From the Section of Molecular Genetics and Microbiology and Institute of Cellular and Molecular Biology, University of Texas, Austin, Texas 78712
Received for publication, September 18, 2000, and in revised form, November 9, 2000
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ABSTRACT |
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Mu DNA transposition from a negatively
supercoiled DNA substrate requires interaction of an enhancer element
with the left (attL) and right (attR) ends of Mu. The orientation of
the L and R ends with respect to each other (inverted) and with respect to the enhancer is normally inviolate. We show that when the enhancer is provided in trans as a linear fragment, the head to head
orientation of the L/R ends is still required. Each functional half of
the linear enhancer maintains the same "cross-wise" interaction
with the subsites L1 and R1, when present in cis or in
trans. In reactions catalyzed by an enhancer-independent
variant of the Mu transposase, the need for negative supercoiling of
the substrate and the inverted orientation of L and R ends is not
relaxed. These results show that the orientation specificity of the
enhancer is not determined by its topological linkage to the Mu ends.
There is a functional asymmetry inherent to the enhancer. Furthermore,
the enhancer does not directly impose topological constraints on the
transposition reaction or specify the reactive orientation of the Mu ends.
Site-specific recombination systems in both prokaryotes and
eukaryotes require interaction of specific DNA sequences. Although some
systems can carry out recombination with the interacting recombination
sites in any orientation (e.g. phage A second objective of this study was to understand the basis of the
orientation specificity of the transposition enhancer itself (8-10).
Since the Mu ends are inverted with respect to each other, the
transposase synapse (in which the enhancer interacts with the L and R
ends to form an LER complex; see Ref. 11; see Fig. 1A) is
expected to be most closely related to the synapse made by the Hin/Gin
invertases, whose enhancers do not display orientation specificity (13,
14). Does the orientation specificity of the Mu enhancer arise as a
result of an inherent asymmetry in its sequence and/or from being
topologically linked to the L and R ends? If the enhancer were free
from the constraints of DNA topology, might it acquire the freedom to
be active in either orientation? Two developments facilitated this
investigation. The first was the demonstration that the enhancer is
functional "in trans" on a linear DNA fragment (15). The
second was the availability of a convenient assay for testing the
orientation specificity of the enhancer by exploiting similarities and
differences between the transposition systems of Mu and the Mu-related
phage D108; these two systems share the same att site specificity but have different enhancer specificities (16). The latter studies found a
highly specific "cross-wise" interaction between the left O1 site
of the enhancer and the right R1 att site and between the right O2 site
of the enhancer and the left L1 att site (Fig. 1B). We have
used these two assays to explore the orientation specificity of both
the enhancer and the L/R ends in this study.
DNA supercoiling plays a critical role in recombination systems that
employ accessory enhancer sites to organize the recombination synapse
(e.g. Mu transposition, Hin/Gin inversion, Tn3/ The enhancer interacts with the L and R ends early in the Mu
transposition reaction to form an unstable nucleoprotein complex LER
(see Fig. 1A; not shown are three sets of individual
transposase-binding sites L1-L3, O1-O3, and R1-R3 within L,
enhancer, and R, respectively). Interactions within LER lead to
formation of a stable type 0 complex in which the Mu transposase (MuA
protein) assumes its active tetrameric form, catalyzing the cleavage
(type I complex) and joining (type II complex) reactions of
transposition (12). Only two subunits within the tetramer, those
located on L1 and R1, have thus far been implicated in catalysis on
supercoiled substrates (Fig. 1B; see Ref. 23). Similar
results have been reported using R1-R2 oligonucleotide substrates
under dimethyl sulfoxide (Me2SO) reaction conditions
(24). The catalytic "DDE" residues of these active subunits work in
trans (25, 26), i.e. DDE+ subunit at
L1 is responsible for cleavage and strand transfer of the opposite R
end whereas the DDE+ subunit at R1 is responsible for these
reactions at the L end (23). The specific function of the other two
subunits is not known (although they also interact with the enhancer).
We report that Mu ends maintain their orientation specificity as well
as their requirement for a supercoiled substrate in reactions with a
topologically unlinked enhancer as well as in those not dependent on
the enhancer. We compare the role of the enhancer in imposing
topological selectivity (defined as the specificity for a particular
orientation of topologically linked reactive sites) in other
recombination systems.
Transposition Substrates--
Plasmids pJMM and pJDD have been
described (16). pJHN (enhancer Linear Enhancer Fragments--
These were prepared by PCR
amplification of template DNA with appropriate primers. They are shown
in Table I. O1L (long) was designed to be
the same size as MM and contains nonenhancer DNA in place of O2. The
numbers in the last column are the starting and ending nucleotide
coordinates of the enhancer fragments encompassing the Mu and/or D108
genome.
Proteins--
Purification of MuA, MuA(E392A), MuA In Vitro Assays for Mu DNA Cleavage--
Strand cleavage (type I
complex) assays were done in 20-µl reaction volumes in 20 mM HEPES-KOH (pH 7.6), 140 mM NaCl, 10 mM MgCl2. Final Me2SO
concentrations were 15% when included. In reactions employing linear
enhancer DNA fragments the molar ratio of the donor plasmid DNA to
enhancer DNA was 1:50 and that of enhancer DNA to IHF protein was 1:1.6
(15). Besides the amounts of transposase proteins (0.1-0.4 µg), the
reaction contained 0.8 µg of donor mini-Mu DNA and 0.2 µg of
E. coli HU protein. Reaction mixtures were incubated at
30 °C for 20 min and analyzed by agarose gel electrophoresis.
