(Received for publication, August 26, 1994; and in revised form, October 19, 1994)
From the
We describe the isolation and characterization of Mu A variants
arrested at specific steps of transposition. Mutations at 13 residues
within the Mu A protein were analyzed for precise excision of Mu DNA in vivo. A subset of the defective variants (altered at
Asp, Asp
, Gly
, and
Glu
) were tested in specific steps of transposition in vitro. It is possible that at least some residues of the
Asp
-Asp
-Glu
triad may have
functional similarities to those of the conserved Asp-Asp-Glu motif
found in several transposases and retroviral integrases. Mu A(D269V) is
defective in high-order DNA-protein assembly, Mu end cleavage, and
strand transfer. The assembly defect, but not the catalytic defect, can
be overcome by precleavage of Mu ends. Mu A(E392A) can assemble the
synaptic complex, but cannot cleave Mu ends. A mutation of Gly
to aspartic acid within Mu A permits the uncoupling of cleavage
and strand transfer activities. This mutant is completely defective in
synaptic assembly and Mu end cleavage in presence of
Mg
. The assembly defect is alleviated by replacing
Mg
with Ca
. Some Mu end cleavage is
observed with this mutant in the presence of Mn
. When
presented with precleaved Mu ends, Mu A(G348D) exhibits efficient
strand transfer activity.
The Mu transposase (Mu A) catalyzes the cleavage of Mu DNA ends
and their subsequent joining (or strand transfer) to target DNA (see
Mizuuchi, 1992). Under standard assay conditions, the cleavage of Mu
ends exhibits a strict requirement for DNA supercoiling, Escherichia coli protein HU and divalent metal ions
(Mg or Mn
). Interactions of
monomeric Mu A protein with six att subsites (three at each
end of Mu constituting attL and attR) and an internal
enhancer element leads to the formation of a tetrameric assembly of Mu
A within a high-order nucleoprotein complex. Ca
ions
can support assembly of this complex, but cannot support strand
cleavage at Mu ends (Mizuuchi et al., 1992). The catalytically
functional form of Mu A is the tetramer.
The Mu A protein is
organized into three principal domains as determined by limited
proteolysis experiments (Nakayama et al., 1987).
Structure-function studies have mapped att and enhancer DNA
binding activities to two separate regions on the
NH-terminal domain I (see Fig. 1A; Nakayama et al., 1987; Leung et al., 1989; Mizuuchi and
Mizuuchi, 1989). The central, proteolytically stable domain II was
inferred to contain the catalytic site since a temperature-sensitive
(ts) mutation that allows cleavage but blocks strand transfer maps here
(Leung and Harshey, 1991). This domain also shows nonspecific (target?)
DNA binding activity (Nakayama et al., 1987). The catalytic
domain must extend into part of COOH-terminal domain III as determined
by the activity of deletions that are localized here (Harshey and
Cuneo, 1986; Bremer et al., 1988; Betermier et al.,
1989; Leung and Harshey, 1991; Baker et al., 1993). The distal
two-thirds of domain III is required for interaction with the accessory
transposition protein Mu B which promotes intermolecular strand
transfer (Harshey and Cuneo, 1986; Leung and Harshey, 1991; Baker et al., 1991).
Figure 1:
Mu A protein (transposase): domain
structure and amino acid alignment with other transposases and a
retroviral integrase. A, on the basis of limited proteolysis,
three domains were assigned to A protein (see text). Amino acid numbers
corresponding to the amino terminus of each major domain are shown
beneath the structure. The NH-terminal domain I contains
subdomains
,
, and
, which encode site-specific DNA
binding activity. I
binds to Mu enhancer and I
binds to
Mu att sites. Domain II is likely the catalytic domain. It
shows nonspecific DNA binding activity, and a sequence stretch
resembling the helix-turn-helix DNA binding motif maps here (open
box between residues 390 and 409; Harshey et al., 1985).
A mutation specifically affecting strand transfer (asterisk)
maps toward the COOH-terminal end of this domain (residue 548; Leung
and Harshey, 1991). The distal two-thirds of domain III is responsible
for important interactions with the Mu B protein, whereas the proximal
one-third is important for assembly and catalysis. The amino acid
residues mutated in this study are indicated above the domain
structure. B, the amino acid alignment shown here is based on
the data from Radstrom et al.(1994). A similar but not
identical alignment has been deduced by Baker and Luo(1994). Aspartic
acid residues at positions 269 and 294 and a glutamic acid residue at
position 392 within Mu A correspond to conserved residues in all
proteins (shown in outlined letters). A third aspartic acid
residue is conserved in all proteins except Mu A. This position is
Gly
of Mu A in our alignment and Phe
of Mu
A in the Baker-Luo alignment. Three other positions, all glycines
(shown in bold letters) are also conserved in all
proteins.
