(Received for publication, April 20, 1995; and in revised form, January 10, 1996)
From the
In an earlier kinetic study (Wang, Z., and Harshey, R. M.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 699-703), we showed
that supercoiling free energy was utilized during Mu transposition to
lower the activation barrier of some rate-limiting step in the
formation of the cleaved Mu end synaptic complex (type I complex). We
report here results from kinetic studies on the assembled but uncleaved
synaptic complex (type 0). Based on the estimated rate constants for
the formation of type 0 and type I complexes, as well as their
temperature and superhelicity dependence, we infer that the type 0
complex is an authentic intermediate in the pathway to Mu end cleavage.
Our results are consistent with type 0 production being the
rate-limiting step in the overall type I reaction. The conversion of
type 0 to type I complex is a fast reaction, does not show strong
temperature dependence, and is apparently independent of substrate
superhelicity. We have explored the DNA structure within the type 0
complex using chemical and enzymatic probes. The observed
susceptibility of DNA outside the Mu ends to single-strand-specific
reagents suggests that a helix opening event is associated with type 0
formation. This structural perturbation could account, at least partly,
for the high activation barrier to the reaction. There is a close
correlation between the appearance of single strandedness near the Mu
ends and the superhelicity of the DNA substrate. It is possible that
supercoiling energy is utilized in effecting specific conformational
transitions within DNA. We have found that Zn and
Co
ions, like Mg
and Mn
ions, can efficiently cleave the type 0 complex. However, unlike
Mg
and Mn
ions, Zn
and Co
ions cannot support assembly of type 0.
We discuss the implications of our findings for the mechanism of Mu
transposition.
Mu DNA transposition occurs by cleavage (nicking) of the left
and right ends of Mu DNA (attL and attR), followed by
joining (strand transfer) to target DNA, a reaction carried out by Mu
transposase (A protein) (reviewed in Mizuuchi(1992)). The functional
configuration of Mu A protein is a tetramer (Lavoie et al.,
1991). Assembly of the active Mu A tetramer is a complex process
requiring negatively supercoiled DNA substrate, a set of six att subsites, a pair of internal enhancer sites, the Escherichia
coli HU protein, and divalent metal ions (Mg or
Mn
) (see Mizuuchi(1992), Lavoie and Chaconas(1993),
Wang and Harshey(1994), and Yang et al. (1995a, 1995b)).
After the cleavage or nicking step of transposition, the cleaved Mu
ends can be located within a stable high-order DNA-protein assembly
called a ``type I complex'' (Fig. 1) (Surette et
al., 1987). In the presence of Ca, an uncleaved
synaptic complex (type 0 complex) accumulates in a reaction that also
requires DNA supercoiling, the internal enhancer sequence, and HU
protein for its formation and resembles the type I complex in harboring
the tetramerized form of Mu A (
)(see Fig. 1)
(Mizuuchi et al., 1992; Surette and Chaconas, 1992).
Figure 1:
The
Mu end nicking step of transposition. Mu ends (L and R, heavy line) on a negatively supercoiled plasmid (left) are brought together (synapsed) in the presence of Mu A
protein, E. coli HU protein, and Ca (Me
, metal ions) to generate a
presumed intermediate (type 0 complex, middle). An internal
enhancer DNA element (dumbell) is required for the formation
of this complex. In the presence of Mg
, Mu ends are
cleaved (single-stranded nick at each 3` end) by Mu A protein to
generate a type I complex (right; non-Mu sequences are
relaxed). The Mu A protein is found as a tetramer in both complexes
(see Introduction for details).
During a kinetic investigation of the role of DNA supercoiling in type I complex formation, we observed a dependence of the reaction rate on superhelical density as well as on the length of DNA outside Mu in the donor plasmid (Wang and Harshey, 1994). A strong temperature dependence (in the range of 15-35 °C) was also observed, and a rather high activation enthalpy (67 ± 15 kcal/mol) was calculated for the reaction. These results could be well explained by a model in which the free energy of supercoiling is used directly to lower the activation barrier of a rate-limiting step. Our results implied the existence of an intermediate in which Mu ends have come together to allow domain closure, i.e. separation of Mu and non-Mu domains, the supercoiling energy from the non-Mu domain being utilized to promote the rate-limiting step of the reaction. We speculated that free energy from DNA supercoiling might be used to drive conformational changes of DNA, such as melting of the ends, and/or conformational changes of Mu A protein.