DNA bands were excised from ethidium bromide-stained agarose gels as
described (16) and digested with either BamHI plus XbaI or with BamHI plus AatII. The DNA
fragments were labeled with [ The "cleavage-in-trans" rule (i.e. the
action of a catalytically active MuA monomer away from the end to which
it is bound) has provided the rationale for mapping the enhancer-att
interactions, whose functional relationships were deduced by assaying
how enhancers of Mu-D108 hybrid specificity respond to mixtures of MuA
and D108A proteins and their catalytically inactive DDE Linear Enhancer Substrates--
Both Mu and D108 enhancers are
composed of three transposase binding regions (O1-O3; see Ref. 16). The
polarity of these sites with respect to each other is not known (see
"Discussion"). Although the O3 site participates in optimal
enhancer function, the O1-O2 sites are sufficient for activity (10,
16). We have therefore used O1-O2 as the enhancer in the present study.
Linear DNA fragments encoding wild-type enhancers from Mu and D108 (MM and DD, respectively) as well as hybrid enhancers (MD and DM) were
generated by PCR amplification as described under "Materials and
Methods." The first and second letters in the enhancer name denote
the source of O1 and O2, respectively: M for Mu and D for D108. The
logic for the design of the hybrid enhancer substrates has been
described (16). The E. coli IHF protein is required for
function of linear Mu enhancer fragments (15).
Strand Cleavage in the Presence of D108-Mu Hybrid Enhancer (DM) in
Trans--
When the enhancer and att sites are all present on the same
plasmid, the O1 site specifies the occupancy of the transposase monomer
at R1, and the O2 site promotes placement of its cognate transposase at
L1 (Fig. 1B). The O1-R1 rule
is rigid; the O2-L1 rule is less so (16). These conclusions were
reached from strand cleavage assays using negatively supercoiled
plasmid substrates containing the hybrid enhancers DM and MD in
cis (16).
Reactions of an enhancer-less plasmid pJHN with the DM enhancer
provided in trans are shown in Fig.
2. Except where indicated, reactions
contained the IHF protein. Wild-type MuA did not yield the cleaved type
I complex (lane b), and the catalytically inactive variant
MuA(E392A) did not produce the uncleaved type 0 complex (lane
d). We know that MuA and its variants are not active on a DM
substrate in cis unless D108A is present (16). Wild-type D108A was only weakly active (lane c; the type I band is
barely visible). Interestingly, this activity was elevated when IHF was omitted from the reaction (lane a*). D108A(E392A) gave type
0 complex (lane h) and, as expected, no type I product. When
wild-type D108A was paired with MuA(E392A), the type I product and,
more prominently, the nicked circular product were formed (lanes
e-g). From previous work (16), we know that single cleavage at
the left end of Mu results in an unstable form of the type I complex. This product migrates as the open circle (OC) during electrophoresis due to relaxation of a substrate domain that is normally held supercoiled in the stable complex. The diffused migration of the OC
band suggests dissociation of MuA from the DNA, perhaps during electrophoresis. Further characterization of the cleavage product is
described below. In reactions that paired MuA with D108A(E392A), stable
type I complex was formed (lanes i-k). The strand cleavage position was mapped in this product as well. Unlike left end cleavage, single cleavage at the right end does not destabilize type I (16). Note
that these reactions also yielded the type 0 complex, its formation
being favored at higher molar ratios of D108A(E392A).
The reason for the higher activity of wild-type D108A in the absence of
IHF, rather than its presence, is not known (Fig. 2, compare
lanes a and c). All the other reactions in Fig.
2, including those containing D108A as one of the binary protein partners, were strictly dependent on IHF (data not shown). In our
previous work (16), we observed that D108A acted efficiently on a
negatively supercoiled substrate containing the DM enhancer in
cis under reaction conditions that did not require IHF. The hybrid enhancer may not have retained all the structural features of
the native enhancer, causing the odd behavior of D108A noted here. For
example, an intrinsic bend or flexibility within the DM enhancer could
be unfavorably modulated by IHF. Subtle differences between wild-type
and mutant proteins in their binding affinities for the enhancer and/or
att sites and in their intersubunit cooperativities could also come
into play. However, these factors do not diminish the significance of
the clear-cut selectivity in strand cleavage observed with specific
protein pairs (see below).
Orientation Specificity of the DM Enhancer Is the Same in Cis and
in Trans--
The scheme for mapping the cleavage positions in the
type I and OC products formed in Fig. 2 is diagrammed in Fig.
3A, and the results are shown
in Fig. 3B. The DNA samples extracted from the excised gel
bands were digested with BamHI and XbaI in one case and with BamHI and AatII in the other. The
3'-hydroxyl ends generated from the digestion as well as those produced
by cleavage of Mu ends were radioactively labeled with 32P
and fractionated by electrophoresis in denaturing polyacrylamide gels.
The diagnostic bands for left end cleavage are LC1 and LC2; those for
right end cleavage are RC1 and RC2 (see Ref. 16 for more details).