Mu transposition shares many common chemical
features with transposition of other transposable elements as well as
integration of retroviruses into their host genomes (see
Mizuuchi(1992)). Most of these elements share a conserved
Asp-Asp-35-Glu, (where 35 indicates the number of residues separating
Asp and Glu) motif (Fayet et al., 1990; Rowland and Dyke,
1990; Kulkosky et al., 1992; Radstrom et al., 1994)
whose importance in catalysis has been established from mutagenesis
experiments (Engelman and Craigie, 1992; Leavitt et al., 1993;
van Gent et al., 1993). Sequence comparison of the Mu
transposase with the equivalent proteins of the above elements
identified residues within domain II of Mu A that aligned with the
Asp-Asp-35-Glu motif (Fig. 1B). ()By analogy
with the exonuclease of E. coli DNA polymerase I which uses a
cluster of acidic residues to position metal ions within its active
site (Beese and Steitz, 1991; Derbyshire et al., 1991), the
Asp-Asp-35-Glu residues are proposed to be involved in coordinating
divalent metal ions and in positioning the target phosphodiester and
the attacking nucleophile for phosphoryl transfer (Kulkosky et
al., 1992; Engelman and Craigie, 1992).
This study focuses on
defining the role of potential active site amino acids within the
presumed catalytic domain II of Mu A by making site-directed mutations
in this region. We show that mutations of Asp (D269V),
Asp
(D294N), and Glu
(E392A) abolish in
vivo activity of the transposase. A mutation at Gly
(G348D) also resulted in loss of activity in vivo. From
a study of the in vitro properties of these mutants, we infer
that these residues play critical and distinct roles in organizing the
transposition complex and in the catalytic steps. It seems plausible
that the acidic residues could be involved in facilitating a two metal
ion mechanism for phosphoryl transfer analogous to that used by the
3`-5` exonuclease reaction of E. coli DNA polymerase I.
While this manuscript was in preparation, Baker and Luo(1994) reported an independent mutational analysis of eleven residues of Mu A. Three of our mutations are at similar positions. The properties of one of our mutants, Mu A(E392A), are comparable with those of Mu A(E392Q) isolated by Baker and Luo(1994). However, the behavior of a second mutant MuA(D269V) characterized by us is distinct from that of Mu A(D269N) described by Baker and Luo(1994). In our analyses, the most interesting step-arrest phenotypes are displayed by Mu A(G348D). Baker and Luo(1994) have not studied an analogous mutant.
For efficient expression of Mu A, the Mu A gene from pRA158 was transferred to another T7 expression vector, pET11-a (Novagen), to generate pET158.
Plasmid
pMK21, used in ``type 0'' and ``type I'' assays,
was constructed as follows. The BamHI site on pRA167 (Leung et al., 1989) was changed to SalI, which upon SalI digestion and ligation eliminated a 7-kilobase SalI trp-lac fragment in the resulting plasmid
pZW167. Restriction sites for BamHI and BglII were
created on this plasmid by site-directed mutagenesis of nucleotide 58
(T C) and nucleotides 224, 228, and 229 (C
A, A
C,
and A
T), respectively. The resulting plasmid pMK21 has 1.8
kilobases of Mu sequences between attL and attR,
which include the enhancer element.
``MeSO
assays'' were carried out using linear DNA fragments obtained by
restriction enzyme digestion from a pUC19 derivative containing R1-R2
(Namgoong et al., 1994).
The plasmid engineered to be cut at the Mu right end by HindIII digestion was a gift from M. Surette and G. Chaconas (University of Western Ontario) and is described in Namgoong et al.(1994).
In this paper we provide a detailed in vitro analysis of three mutant A proteins that were functionally defective as judged by the excision assay: Mu A(D269V), Mu A(G348D), and Mu A(E392A). Particularly interesting was the characteristic ``step-arrest'' behavior of the variant proteins in the transposition pathway and the differential response of two of these proteins to different divalent cations in the reaction (see below). Mu A(D294N) was uniformly defective in all in vitro assays detailed below, retaining approximately 5-10% of the wild-type activity (data not shown). Hence this variant is not revealing in regard to the mechanism of transposition and is not discussed in detail.
Figure 2:
The rationale for the assay for
synapsis. Synapsis between attL and attR within the
circular plasmid in presence of Ca
leads to the type
0 complex within which Mu A is shown to exist as a tetramer (Lavoie et al., 1991; Mizuuchi et al., 1992). The enhancer
element is located across the HindIII (H) site (not
shown). HindIII plus XbaI (X) digestion
would give rise to the
form (Surette et al., 1991).