In this study we show first that the
uncleaved type 0 complex assembled in the presence of Ca is likely a bona fide intermediate in formation of the type I
complex. We present evidence suggestive of the presence of
single-stranded DNA just outside the Mu ends in the type 0 complex and
its dependence on DNA supercoils.
In experiments using different metal ions, type 0 complexes were directly assayed according to the procedure recently described by Kim et al.(1995), where treatment of type 0 reaction mixtures with 10 µg/ml heparin before electrophoresis results in a distinct type 0 complex band, migrating just above the supercoiled donor plasmid.
Primer
extension was carried out in two alternate ways. 1) Primers were first
labeled with [-
P]ATP to a specific activity
of
1 Ci/µmol. Reaction mixtures containing 1 µl (0.1
µg) of template DNA, 0.5 µl (0.5 µM) of labeled
primer, and 1.9 units of
Taq polymerase in 30 mM Tris, pH 9.5, 7.5 mM MgCl
, were incubated at
90 °C for 2 min and immediately cooled on ice. Next, 2 µl of
extension mix (15 µM dNTP) was added, and incubation
continued at 50 °C for 20 min. DNA chain extension was stopped by
the addition of 3.5 µl of formamide stop solution. The resulting
DNA fragments were resolved on 6% polyacrylamide sequencing gels. 2)
Primers were extended using the Sequenase sequencing kit, with
[
S]dATP to detect the extended primer chains.
Reactions were stopped and analyzed as in method 1.
Data from a typical kinetic experiment at 25 °C are shown in Fig. 2. Under these assay conditions, where the DNA substrate was the only limiting reaction component, a pseudo-first-order reaction with respect to DNA is expected (Wang and Harshey, 1994). When the kinetic data were fitted with a pseudo-first-order equation (see Fig. 2legend), we obtained an apparent rate constant of 0.38/min and 0.75/min for type 0 formation and for conversion from type 0 to type I, respectively. These two rate constants correspond to a half-time of 1.8 and 0.92 min for the two reactions, respectively.
Figure 2:
Kinetics of formation and conversion of
type 0 and type I complexes at 25 °C. Mini-Mu donor plasmid pZW140
was assayed for formation of type 0 (), conversion from type 0 to
type I (
) and type I (
) from 0 to 16 min at 25 °C, as
described under ``Experimental Procedures.'' The final stages
of all reactions were stopped by the addition of 0.2% SDS followed by
electrophoresis on agarose gels. DNA band intensities were quantified
as described under ``Experimental Procedures,'' and the yield
of cleaved product (relaxed DNA species) was plotted as a percentage of
the total DNA against time. Superimposed on the kinetic data for type 0
complex formation and its conversion are the best fitting traces (thick and thin lines, respectively) for an
exponential rise ([product] = S
(1 -
exp(-kt)), from which the rate constants were obtained.
The dotted line is the computed time course for two
irreversible, consecutive reactions (), using k
= 0.38/min and k
= 0.75/min.
From the two rate constants calculated above, we computed a theoretical trace (Fig. 2, dotted line) for the time course of type I formation using the following standard equation for two irreversible, consecutive reactions of first order,
where S is the starting concentration of substrate.
The experimentally obtained kinetic data (open triangles) for
the overall type I reaction showed a fairly good fit to the theoretical
curve.
It should be pointed out that the DNA substrates employed here, as isolated from E. coli, consist of different topoisomers and are thus kinetically heterogeneous. A rigorous quantitative analysis of the reaction scheme is therefore not strictly meaningful unless each individual topoisomer population is analyzed. Although large differences in the reaction rates among the DNA substrates is not expected here, given the average superhelicity of the plasmids (estimated to be -0.06), we still caution against the literal interpretation of the kinetic constants observed.
Figure 3: Kinetics of formation and conversion of type 0 and type I complexes at 20 °C. Experiments were performed at 20 °C and analyzed as in Fig. 2. The best fitting curves for the data points were obtained as described in Fig. 2. Symbols are as in Fig. 2.
We caution the reader against possible misinterpretation of the type 0 to type I conversion data in Fig. 3. The plateau of the kinetic curve represented by the filled circles denotes 100% conversion of type 0 to type I. The 40% value on the ordinate corresponds to the total yield of the type 0 complex (prior to cleavage) in this experiment. Recall that our assays measure type 0 indirectly following DNA cleavage. The validity of the interpretations based on this indirect estimate of type 0 has been verified by the subsequent development of a direct gel mobility assay for type 0 (Kim et al.(1995); see also Fig. 10).