The results of the cleavage analysis from reactions in Fig. 2,
lanes f (containing equimolar D108A and MuA(E392A)) and
lane j (containing equimolar MuA and D108A(E392A)), are
displayed in Fig. 3B. The OC product from the f reaction was
cleaved almost exclusively at the left end (LC1 and LC2 in lane
2 and undetectable RC1 and RC2 in lane 6). The type I
product from the f reaction also showed predominant cleavage at the
left end (LC1 and LC2 in lane 3 and faint bands of RC1 and
RC2 in lane 7). By contrast, the type I product from the j
reaction shown in Fig. 2 (MuA/D108A(E392A)) showed nearly exclusive
cleavage at the right end (RC1 with undetectable LC1 and LC2 in
lane 4; RC1 and RC2 in lane 8).
The conclusions from the data in Fig. 2 and Fig. 3B are
summarized in Fig. 3C. The placement of the wild-type or
mutant MuA or D108A monomers is dictated by the trans rule
for DDE donation during MuA active site assembly; left end and right
end cleavages require the DDE-containing monomer to be positioned at
the R1 and L1 sites, respectively (23). The DM enhancer strictly
specifies the occupancy of D108A at R1, either wild-type (Fig.
3C, left) or the DDE mutant (Fig. 3C, right).
Left end cleavage is promoted in the former case, whereas it is blocked
in the latter. The DM enhancer also promotes the occupancy of MuA (over
D108A) at the L1 end, but the specificity is less rigorous. L1 is
primarily occupied by MuA(E392A) when paired with D108A (Fig.
3B; the preponderance of cleavage at the left over the right
end in lanes 2 and 3 and 6 and
7 corresponding to reaction f in Fig. 2) and incorporates wild-type MuA when paired with D108A(E392A) (right end cleavage in
lanes 4 and 8 in Fig. 3B corresponding
to reaction j in Fig. 2). However, the presence of D108A(E392A) at L1
in a fraction of the molecules is denoted by the formation of the type
0 complex (for example, reaction j in Fig. 2). Similarly, D108A is not
totally excluded from L1, as indicated by the faint cleavage observed at the right end (lane 7 in Fig. 3B corresponding
to reaction f in Fig. 2).
In summary, our results with the DM enhancer in trans are
essentially identical to those obtained by Jiang and co-workers (16)
for the same enhancer in cis. Together, these findings ascertain that the distribution of the transposase subunits at the Mu
ends is determined by their cross-wise specificities for the enhancer
elements. The left O1 region of the enhancer specifies the right R1
site; and to a lesser degree, the right O2 region specifies L1. This
rule for the distribution of transposase molecules is the same
regardless of whether the enhancer is present in cis or in
trans.
We have carried out assays similar to those in Fig. 2 with the same
substrate plasmid pJHN and the MD enhancer supplied in trans. The efficiency of the MD reactions was very poor.
Orientation Specificity of Natural Enhancers (MM and DD) Is Also
the Same in Cis and in Trans--
The action of the native enhancers
MM or DD in trans on the plasmid pJHN was assayed in
presence of IHF, and the points of strand cleavage were mapped (Fig.
4). A control set of reactions was done
with plasmid pJMM containing MM in cis (Fig. 4A)
and plasmid pJDD containing DD in cis (data not shown). The
cis reactions used standard conditions for supercoiled
substrates that did not include IHF.
Wild-type MuA was active and D108A was inactive in the type I reaction
with the MM enhancer in cis (Fig. 4A, lanes a and
b) or in trans (lanes d and
e). An equimolar mixture of D108A and MuA(E392A) yielded
both type I and type 0 products (lanes c and f).
The type 0 complex would be expected to contain MuA(E392A) monomers at
R1 and L1. The results were similar for the DD enhancer in
trans (lanes g-i). Type I reaction was obtained
with D108A (lane g) but not MuA (lane
h), and both type I and type 0 were obtained with a mixture
of MuA and D108A(E392A) (lane i). The reaction profile was
the same for the plasmid containing DD in cis (not shown).
The type I complex from the wild-type reactions (Fig. 4B, lanes
a, d, and g) contained both left and right end
cleavages as expected (LC1, LC2, and RC1 in lanes 2, 5, and
8; RC1, RC2, and LC1 in lanes 11, 14, and
17). On the other hand, type I from the mixed reactions
(lanes c, f, and i) harbored only right end
cleavages (RC1 alone in lanes 3, 6, and 9; RC1
and RC2 alone in lanes 12, 15, and 18). These
results are in accordance with the strict O1-R1 rule and the less
stringent O2-L1 rule. With the MM enhancer, MuA(E392A) would occupy R1,
thereby negating left end cleavage. When MuA(E392A) occupies L1 as
well, neither end can be cleaved, yielding the type 0 complex. When
D108A occupies L1, right end cleavage can occur. These arguments apply
to the DD enhancer as well.
Table II summarizes the activities of
various cis and trans arrangements of the Mu
enhancer (see "Materials and Methods: for details). Some of these
experiments have been previously reported (10, 15), but we present them
here for the sake of completeness. The negatively supercoiled pJMM
plasmid (harboring MM in cis) was active in the type I
reaction with or without added IHF and provides a reference for the
other reactions. The enhancer-less pJHN could be complemented in
trans by MM in presence of IHF. No complementation was
observed with O1 alone, O2 alone, O1 plus O2 supplied as separate DNA
fragments and by O1L (containing O1 + IHF-binding site + nonspecific
DNA) even at a 50-fold molar excess over the att sites (see also Ref.