Digestion with either HindIII or XbaI alone would
yield one of two
forms. When the chi is treated with SDS, two
linear DNA fragments (one harboring attL; the other harboring attR) will be generated.
Figure 3:
Assembly of the type 0 complex in presence
of Ca. A, after incubation of the substrate
plasmid with Mu A or Mu A variants (approximately 2 pmol of
protein/pmol of att subsite) in the presence of HU and
Ca
followed by DSP treatment, extensive digestion
with HindIII plus XbaI was carried out prior to
electrophoretic fractionation in agarose. Lane designations are as
follows: lane 1, linearized plasmid; lane 2, plasmid
cut with HindIII plus XbaI; lane 3, uncut
plasmid; lanes 4-7, reactions with Mu A, Mu A(D269V),
MuA(G348D), and Mu A(E392A), respectively. Lanes 8-11,
reactions as in lanes 4-7 treated with SDS. B,
unlike the assays shown in A, reactions here were not digested
with restriction enzymes, nor treated with SDS. 10 µg/ml of heparin
was added to the samples prior to electrophoresis. CCP, closed
circular plasmid; OCP, open circular plasmid; LP,
linear plasmid; R, attR DNA fragment; L, attL DNA fragment. The species above the open circular form of
the substrate plasmid in B is probably a trace of the
supercoiled dimer form of the plasmid.
When samples were fractionated without
restriction digestion but with addition of heparin prior to
electrophoresis, the pattern shown in Fig. 3B was
obtained. The band that migrates just above the covalently closed
circular form (CCP) of the plasmid is the type 0 complex, the
precursor of the band seen in Fig. 3A. When
these reaction mixtures were treated with HindIII and XbaI under partial digestion conditions, there was a strong
correspondence between the disappearance of type 0 and the appearance
of
together with ``alpha'' bands (
arises from a
single restriction cut within the synapsed complex; see Fig. 2)
(data not shown). The type 0 band was present in the Mu A (lane
2, Fig. 3B), Mu A(G348D) (lane 4, Fig. 3B), and the Mu A(E392A) (lane 5, Fig. 3B) reactions and absent in the Mu A(D269V)
reaction (lane 3, Fig. 3B). The level of type
0 complex yielded by Mu A(G348D) was lower than that obtained with Mu A
or Mu A(E392A), possibly because the synapsed complex produced by Mu
A(G348D) in presence of Ca
was less stable than the
normal complex.
Thus, while Mu A(G348D) and Mu A(E392A) can assemble the type 0 complex, MuA(D269V) is defective in assembly.
Figure 4:
Reactions of Mu A variants in
Mg, in Ca
followed by
Mg
and in Mn
. A, standard
type I assays were done in presence of Mg
. Samples
were treated with SDS prior to electrophoresis. Lane 1 is a
control reaction without addition of Mu A protein. For each reaction
set, reactions from left to right contained 1, 2, and
4 pmol of Mu A or A variant/att subsite. B, reaction
mixtures were first incubated with Ca
under type 0
assay conditions. Incubations were then continued in presence of added
Mg
(10 mM). Samples were fractionated
without SDS treatment. 1. C, reactions were done as in A, except that Mg
was replaced by
Mn
, and samples were fractionated without SDS. Lane 1 is a reaction without added Mu A protein. Lane 2 is a wild-type Mu A reaction in 2 mM Mn
. Lanes 3-14 represent
reactions with Mu A variants. For each variant, reactions from left to right contain 5, 10, 20, and 40 mM Mn
, respectively. All other symbols as in Fig. 3. The bands migrating below the open circular plasmid (OCP) forms in some lanes are likely to be a trace amounts of
the linear form of the substrate plasmid. CCP, closed circular
plasmid.
Thus, in presence of Mg, Mu
A(E392A) is normal and Mu A(G348D) is defective in the assembly of a
stable precleavage complex (equivalent to type 0).
Thus, neither Mu A(G348D) nor Mu A(E392A) can mediate att-specific DNA cleavage within the pre-organized type 0 complex.
Thus, Mn but not Mg
ions can support att cleavage by Mu A(G348D) at concentrations comparable to those in
the wild-type reaction. The cleaved ends can be used for strand
transfer. Mn
-supported att cleavage by Mu
A(E392A) must be quite low as inferred from the trace amounts of strand
transfer yielded by this mutant.
