(Received for publication, April 18, 1995)
From the
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
and
; MRF
and MRF
), are required to disassemble
the MuA complex and initiate DNA synthesis. MRF
modifies the
protein content of the STC, removing MuA from the DNA in the process.
The MRF
promotes initiation of Mu DNA synthesis on the STC altered
by the MRF
. 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 MRF
,
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.
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). ()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.
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
[
-
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
-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 MRF
, and 240 units/ml MRF
.
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 MRF
or MRF
activity is
the amount required to promote the incorporation of 20 pmol of
deoxynucleotides under standard reaction conditions, using 6 units of
fraction III
or fraction III
(preparation described below) as
a source of the complementing MRF
or MRF
activity.
Fraction III 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 MRF
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 III was
dialyzed against buffer C to a conductivity equivalent to buffer C
containing 100 mM NaCl. One-fifth of fraction III
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 MRF
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 MRF and MRF
activities
was prepared as described previously(12) .
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.
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, ()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
, 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
, covalently closed circular simple
insert product; SI
, linear form of the simple
insert product (arising from denaturation of DNA); CO
, covalently closed circular form of the
cointegrate; CO
, 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.
A host
enzyme fraction (fraction II) containing MuA releasing activity was
resolved by heparin-agarose chromatography into two enzyme fractions,
fraction III and fraction III
. 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
and
(MRF
and MRF
; Fig. 3). This reconstitution assay was
used to further fractionate MRF
by gel filtration chromatography
(MRF
, fraction IV) and MRF
by Q-Sepharose chromatography
(MRF
, 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 III and
III
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 III
with (closed squares) or
without (open squares) 6 units of fraction III
, and
fraction III
with (closed triangles) or without (open
triangles) 6 units of fraction III
(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.
Figure 4:
Removal of MuA from the STC in the
presence of MRF. The STC was incubated with E. coli replication proteins supplemented with MRF
or MRF
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
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 MRF 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 MRF
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 MRF did not require the
addition of the E. coli replication proteins necessary to
catalyze Mu DNA synthesis (Fig. 5, lane 6). Treatment
with MRF
(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 MRF. The STC formed with
MuA or a mixture of MuA211 and MuA (30-µl reaction mixture) was
subjected to treatment with MRF
. 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
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 MRF (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 MRF
, 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 MRF
to remove the mixed
MuA tetramer from DNA. In the following experiments we demonstrate that
the removal of MuA by MRF
is an important step for the initiation
of Mu DNA replication.
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 MRF were a relevant step for initiation of Mu DNA synthesis,
then MRF
and the specific replication enzymes should be required
for replication of the altered STC.
The STC that was treated with
MRF and isolated by gel filtration to remove unbound proteins was
unable to replicate in the 8-protein system unless it was supplemented
with the MRF
(Table 3). MRF
was not necessary to
replicate the altered STC (Table 3); the presence of MRF
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 MRF
. These results indicate that the STC
altered by MRF
is not identical to the deproteinized strand
transfer product. In fact, treatment of the deproteinized strand
transfer product with MRF
was not sufficient to form the altered
STC, which requires MRF
for its replication. The deproteinized
strand transfer product treated with MRF
could be replicated in
the absence of MRF
(Table 4). Even in the presence of
MRF
, 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 MRF
is an important step in initiating
DNA replication by MRF
, DnaB protein, DnaC protein, and pol III
holoenzyme.
On the other hand, treatment of the STC with MRF
even in the presence of the 8-protein system did not form an altered
STC that could be replicated in the absence of MRF
(Table 5). The STC that had been incubated with MRF
and then
isolated by gel filtration could only be replicated in the 8-protein
system supplemented with both MRF
and MRF
.
Our results
suggest that the STC altered by MRF has bound protein that
inhibits the initiation of Mu DNA replication, a blockade that may be
removed by MRF
. Upon treatment of STC isolated by gel filtration
with MRF
, 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 MRF. 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 MRF
(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).
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 MRF.
Nonetheless, these functions of MuA, interaction with MuB and
MRF, 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. (
)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 MRF
for the initiation of Mu DNA
replication.
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. MRF
(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 MRF
. Other factors in the
MRF
fraction are also needed to reconstitute Mu DNA replication. (
)These results suggest that ClpX protein is necessary but
not sufficient for MRF
activity. Indeed, MRF
and MRF
may
each consist of multiple factors that can be further separated. For
instance, MRF
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 MRF and MRF
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.