15). An inactive derivative of pJMM lacking O1 (pJH28) could be
partially rescued by O1-O2 (MM) in trans but not by O1 alone
or by O1L. In a 30-min period, a plasmid deleted for O2 alone
(pJH27) retained ~60% of wild-type pJMM activity, which saturates
more rapidly (see also Ref. 10). By comparison, the corresponding
relative activity at 5' was only 20%. The pJHN reactions with the MM
enhancer in trans also displayed similar slow kinetics (data
not shown; see Ref. 15). Addition of MM in trans in the
presence or absence of IHF did not improve the reactivity of pJH27.
Deletion of the IHF-binding site in addition to O2 (but leaving O1
intact) did not change the activity of the resulting substrate
(pJH29).
We noticed that addition of IHF (in amounts used for the enhancer in
trans experiments) inhibits the intrinsic activity of plasmid pJH27 that lacks O2 (but retains O1 and the adjacent IHF site)
(Table II). The inhibition was relieved with added MM, probably by
titration of IHF. Note that IHF had no inhibitory effect on pJMM that
contains both O1 and O2. Some of the unexpected results with IHF might
be due to its architectural effects on DNA in a context-dependent manner. Recall that IHF also inhibited
the activity of the DM enhancer in trans in the type I
reaction with pJHN (Fig. 2A, lane a versus
c).
The sum of the data in Figs. 2-4 and Table II corroborate and extend
the rules for enhancer-att interactions previously deduced (16). Our
current results demonstrate that both hybrid and natural enhancers obey
these rules in cis and in trans reactions.
Consistent with its relaxed specificity, O2 is not essential for
activity in cis. In pJH27 and pJH29 lacking O2, the normal
L1 preference for MuA over D108A in mixed reactions is lost, and the
placement of protein monomers on L1 become completely randomized
(results not shown).
A Normal Orientation of Mu Ends as Well as DNA Supercoiling Is
Required When the Enhancer Is Topologically Unlinked or Is Completely
Bypassed--
Since the organization of the transposition structure
requires at least a transient three-site complex (LER), their precise alignment in a circular supercoiled molecule would be subject to large
entropic constraints. We have tested whether the topological and
geometric requirements of the reaction may be relaxed by unlinking the
enhancer from the att sites in two ways. First, we provided the
enhancer in trans to plasmid substrates containing the attL and attR sites in direct and indirect orientation. We note that whereas
Surette and Chaconas (15) have reported that the enhancer does not
function in trans when the ends are inverted, their
experiments were conducted on the relaxed and linear form but not the
supercoiled form of the inverted substrate; we now know that DNA
supercoiling is essential for both pre- and post-synaptic events in
transposition under normal reaction conditions (20-22). Second, we
assayed the activity of an enhancer-independent MuA variant (MuA
Type I complex was formed from pJHN with MuA and the MM enhancer in
trans (Fig. 5, lane 1) and with MuA Loss of Topological Specificity with Me2SO Cannot Be
Due to Enhancer Independence Alone--
It has long been known that
addition of 15% Me2SO to the transposition reaction
results in enhancer independence and abrogates the need for circularity
or supercoiling of the substrates (2, 9). The plasmids pJHN, pJH25, and
pJH26 (see Fig. 5) were reacted with either MuA or MuA
We note that cleaved inverted end complexes are unstable in the absence
of the enhancer, giving rise to material at the OC position
(pJH25, lanes 5 and 6). The absence of
OC products in the presence of the enhancer in complexes formed with
MuA but not MuA
In attempts to mimic the Me2SO effect under the standard
Me2SO-free reaction condition, we carried out MuA/pJH26 and
MuA In this study, we have examined whether the topological
selectivity of Mu transposition might arise from the restrictive nature of aligning three DNA sites (attL, attR, and the enhancer) for productive synapsis on supercoiled DNA. We have found that unlinking the enhancer from the att sites or carrying out the reaction in an
enhancer-independent manner fails to override the requirement for
supercoiling or for the inverted orientation of the att sites. We have
also found that the cross-wise interactions observed between the
enhancer and att sites on supercoiled DNA are maintained when the
enhancer is provided in trans. We discuss these results in detail below.
Functional Asymmetry of the Mu Enhancer Is Independent of Its
Linkage to the Att Sites
In a supercoiled plasmid, the enhancer cannot be transferred
outside the Mu domain or reversed in orientation inside it without markedly affecting transposition, signifying a defined order and direction of linkage among attL, attR, and the enhancer (8-10). An
important outcome from our present study is the demonstration that the
O1-R1 and O2-L1 relations are maintained in both the cis and
trans configurations of the enhancer (Figs. 2 and 3), arguing for an inherent functional asymmetry of the enhancer, independent of its topological linkage to Mu ends.
What is the significance of the functional asymmetry of the enhancer?
The enhancer DNA segment is relatively large. The O1-O2 region is
~150 bp in Mu and 100 bp in D108 (see Ref. 16). Consensus sequences
of 14 and 8 bp have been identified as potential transposase-binding sites in the Mu and D108 enhancer regions, respectively (19, 27). Since
these sequences appear repeatedly and in both orientations, the
organization of the enhancer per se does not suggest an
obvious functional polarity to it. A single IHF-binding site present
between O1 and O2 could, in principle, introduce asymmetry when bound by IHF. However, IHF is not an obligatory transposition factor and is
required only when the supercoiling density of the substrate is well
below Mu Enhancer Function Requires O1 to Be in Cis with O2
We have shown in this study that O1 is indispensable in
cis or in trans (Table II). O2 can be substituted
with nonspecific DNA, at least in supercoiled substrates, with
retention of greater than half the normal activity (see also Ref. 10).