Figure 10:
Zn and Co
ions can substitute for Mg
in cleavage but not
assembly of type 0. In a two-step cleavage reaction, type 0 complexes
of pZW140 were first assembled (lane 2) and then incubated
with 10 mM MgCl
(lane 3), 2 mM MnCl
(lane 4), 1 mM ZnCl
(lane 5), or 1 mM CoCl
(lane
6) at 30 °C for 20 min. The addition of 10 µg/ml heparin
to the reaction mixtures before electrophoresis in an agarose gel
allowed a small but significant separation of the type 0 complex from
unreacted supercoiled donor substrate plasmid (Kim et
al.(1995); see ``Experimental Procedures''). Lanes
7-10 are as 3-6 except that the
assembly-cleavage reactions were carried out in one step with the
indicated metal ions. Lane 1 is a control reaction with no
proteins added. The band migrating below type I in lanes 4 and 8 is the product of intramolecular strand transfer. The band
above R is a supercoiled dimeric form of the
plasmid.
We note that the kinetics of type 0 formation (in
the presence of Ca) or type I formation (in presence
of Mg
) show greater variability from experiment to
experiment than the conversion of type 0 (formed in the presence of
Ca
) to type I in the presence of
Mg
. The reasons for this are not clear. One potential
contributing factor is the nonuniform recovery of the type 0 complex
from individual reaction mixtures using the ``spin dialysis''
protocol (see ``Experimental Procedures''). Nonetheless, the
data from Fig. 2and Fig. 3reveal important
rate-determining aspects of the strand cleavage reaction by the Mu A
protein. First, the conversion of type 0 to type I is a fast reaction,
showing only a modest rate acceleration with temperature (ratio of t
values at 20 and 25 °C is approximately
2). On the other hand, Ca
-mediated formation of the
type 0 complex or Mg
-mediated formation of the type I
complex are slower reactions that show markedly higher
temperature-dependent rate enhancements (ratio of t
values at 20 and 25 °C is approximately 4 for the type 0 and
approximately 6 for the type I reaction). Second, the significantly
lower t
for the type 0 reaction
(Ca
-mediated) compared with the type I reaction
(Mg
-mediated) at both 25 and 20 °C suggests that
the assembly of the precleavage synaptic complex (type 0) has a lower
activation barrier in the presence of Ca
than in the
presence of Mg
. Thus we conclude that the strong
temperature dependence of the type I reaction is contributed mainly by
the step of assembling a complex equivalent to type 0. It should be
noted that, despite the drop in the rate of type 0 formation upon
temperature shift from 25 to 20 °C, there was little change in the
final yield of type 0 (
)(compare Fig. 2with Fig. 3). All of the above results are consistent with a model in
which the formation of a type 0 equivalent is the rate-limiting step of
the overall strand cleavage reaction, at least for the DNA substrate
tested here.
Figure 4:
Superhelical density dependence of type 0
and type I formation. Topoisomers of mini-Mu plasmid pZW140 were
assayed for type 0 and type I formation at 30 °C as described in Fig. 2. Type 0 reactions were incubated for 15 min followed by
spin dialysis and cleavage with Mg, and type I
reactions were incubated for 20 min. The reactions were stopped with 20
mM EDTA before electrophoresis. DNA bands were quantified as
described in Fig. 2, and the percentage of type I (cleaved
product) formed was plotted against superhelical density, after
normalizing to the product yield at
= -0.069. Drawn
through the data points is a 5th order spline function. Symbols are as in Fig. 2.
Since we measure type 0 after converting it into type I, one might argue that the apparent dependence of type 0 formation on superhelicity is due to the requirement of supercoils during conversion of type 0 to type I complex. However, as shown below, supercoils appear not to be required for this conversion step.
Thus, the demonstration that the kinetic dependence of the formation of type 0 on superhelicity parallels that of type I lends further support to the inference that the type 0 complex is a real intermediate in the type I reaction pathway.
