuA protects the forked junctions of the strand transfer product until the binding of a host factor that can effectively regulate access of host enzymes to the Mu fork, recruiting specific replication enzymes needed to create a replication fork. Most likely, MuA recruits such a host factor by direct protein-protein interactions. We also cannot rule out the possibility that MuA's function is to hold the DNA in a configuration which allows binding of such a host factor.

We postulate that a host factor which can serve as initiator for DNA replication is loaded onto the strand transfer product during removal of MuA from the STC. Possibly, MuA is designed to recruit such a factor in Mu's wide range of hosts(33) , allowing use of replication proteins from various bacterial species for Mu transposition.

The number of factors needed in addition to the 8-protein system to catalyze cointegrate formation from the STC is unclear. The results of Mhammedi-Alaoui et al.(34) have indicated that Mu cannot readily replicate in the ClpX host and that transposition is blocked after strand transfer. We have found that the level of Mu DNA synthesis is reduced 8-fold or more using a cell extract of a ClpX strain as a source of host factors. MRFalpha (fraction IV) does contain ClpX protein, which was detected using polyclonal antibodies, and it is able to complement the ClpX cell extract to restore high levels of Mu DNA synthesis. ClpX protein by itself can destabilize the synaptic complex of Mu ends, but the resulting nucleoprotein complex cannot be converted to cointegrates in the 8-protein system supplemented with MRFbeta. Other factors in the MRFalpha fraction are also needed to reconstitute Mu DNA replication. (^4)These results suggest that ClpX protein is necessary but not sufficient for MRFalpha activity. Indeed, MRFalpha and MRFbeta may each consist of multiple factors that can be further separated. For instance, MRFalpha may consist of factors that remove MuA from the STC as well as factors that are bound to the altered STC.

How MuA disassembly characterized in this work is applicable to simple insert formation is yet unclear. We have not been able to detect any reduction in the ability of the pMK108 STC formed with MuA211 and MuA to be converted to a simple insert. At this time it is difficult to interpret this result since we cannot be sure whether the pathway for simple insert formation in vitro reflects the mechanism for its formation in vivo. Our results are consistent with the ability of MuA211 to direct simple insert formation in vivo to establish Mu lysogeny(18) . One may postulate that other factors, which can interact with MuA211, could be involved in removing MuA from the STC for nonreplicative transposition.

The present work establishes the critical function of MuA after strand transfer to complete replicative transposition. The results of our work indicate that the functions associated with MRFalpha and MRFbeta include removal of MuA from the STC, formation of an altered STC that is free of MuA and yet is protected from action by the replication enzymes, and the loading of specific replication proteins at the Mu fork to initiate DNA replication. These functions provide us with the criteria for further defining host components necessary for Mu transposition.


FOOTNOTES

*
This investigation was supported by National Institutes of Health Grant GM49649 and American Cancer Society Grant NP-769B. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 202-687-1442; Fax: 202-687-7186.

(^1)
The abbreviations used are: STC, strand transfer complex; pol, polymerase; SSB, single-strand binding protein; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; RFI, closed circular duplex DNA; MRFalpha and MRFbeta, Mu replication factor alpha and beta; AMP-PNP, 5`-adenylyl-beta,-imidodiphosphate; ATPS, adenosine 5`-O-(thiotriphosphate).

(^2)
M. Mizuuchi and K. Mizuuchi, manuscript in preparation.

(^3)
M. Mizuuchi and K. Mizuuchi, manuscript in preparation.

(^4)
R. Kruklitis and H. Nakai, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Bonnie Fijal for purification of MuA211 protein for this work. We thank our collaborators Elliott Crooke, Kirsten Skarstad, and Nick Dixon, with whom we maintain and share our supplies of E. coli replication proteins. We thank Rasika Harshey for providing us with the MuA211 overproducer and for suggesting use of the mutant transposase for our studies. We thank Kiyoshi and Michiyo Mizuuchi for helpful discussions and for communicating results prior to publication. Finally, we thank Sam Rabkin and E. Crooke for critically reading the manuscript.