However, lack of O2 results in slower kinetics of the type I reaction
and a loss of the O2-specified preferential placement of transposase subunits at L1. Although the enhancer can be unlinked from the att
sites as the O1-O2 entity, attempts to unlink O1 and O2 have not been
successful. The functional relevance of the physical coupling between
O1 and O2 is supported by a number of observations (see Table II). It
is significant that a plasmid lacking the Mu enhancer (pJHN) is
restored to near normal activity by MM supplied in trans,
whereas MM in trans has only a weak effect on a plasmid lacking O1 but retaining O2 (pJH28). So, the modest activity of a
plasmid missing O2 but containing O1 is not improved by MM in trans. Thus, the presence of either half of the functional
enhancer in cis effectively blocks the effects of the full
enhancer in trans. These results are indicative of strong
cooperativity between O1 and O2, disruption of which results in loss of
enhancer function.
Orientation Specificity of the Mu Ends Is Independent of the
Enhancer, A Common Role for Enhancers in Site-specific
Recombination?
Our finding that the requirement for an inverted orientation of Mu
ends is not alleviated by unlinking the enhancer or by eliminating it
entirely through use of an enhancer-independent variant (Fig. 5,
MuA We discuss below the plausible contribution of the Mu enhancer to
plectosome assembly by considering the properties of
enhancer-independent reactions in other site-specific DNA recombination systems.
The Hin/Gin System--
In contrast to the Mu enhancer, the
enhancers in the Hin/Gin invertase systems function in either
orientation. This is likely due to the 2-fold symmetry present in the
enhancers (bound by Fis) as well as in their recombination target sites
hix/gix (see Ref. 17). However, as with Mu, supercoiling in the Gin
system is essential only at the gix site and not at the Fis site (38). Point mutations that confer enhancer independence relieve topological constraints, permitting the mutant recombinases to carry out the normally prohibited deletion reaction and intermolecular transposition (5, 6). Although wild-type Gin (and Hin) can form synaptic complexes
with both orientation of sites (14, 39), the mutant reaction allows
processive recombination and hence loss of the normal discrimination
that disallows joining at directly oriented sites after 180° rotation
due to the mispaired core sequence. Unlike the wild-type Gin reaction,
which occurs exclusively from a synapse with two negative supercoil
nodes, the mutant reaction occurs from a broad spectrum of synapses
yielding topologically complex products (40). The ability of an
enhancer-independent Gin to induce local DNA unwinding and effect
strand cleavage at a gix site in the absence of Fis, DNA supercoiling,
or synapsis with a second gix site, suggests that the role of the
enhancer is to normally promote Gin-Gin contacts to effect DNA
unwinding at the gix sites (41). Thus, the role of the enhancer in
these systems is to organize a synapse with a unique topology,
promoting protein-protein interactions that facilitate DNA unwinding at the recombination sites, assisted by the energetics of DNA supercoiling.
The Resolvase System--
The complex organization of the Mu att
sites and the functional asymmetry of the Mu enhancer are perhaps more
similar to the Tn3/
Point mutations of Tn3 resolvase have been obtained that allow
recombination between sites I, independent of sites II and III (4).
Like in the Gin system, these mutant proteins promote recombination
between normally disallowed configuration of res sites. Product
topologies indicate that, in contrast to the The Mu System--
DNA supercoiling has varied pre- and
post-synaptic roles in Mu transposition (see Introduction), some
similar to those found in the invertase and resolvase systems. With
MuA, two types of enhancer independence have been revealed. The
Me2SO-mediated enhancer independence (see Ref. 9 and under
"Results" for this study) results in concomitant independence from
the topological and directional restrictions, similar to that seen in
the invertase and resolvase systems. Because of its amphipathic
properties, Me2SO can influence both DNA and protein
conformations. By analogy with the Gin system, Me2SO might
promote functional MuA-MuA contacts and/or induce DNA distortion at the
Mu ends, which is normally facilitated by negative supercoiling (22).
It has been reported that even a single att site is sufficient to
promote formation of the MuA tetramer under Me2SO
conditions (45). There is also evidence that, in presence of
Me2SO, strand transfer can occur from nonstandard alignment
of linear att sites (see Refs. 23 and 46). Since a defined topology to
the normal Mu transposition complex can now be experimentally
deciphered,3 it should be
possible to test whether the Me2SO effects include the
disordering of a unique synapse into a random set of synapses, like
that observed for the mutant invertases and resolvases.