Figure 5:
High-order DNA-protein assembly and strand
transfer under MeSO assay conditions. A and B, reactions were carried out in Me
SO with a
linearized plasmid containing attR subsites R1-R2 (Namgoong et al., 1994). C and D, the substrate was a
linear DNA fragment containing precleaved attR (R1-R2-R3)
(Namgoong et al., 1994). Samples were fractionated by
electrophoresis without SDS treatment (A and C) or
after SDS treatment (B and D). R, linear DNA
fragment containing R1-R2; RC; linear DNA containing
precleaved attR; HC, high-order DNA-protein complex; STP, strand transfer products.
The results
when linear DNA precleaved at attR was used as the substrate
in the MeSO reaction are shown in Fig. 5, C and D. Surprisingly, Mu A(D269V), which tested negative
in all assays so far, was seen to assemble a complex (lane 3, Fig. 5C) in amounts comparable with that formed by Mu
A(E392A) (lane 5, Fig. 5C). Furthermore, Mu
A(G348D) also yielded a similar complex with the precleaved DNA (lane 4, Fig. 5C). The formation of this
complex with all three mutants as well as wild-type Mu A was
independent of the addition of metal ions (not shown). Among the
mutants, only Mu A(G348D) (lane 4, Fig. 5D)
and not the other two mutants (lanes 3 and 5, Fig. 5D) could yield strand transfer products from the
prenicked substrate. This strand transfer activity was absolutely
dependent on added metal ions (Mg
or
Mn
) in contrast to the strand transfer activity of
wild-type protein which did not require an exogenous supply of either
of these two metals under the Me
SO assay conditions (data
not shown).
The properties of Mu A(E392A) described here
are consistent with those of Mu A(E392Q) reported by Baker and
Luo(1994). However, Mu A(D269V) is distinct from Mu A(D269N) studied by
Baker and Luo(1994). In our assays, Mu A(D269V) shows a severe defect
in DNA-protein assembly and does not show att cleavage or
strand transfer activity in presence of Mn.
Divalent metal ions play a central role in phosphoryl transfer reactions in nucleic acids. Examples include reactions mediated by polymerases, nucleases, restriction enzymes, DNA-dependent ATPases, and ribozymes, among others. It is reasonable to expect that key acidic residues within the ``phosphoryl transferase'' family might be involved in coordinating the metal and orienting it within the active site. The x-ray structure of the Klenow fragment of E. coli DNA polymerase I, together with directed mutational analysis, provides insights into how two metal ions can be positioned within the transition state for the 3`-5` exonuclease activity of this enzyme (Beese and Steitz, 1991; Derbyshire et al., 1991). Properties of residue-specific mutants of retroviral integrases have led to the hypothesis that key acidic amino acids (forming a Asp-Asp-35-Glu motif), critical for catalysis, may function by coordination of the metal cofactor (Kulkosky et al., 1992; Engelman and Craigie, 1992; van Gent et al., 1993; Leavitt et al., 1993). In this study we describe variants of Mu A protein that are arrested at specific steps of the transposition pathway in a metal-specific manner.
Our studies reveal specific effects of these mutations
in individual steps of the transposition pathway and their distinct
responsiveness to a set of divalent metal ions, Ca,
Mg
or Mn
. Quite striking is the
observation that changing Gly
(which aligns with the
second Asp of the retroviral Asp-Asp-35-Glu motif (see Fig. 1B)) to aspartic acid results in bi-specificity of
metal ions: Ca
but not Mg
can be
utilized in the assembly step, only Mn
can function
in cleavage, and Mg
or Mn
can
function in the strand transfer steps. This mutation, in conjunction
with Glu
, artificially creates an analog of the
Asp-35-Glu motif that is absent in wild-type Mu A. The novel assembly
and catalytic potentials revealed by the mutation strongly implies its
location within the active site pocket or its influence on the active
site configuration. Overall, the properties of the mutants suggest that
the transposase and the retroviral integrases may utilize analogous
metal coordinating mechanisms although the amino acid residues that
perform this function may not conform to an obvious primary sequence
motif.
The differential
effects of various divalent cations on Mu A and Mu A variants in the
steps of transposition would be consistent with a two metal or multiple
metal ion mechanism, although a mechanism involving a single metal ion
(Suck, 1992) cannot be ruled out. The requirement for Ca (and not Mg
or Mn
) for type 0
assembly and that of Mn
(rather than
Mg
) for strand cleavage and stransfer by Mu A(G348D)
is suggesstive of the involvement of more than one metal ion during
transposition. The extreme simplicity of a two metal ion mechanism in
phosphoryl transfer (one to generate the oriented nucleophile and the
other to stabilize the leaving group) suggests that this catalytic
strategy may be utilized by a number of enzyme systems. The
evolutionary design of their active sites would be guided by the need
to achieve precise orientation of the metal ions with respect to the
substrate. Structural analyses of the active site configurations of Mu
A and its step-arrest variants should be insightful in this regard.