REFERENCES

  1. Mizuuchi, K. (1992) Annu. Rev. Biochem. 61,1011-1051 [CrossRef][Medline] [Order article via Infotrieve]
  2. Craigie, R., Mizuuchi, M., and Mizuuchi, K. (1984) Cell 39,387-394 [Medline] [Order article via Infotrieve]
  3. Mizuuchi, M., and Mizuuchi, K. (1989) Cell 58,399-408 [Medline] [Order article via Infotrieve]
  4. Mizuuchi, M., Baker, T. A., and Mizuuchi, K. (1992) Cell 70,303-311 [Medline] [Order article via Infotrieve]
  5. Surette, M. G., and Chaconas, G. (1992) Cell 68,1101-1108 [Medline] [Order article via Infotrieve]
  6. Baker, T. A., and Mizuuchi, K. (1992) Genes & Dev. 6,2221-2232
  7. Kuo, C.-F., Zou, A., Jayaram, M., Getzoff, E., and Harshey, R. (1991) EMBO J. 10,1585-1591 [Abstract]
  8. Lavoie, B. D., Chan, B. S., Allison, R. G., and Chaconas, G. (1991) EMBO J. 10,3051-3059 [Abstract]
  9. Mizuuchi, M., Baker, T. A., and Mizuuchi, K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,9031-9035 [Abstract]
  10. Lavoie, B. D., and Chaconas, G. (1990) J. Biol. Chem. 265,1623-1627 [Abstract/Free Full Text]
  11. Surette, M. G., Buch, S. J., and Chaconas, G. (1987) Cell 49,253-262 [Medline] [Order article via Infotrieve]
  12. Kruklitis, R., and Nakai, H. (1994) J. Biol. Chem. 269,16469-16477 [Abstract/Free Full Text]
  13. Toussaint, A., and R é sibois, A. (1983) in Mobile Genetic Elements (Shapiro, J. A., ed) pp. 105-158, Academic Press, New York
  14. Toussaint, A., and Faelen, M. (1974) Mol. & Gen. Genet. 131,209-214
  15. R é sibois, A., Pato, M., Higgins, P., and Toussaint, A. (1984) in Proteins Involved in DNA Replication (Hubscher, U., and Spadari, S., eds) pp. 69-76, Plenum Press, New York
  16. Mizuuchi, K. (1983) Cell 35,785-794 [Medline] [Order article via Infotrieve]
  17. Wolffe, A. P., Jordan, E., and Brown, D. D. (1986) Cell 44,381-389 [Medline] [Order article via Infotrieve]
  18. Harshey, R. M., and Cuneo, S. D. (1986) J. Genet. 65,159-174
  19. Craigie, R., and Mizuuchi, K. (1985) J. Biol. Chem. 260,1832-1835 [Abstract]
  20. Maki, H., Maki, S., and Kornberg, A. (1988) J. Biol. Chem. 263,6570-6578 [Abstract/Free Full Text]
  21. Dixon, N. E., and Kornberg, A. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,424-428 [Abstract]
  22. Bryant, F. R., Johnson, K. A., and Benkovic, S. J. (1983) Biochemistry 22,3537-3546 [Medline] [Order article via Infotrieve]
  23. Fuller, R. S., Kaguni, J. M., and Kornberg, A. (1981) Proc. Natl. Acad. Sci. U. S. A. 78,7370-7374 [Abstract]
  24. Laemmli, U. K. (1970) Nature 227,680-685 [Medline] [Order article via Infotrieve]
  25. Nakai, H. (1993) J. Biol. Chem. 268,23997-24004 [Abstract/Free Full Text]
  26. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 [CrossRef][Medline] [Order article via Infotrieve]
  27. Baker, T. A., Mizuuchi, M., and Mizuuchi, K. (1991) Cell 65,1003-1013 [Medline] [Order article via Infotrieve]
  28. Leung, P. C., and Harshey, R. M. (1991) J. Mol. Biol. 219,189-199 [Medline] [Order article via Infotrieve]
  29. Surette, M. G., Harkness, T., and Chaconas, G. (1991) J. Biol. Chem. 266,3118-3124 [Abstract/Free Full Text]
  30. Adzuma, K., and Mizuuchi, K. (1988) Cell 53,257-266 [Medline] [Order article via Infotrieve]
  31. Baker, T. A., Mizuuchi, M., Savilahti, H., and Mizuuchi, K. (1993) Cell 74,723-733 [Medline] [Order article via Infotrieve]
  32. Chaconas, G., Gloor, G., and Miller, J. L. (1985) J. Biol. Chem. 260,2662-2669 [Abstract]
  33. Koch, C., Mertens, G., Rudt, F., Kahmann, R., Kanaar, R., Plasterk, R. H. A., Van de Putte, P., Sandulache, R., and Kamp, D. (1987) in Phage Mu (Symonds, N., Toussaint, A., Van de Putte, P., and Howe, M. M., eds) pp. 75-91, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  34. Mhammedi-Alaoui, A., Pato, M., Gama, M.-J., and Toussaint, A. (1994) Mol. Microbiol. 11,1109-1116 [Medline] [Order article via Infotrieve]

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