We have shown in this study that enhancer independence conferred by an
N-terminal deletion of MuA (MuA
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
integration/excision, phage P1 Cre/lox recombination, yeast
2-µm plasmid Flp/FRT recombination), other systems have a
strict requirement for just one orientation (e.g. Mu
transposition, Tn3/
resolvase reactions, Hin/Gin inversion reactions) (see Ref. 1). A hallmark of the latter systems is the
employment of enhancers (or accessory DNA sites) and the requirement for negative DNA supercoiling. Either one or both of these elements (enhancers and DNA supercoiling) could contribute to the orientation specificity of the recombination sites in these systems. For the Mu
transposition system, the role of supercoiling was explored by Craigie
and Mizuuchi (2) prior to the discovery of the enhancer. Transposition
of the L and R ends of Mu present in two different orientations on
catenated or knotted supercoiled DNA substrates was monitored. The
pattern of reactivity of Mu ends on these novel substrates led the
authors to suggest that the normally inverted L and R ends might
juxtapose most easily in an "intertwined" parallel configuration on
negatively supercoiled DNA; for directly oriented sites an energy
barrier must be overcome to bring them together in this parallel
configuration. Thus, DNA topology was responsible for sensing the
relative orientation of the Mu ends. Experiments with Tn3 resolvase
were consistent with the Mu results in suggesting the existence of a
"topological filter" (defined as a combined effect of constraints
derived from both the circularity of the DNA substrate and the
requirement for a highly specific interwrapping of the recombining
sites; see Ref. 1) that limits productive interaction to a particular
orientation of sites (3). Recent experiments with Tn3 resolvase have
implicated interwrapping at the enhancer in determining the topological
selectivity of the reaction (4); enhancer-independent resolvase mutants
simultaneously lost their normal specificity for directly oriented
sites and for supercoiled substrates. Similar results have been
reported with the Gin invertase family (5, 6). Since the Mu experiments implicating DNA topology in determining the orientation specificity of
the ends (2) were done before the discovery of the enhancer, one
objective of the present study was to dissect the contribution of the
enhancer, if any, to the topological filter. These studies were
facilitated by the existence of an enhancer-independent variant of the
Mu transposase (7).
resolution; see Refs. 12, 17, and 18). Varied roles for supercoiling have been deduced in these systems (see "Discussion"). In the Mu
transposition system supercoiling is not essential at the enhancer, which can function in trans on a linear DNA fragment when
the L/R ends are present on supercoiled DNA (15). These results do not
rule out a role for supercoiling at the enhancer when it is present in
cis. It is clear that a cis location increases
the effective concentration of the enhancer, since a 50-fold molar excess is required for function when the enhancer is supplied in
trans in the presence of Escherichia coli protein
IHF (15). A requirement for the DNA bending protein IHF (which
binds between the O1 and O2 sites of the enhancer; see Ref. 19) on
linear enhancer DNA fragments, or on circular DNA with low superhelical densities (10), suggests that supercoiling may also favor DNA bending at the enhancer. In addition, supercoiling has been shown to be
important for binding HU protein at the L end (20) as well as for a
critical post-synaptic event (21, 22).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) was created by deleting a
SalI-EcoRI fragment encoding the enhancer from
pJMM. pJH25 (enhancer
, inverted attL) was created by
inverting a 233-bp1
XbaI-BglII fragment encompassing attL in pJHN.
pJH26 (enhancer+, inverted attL) was constructed in a
manner similar to pJH25, except that pJMM was used as the substrate.
pJH27 (O2
) was constructed by first incorporating an
EcoRI site at nt 964 (between IHF and O2 sites) in a PCR
primer used to amplify the O1-IHF region on pJMM. A
BglII-EcoRI digest of the PCR product was
exchanged into similar sites on pJMM, resulting in deletion of O2.
pJH28 (O1
) was constructed in a manner similar to pJH27,
except by introducing a SalI site at nt 938 (between O1 and
IHF sites) into the PCR primer used for amplifying the IHF-O2 region,
followed by exchange of the SalI-EcoRI-digested
PCR product into these sites on pJMM. pJH29 (IHF
O2
) was constructed similarly, by introducing the
EcoRI site at nt 943 between O1 and IHF sites.
Linear enhancer fragments derived by PCR amplification
84, D108A,
D108(E392A), and HU proteins has been described (7). IHF was a generous gift from Steve Goodman, University of Southern California.
-32P]cordycepin phosphate
using terminal nucleotidyltransferase, electrophoresed on 6%
denaturing polyacrylamide gels, and detected by autoradiography as
described (16).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
variants (see Ref. 16; these two transposases bind to each other's att
sites but are highly specific for their cognate enhancers). We have now
employed the same system for testing the orientation specificity of an
enhancer when supplied on a linear fragment (in trans) to
attL and attR ends present on supercoiled DNA. These studies will be
presented first, followed by those employing a variant transposase,
which functions in the absence of the enhancer.
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Fig. 1.
Nucleoprotein complexes in Mu
transposition. A, monomeric MuA protein binds to the
two Mu ends L and R (each composed of three att sites), as well as
interacts with an enhancer element E on a negatively supercoiled
plasmid, to promote rapid formation of the LER complex in the presence
of divalent metal ions and E. coli HU protein.
Ca2+ ions support conversion of LER to type 0, in which MuA
has tetramerized and the enhancer is no longer associated with the
ends. Mg2+ or Mn2+ ions also support formation
of type 0, promoting cleavage of the synapsed Mu ends to produce the
type I complex. MuB protein modulates the activity of MuA at each stage
of the reaction and captures target DNA in the presence of ATP to
generate the type II strand transfer complex (12). B, a
model for the arrangement of catalytic MuA subunits within LER.
Subunits bound through their DNA-binding domains to L1 and R1 interact
cross-wise with O1 and O2 regions of the enhancer. The central domain
(containing catalytic DDE residues) of each subunit acts in
trans to cleave (white dot) and subsequently
strand transfer (not shown) specific phosphodiester bonds at the two Mu
ends. There are two other MuA subunits in the tetramer, not shown
because specific structural/catalytic functions have not yet been
assigned to them.
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Fig. 2.