Figure 5:
Removal of supercoils destabilizes the
type 0 complex but allows Mu end cleavage. Type 0 complexes formed with
pZW140 (lane 1) were treated with P1 nuclease alone (lane
2) or P1 followed by Mg (lane 3). The
type 0 complexes migrated at the same position as the supercoiled (S) donor substrate under these experimental conditions. When
P1 cleavage was followed by the addition of Mg
, forms
labeled TL (twin loop) and L* (linear, single loop) and type I were
generated. Lane 4 was a control reaction in which type I
complexes were treated with P1. See text for details. R and L refer to the relaxed and linear forms of the
plasmid.
Figure 7: Superhelicity dependence of generation of the P1-sensitive region outside the left end of Mu in type 0 complexes. Type 0 complexes generated with plasmid pZW140 substrates of varying superhelicity were treated with P1 nuclease. Primer extension using the L2 primer was performed as in Fig. 6. A, C, G, and T, dideoxy sequencing lanes; lane 1 (unlabeled), control plasmid DNA (superhelical density, about -0.06); lanes 2-13, type 0 complexes with DNA of increasing superhelicity (-0.020, -0.024, -0.027, -0.032, -0.036, -0.041, -0.046, -0.051, -0.055, -0.060, -0.065, -0.069), as described in Fig. 4.
Figure 6:
P1
cleaves the top strand outside the left end of Mu in type 0 complexes. A, four different primers (LT, L2, R3, RB) were used to probe
the top and bottom strands near the left and right ends of Mu in type 0
complexes of pZW140 cleaved with P1. Thick lines, Mu DNA; thin lines, non-Mu DNA; diamonds, Mu A protein
cleavage sites. L and R, left and right ends of Mu. B, for each primer, dideoxy sequencing reactions are shown
first (A, C, G, and T). Primer
extensions were carried out on the following substrates as described
under ``Experimental Procedures'' (method 1): untreated type
0 complexes (lane 1); type 0 complexes treated with P1
nuclease (lane 2); type 0 complexes treated first with P1 and
next with Mg (lane 3); type I complexes (lane 4); type I complexes treated with P1 (lane 5). Arrowheads indicate Mu (black)/non-Mu (white) DNA junctions.
In
summary, although removal of supercoils from the type 0 complex with P1
nuclease destabilizes the complex under gel electrophoresis conditions,
cleavage with Mg (prior to gel electrophoresis)
retains a significant fraction as stable forms that are consistent with
having arisen from type 0 substrates lacking supercoils in either the
Mu or the non-Mu domains of the mini-Mu plasmid. This result shows that
substrate supercoiling is required only for assembling the
cleavage-competent protein-DNA architecture (type 0) and not for the
execution of the cleavage reaction per se.
Figure 8:
KMnO cleaves the continuous
strands outside both ends of Mu in type 0 complexes. Type 0 complexes
made with plasmid pZW140 were treated with KMnO
, followed
by DNA strand breakage at the modified sites as described under
``Experimental Procedures.'' Primer extension reactions were
carried out as described under ``Experimental Procedures''
(method 2). LT, L2, RB, and R3 refer to primers used in sequencing
reactions to probe both strands at the two ends of Mu (see Fig. 6A). For each primer, the first four lanes are reactions with KMnO
-treated naked supercoiled DNA,
and the next four lanes those with type 0 complexes. All other
symbols are as in Fig. 6.
Fig. 9summarizes the cleavage sites of P1 outside the left
end ( Fig. 6and Fig. 7), and KMnO outside
both ends of Mu (Fig. 8) in type 0 complexes. We interpret the
Mu end cleavages seen with these two single-strand specific probes as
indicators of the generation of single-stranded DNA at both ends of Mu
in the type 0 complex.
Figure 9:
Summary of P1 and KMnO cleavages outside Mu ends in type 0 complexes. A,
sequence spanning the Mu (highlighted area)/non-Mu junction at
the left (L) end of Mu. The diamond indicates the
cleavage site of Mu A, arrowheads the cleavage sites of P1
nuclease, and ovals the cleavage sites of KMnO
in
the type 0 complexes. The heights of these symbols roughly
correlate with the relative cleavage intensities. A 5-base pair stretch
directly duplicated outside each Mu end is underlined. B, sequence spanning the Mu/non-Mu junction at the right (R) end of Mu. Symbols are as in A.