Reaction of MuA, D108A, and their variants
with enhancer-less plasmid pJHN and the DM hybrid enhancer supplied in
trans. The DM substrate was incubated with
mixtures of indicated amounts (in µg) of wild-type MuA or D108A
proteins and their DDE variants and assayed for type I or
type 0 complex formation. Except for lane a* all reactions
included IHF. The supercoiled (SC), open circular
(OC), and linear (L) forms of the plasmid are
indicated. Intramolecular strand transfer complexes migrate slightly
ahead of the type I complex (see lane g).
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Fig. 3.
Analysis of cleaved ends in pJHN when DM is
supplied in trans. A, cleavage at Mu
ends yield free 3'-hydroxyl groups, hence a 3' end labeling strategy
(described by Jiang and co-workers (16)) was used to assay left or
right end cleavages in the type I complex. The top and bottom strands
of the Mu genome are represented by unfilled and
filled bars, respectively. The diamonds indicate
the strand cleavage positions. Double digestion with
BamHI-XbaI or BamHI-AatII,
followed by 3' end labeling (indicated by the asterisk)
would give rise to the indicated radioactive products. They can be
revealed by electrophoresis in denaturing polyacrylamide gels (see
B). Uncleaved attL generates an LU doublet consisting of a
95-nt fragment from the bottom strand, which has the same length as the
fragment from the top strand; uncleaved attR generates RU1 from the top
strand, which differs in length by 8 nt from the corresponding bottom
strand fragment RU2. The products specific to left and right end
cleavages are denoted by LC and RC, respectively. (U and
C indicate uncleaved and cleaved fragments, respectively.)
B, the reactions in lanes f and j of
Fig. 2 were analyzed by the strategy outlined in A. Lanes 1 and 5 represent the substrate DNA that
was not treated with MuA or D108A proteins.
XbaI-BamHI and BamHI-AatII
restriction digestions are indicated. The symbols OC and
I correspond to the isolated open circular product and the
type I complex, respectively. DM denotes the contaminating
linear enhancer fragment that is found distributed throughout the lanes
in Fig. 2 likely due to its high concentration. C, deduced
position of transposase subunits at L1 and R1. DDE = DDE+ subunit; X = DDE
subunit. Gray oval = D108A or its variant; white
oval = MuA or its variant. The disposition of all six att
sites (L1-L3 and R1-R3) is shown. The occupancy of L1 by either of
the two transposases is represented by the half-white/half-gray
oval. The two subunits in the tetramer whose enhancer specificity
could not be addressed in these experiments are indicated as
dotted ovals. Gray and white O1 and O2
sites represent D and M enhancers, respectively.
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Fig. 4.
Reactions of the natural enhancer substrates
MM and DD with MuA, D108A, and their variants both in cis
and in trans. A and
B, as in Fig. 2 and Fig. 3B, respectively, except
with the MM and DD substrates. Position of contaminating linear
enhancer fragments from lanes in A is indicated as MM and DD
in B.
Activity of cis and trans configurations of complete and partial
enhancers
84;
Ref. 7) on these plasmids. The results are shown in Fig.
5. Among the three test plasmids, pJHN is
the control in which the att sites are in their correct orientation. In
pJH25 and pJH26, the sites are in a head to tail arrangement. Only
pJH26 contains the enhancer in cis.
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Fig. 5.
Contribution of the enhancer to topological
selectivity of the Mu ends. Disposition of att (L and
R) and enhancer (E) sites on plasmid substrates
is indicated on the top panel. Reactivity of these plasmids
in the presence of wild-type MuA with MM enhancer in trans
(lanes 1 and 3) or in cis (lane
6) or MuA 84 in the absence of added enhancer (lanes
2 and 4) or presence of enhancer in cis
(lane 7) is shown in the bottom panel. Lane
5 is a control with IHF alone. Other symbols as in Fig. 2.
84 in the
absence of MM (lane 2). When the pJHN substrate was nicked
or linearized, no cleavage at the left or right Mu end was detected in
the MuA plus MM (also reported in Ref. 15) or MuA
84 reactions (data not shown). Neither pJH25 nor pJH26 was reactive under any of the
conditions tested (lanes 3-7). Thus, the correct
orientation of Mu ends and DNA supercoiling are essential even when the
enhancer is unlinked from the att sites or when the reaction is
enhancer-independent.
84 under
Me2SO conditions and were analyzed directly (Fig.
6,
SDS) or after
deproteinizing (Fig. 6, +SDS). All three plasmids, including
the relaxed form of pJHN, were reactive with both MuA and MuA
84.
Under these conditions, the type I reaction proceeded nearly
quantitatively to type II, resulting in multiple intramolecular strand
transfer product bands. An exception was the MuA
84 reaction with
pJH26. In this case, the predominant product was the cleaved type I
complex, and the type II product bands were weak (lane 9).
After protein removal (+SDS, lanes 13-20), the
type II strand transfer products from pJHN migrated below the
supercoiled plasmid band (lanes 13 and 14; ST),
whereas those from pJH25, pJH26, and relaxed pJHN migrated close to the
open circular form (lanes 15-20; ST). This difference in
topology is expected. In pJHN, with the correct orientation of att
sites, only the vector domain will be relaxed following cleavage of Mu ends. The supercoils present within the Mu domain of the plasmid will
therefore be trapped in the strand transfer products. In pJH25 and
pJH26, with incorrectly oriented att sites, the strand nicks will be
located in both plasmid domains. The plasmid would thus be relaxed
fully prior to strand transfer.