In this report, we have presented kinetic studies on the type
0 complex, its formation and conversion to type I complex. The kinetics
of type 0 formation in the presence of Ca fits well
with the expectation that it precedes type I in the Mu A mediated DNA
cleavage reaction (Fig. 2). The rate of production of type 0 and
type I show close parallels in their dependence on temperature (Fig. 3) and on the superhelicity of the DNA substrate (Fig. 4). The simplest explanation for these observations is
that type 0 complex is a bona fide intermediate in the pathway leading
to the type I complex. Our results also suggest that the formation of
the pre-cleavage synaptic complex (type 0 or type 0 equivalent) has a
lower activation barrier in presence of Ca
than in
presence of Mg
Furthermore, we find that the
conversion of type 0 to type I complex is a relatively rapid reaction,
and is fairly insensitive to temperature. Overall, these observations
suggest that type 0 formation is the rate-limiting step in type I
formation, at least for a DNA substrate with superhelicities at or
below -0.06.
Given that the formation of the type 0 complex is the rate-limiting step in generating the type I complex, the observed dependence of type 0 production on DNA superhelicity likely reflects a true rate dependence on supercoils. However, we cannot rule out the possibility that supercoiling may determine the equilibrium of the reaction as well. Indeed, if supercoils are used to elevate the energy level of the substrates, instead of lowering the energy level of the transition state, supercoiling will influence both the rate and equilibrium of type 0 complex formation. If formation of type 0 complex is already overwhelmingly favored, as seems to be the case for plasmids with a superhelicity near -0.06, further increase in superhelicity would result in no detectable difference in the yield but could lead to higher reaction rates.
We had proposed that in the Mu
transposition reaction, the free energy of supercoils might be used to
promote energetically costly events such as the structural transition
of DNA (e.g. melting) and/or of proteins (Wang and Harshey,
1994). It may well be that the target for Mu nicking is single- rather
than double-stranded DNA. If Mu DNA ends must melt before they can be
cleaved, then the supercoiling, in principle, could facilitate this
event. We have now shown that DNA at the ends of Mu in the type 0
complex is susceptible to cleavage by single-strand-specific agents
(nuclease P1 and KMnO; Fig. 6Fig. 7Fig. 8Fig. 9). Furthermore, P1
cleavage near the left end of Mu is dependent upon supercoils, and this
dependence is correlated with formation of the type 0 complex (Fig. 4Fig. 5Fig. 6Fig. 7). Cleavage of
type 0 by P1 does not abolish cleavage by Mu A protein at the DNA ends (Fig. 5, 6), although we do not know whether the conformational
changes in DNA, as detected by P1 nuclease and KMnO
, are
removed along with the presumed removal of supercoils upon P1 cleavage.
Since Mu A protein contacts DNA outside Mu in the type 0 complex
(Mizuuchi et al., 1992), it is possible that the structural
distortions within the type 0 remain after P1 cleavage, even though
they may not be exactly the same.
Both single strand DNA probes used
in this study were seen to preferentially cleave the continuous strands
of DNA (the strands not cleaved by Mu A) in type 0 complexes (see Fig. 9). These cleavages extended from 2 nucleotides within Mu
to 8 nucleotides outside Mu. It is very likely that this specificity
for the continuous strand is a result of protection of the
complementary strand by Mu A protein. We note that although
footprinting of type I complexes with DNase I and
methidiumpropyl-EDTAFe(II) showed protection of 12-13 base
pairs outside Mu ends (Lavoie et al., 1991; Mizuuchi et
al., 1991), dimethyl sulfate modification experiments failed to
reveal methylation protection of the continuous strands outside Mu (Kuo et al., 1991). It is not clear why P1 did not cleave DNA
outside the right end of Mu, a region readily detected by
KMnO
. This preference may be a result of preferential
access of P1 to only the left end of Mu. Alternatively, the
single-stranded features at the two ends are a result of structural
distortions that are not equivalent. Of interest in this regard are
results of hydroxyl radical footprinting of type I complexes, which
showed enhanced cleavages 2 base pairs outside the Mu ends on the
continuous or unnicked strands (Lavoie et al., 1991). However,
the hydroxyl radical hyperreactivity was higher at the right end. The
asymmetric DNA conformation outside the two ends of Mu as detected by
the various DNA structure probes should not be surprising in view of
the fact that, except for the two terminal base pairs at the ends of
Mu, the sequence and arrangement of the att subsites (Mu A
protein binding sites) are quite different at attL and attR (see Fig. 9) (Craigie et al., 1984; Zou et al., 1991). Also, the sequences outside the two Mu ends (in
their integrated state on the host genome) are normally unrelated
(except for the 5-base pair target duplication bordering each end). The
asymmetry of DNA conformation outside the Mu ends may be of
significance to the biology of phage Mu replication, which is thought
to proceed asymmetrically, from the left to the right end of Mu (see
Harshey(1988)). Further experiments are needed to explore the DNA
structure in this region. We note that KMnO
reacts not only
with bases in denatured DNA but has also been reported to selectively
react with B-Z or Z-Z junctions in supercoiled plasmid DNA (Jiang et al., 1991). Similarly, P1 nuclease is capable of
recognizing a range of non-A/non-B/non-Z conformations in
double-stranded DNA (see Pulleybank et al.(1988)).
We
conjecture that the single-stranded DNA regions in a type 0 complex may
have specific interactions (such as hydrogen bonding and hydrophobic
interactions) with Mu A protein to stabilize the melted/distorted
structure. This could account for the sequence preference exhibited by
Mu A beyond the cleavage sites at the Mu ends (see Wu and
Chaconas(1992)). However, considerable flexibility must exist to
accommodate a variety of base combinations. We note that a change of
even one hydrogen bonding interaction within the type 0 complex could
have an apparently dramatic consequence for its stability. The type 0
complex formed with pZW140 has an apparent half-time of decay of
16 min at 30 °C (expected to be around 90 min at room
temperature; the decay rate was estimated by timed incubation of type 0
complex in the absence of divalent metal ions followed by quantitating
the amount of type I complex generated after the addition of
Mg
). The average free energy for a hydrogen bond (or
more precisely, the average free energy change for a hydrogen bond
exchange), is about 1.2 kcal/mol (Fersht, 1987). Thus, change of a
single hydrogen bond within the type 0 complex could extend its
half-life to 12 h or shorten it to just 11 min at room temperature, on
average. (
)If the rate-limiting step of type 0 formation is
the opening of the DNA helix outside the Mu ends, then it is also
reasonable to expect that the sequence of this DNA will influence the
activation barrier to the transition state, and therefore the rate of
type 0 formation (see Cantor and Schimmel(1980) and Murchie et
al.(1992)).
We have shown in this study that Zn and Co
metal ions (in addition to
Mg
and Mn
) can support cleavage of
the type 0 complex (Fig. 10). Divalent metal ions play a central
role in phosphoryl transfer in nucleic acids. Structural analysis of E. coli alkaline phosphatase (Kim and Wyckoff, 1991) and of
the Klenow polymerase (Beese and Steitz, 1991) complexed with a
deoxynucleoside monophosphate and a single-stranded DNA substrate
strongly suggest that two metal ions are liganded to specific amino
acids. One metal ion is proposed to function as a Lewis acid and to
form the attacking hydroxide ion, whereas a second metal ion is
proposed to coordinate the bridging 3`-oxygen atom at the cleavage
site, stabilizing the transition state and facilitating the leaving of
the 3`-OH group. It is likely that metal ions play several roles in the
Mu transposition reaction, including one similar to that postulated for
the nuclease activity of the Klenow fragment (see Baker and Luo(1994)
and Kim et al.(1995)). For example, the inability of
Ca
ions to support cleavage of the type 0 complex
suggests at least two roles for divalent cations, one in assembly of
the complex and the other in cleavage (Mizuuchi et al., 1992).
The ability of Zn
and Co
to support
only cleavage but not formation of type 0 (Fig. 10), lends
further credence to this notion. Metal ions such as cobalt, zinc, and
copper can have differential, sequence-specific effects on DNA (see
Kang and Wells(1994) and references cited therein). It is likely that
during assembly, metal ions may play a role in promoting and/or
stabilizing the altered DNA conformation in the type 0 complex. We have
recently described the isolation of a mutant of Mu A that shows a
differential response to different divalent cations (Kim et
al., 1995). Mu A(G348D) was seen to assemble a type 0 complex only
in the presence of Ca
(and not Mg
or Mn
) and cleave a preassembled type 0 only in
the presence of Mn
(and not Mg
),
but strand transfer precleaved Mu ends in the presence of either
Mg
or Mn
. All of these results
suggest not only a multiple role for metal ions in Mu transposition,
but also a different role in each of the three observable stages of
transposition: assembly, cleavage, and strand transfer. The combination
of variant proteins and metal ions should provide tools for the
dissection of the role of metal ions in the various steps of the
transposition reaction.