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Fig. 6.
Loss of topological selectivity in
Me2SO. Reactivity of plasmids shown in Fig. 5 to
wild-type MuA or to MuA 84 under Me2SO reaction
conditions (see "Materials and Methods"). Lanes 1, 4, 7, and 10 are controls with no transposase protein added. The
relaxed form (R) of pJHN was generated by topoisomerase I
treatment. Samples were electrophoresed in the absence (
) or presence
(+) of SDS. Type II strand transfer complexes or ST strand transfer
products are indicated. Other symbols as in Fig. 2.
84 (pJH26, lanes 8 and
9) suggests that the enhancer stabilizes inverted end
complexes in Me2SO conditions, allowing them to mature into
strand transfer products.
84/pJH25 reactions under combinations of different ionic
strengths (100-200 mM NaCl or KCl; 4-16 mM
Mg2+) and varying superhelical plasmid densities (
of
0.015 to
0.075). Under none of these conditions did we observe any
activity (data not shown). In summary, comparison of data presented in
Figs. 5 and 6 shows that the breakdown of topological and geometric restrictions in the presence of Me2SO cannot be due to
enhancer independence alone.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
0.06 (10). Our results suggest that the functional asymmetry of
the enhancer is a reflection of its physical asymmetry and that the
specific interacting elements within the large O1-O2 regions must be
positioned to match the asymmetry of the ends with which they interact.
It is known that two left ends or two right ends are inefficient in
transposition compared with the normal attL/attR configuration (9). We
have also determined that substitution of a single att site L1 for R1
does not alter the O1-right/O2-left
interactions.2 Thus, not only
are interactions with the enhancer unique at each end, but they also
involve more than one att site, consistent with the network of
end-enhancer interactions deduced from genetic studies (28) and from
the observation that efficient transpososome assembly requires that all
four subunits in the MuA tetramer carry the enhancer-binding domain
(29). The unique end-enhancer interactions are likely designed to build
the MuA tetramer along specific protein interfaces, thus precisely
regulating assembly of the active transpososome.
84) suggests that the enhancer is not the primary determinant of the orientation specificity of Mu ends on
supercoiled substrates. This finding is somewhat surprising in view of
the fact that systems requiring only two recombination target sites
enjoy greater topological freedom than those that require a third site
(30-33). This is because a three-site synapse in a circular,
supercoiled substrate is thought to occur by DNA slithering or
branching, rather than by random collision (34-37), and can be highly
restrictive with respect to the permissible orientation of the
interacting sites. Although our results suggest that the orientation
specificity of the Mu system is a property of the two ends themselves,
they do not directly address its mechanism. The asymmetric orientation
of the multiple att sites at the two ends, combined with the trans
activity of the transposase, are likely to favor stable transpososome
assembly only within a unique synapse on negatively supercoiled DNA, an
idea compatible with the original proposal by Craigie and Mizuuchi (2)
of a special "plectosome" structure in which two double-stranded
DNA segments are associated with a specific superhelical geometry.
resolvase system whose recombination target
site "res" is a composite of three sites, I, II, and III, all of
which bind resolvase dimers (18). Only the dimer at site I is catalytic and acts as the recombinase. Those bound at sites II and III mediate the enhancer effect and promote correct alignment of sites I (42, 43).
As in the Mu and invertase systems, supercoiling has two distinct roles
here, both pre- and post-synaptic. Either supercoiling or catenation of
res-containing circles in the absence of supercoiling supports
synapsis. Supercoiling is also required after synapsis since relaxed
synaptic complexes or relaxed catenanes cannot proceed through
recombination (44).
3 wild-type synapse,
site alignment during the mutant reaction occurs by random collision.
Thus, in both the Hin and the resolvase systems, the enhancer appears
to play a similar role in organizing a unique synapse.
84; see Ref. 7), unlike the
Me2SO case and contrary to the invertase/resolvase examples, does not relieve the dependence on DNA supercoiling and on
the correct orientation of Mu ends. Do the contrasting properties of
MuA(
84) with those of the enhancer-independent mutants of Gin (or
Cin) and Tn3 resolvase suggest functionally distinct roles for the
enhancer in the transposition and recombination reactions? Not
necessarily. It is plausible that the topological and geometric
features of a system are intrinsic to the target site and the cognate
protein (the transposase or recombinase) of that system (for example,
asymmetry of the Mu att sites and the trans activity of MuA)
and are independent of the enhancer. The apparent role of the enhancer
in each system is to use DNA supercoiling to facilitate precise
assembly of the synaptic complex and/or promote the chemical competence
of the corresponding DNA-protein complex. Although a potential
implication of this model is that conditions that overcome
topological/orientation specificities would lead to enhancer
independence, the converse is not necessarily true, as demonstrated
here for the MuA reaction. Available evidence from the transposition
and recombination systems poses no contradiction.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Steve Goodman for IHF and M. Jayaram and Shailja Pathania for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM33247 and in part by Robert F. Welch Foundation Grant F-1531.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 512-471-6881;
Fax: 512-471-7088; E-mail: rasika@uts.cc.utexas.edu.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M008523200
2 H. Jiang and R. M. Harshey, unpublished results.
3 S. Pathania, M. Jayaram, and R. M. Harshey, unpublished results.
![]() |
ABBREVIATIONS |
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The abbreviations used are: bp, base pair; nt, nucleotide; PCR, polymerase chain reaction; OC, open circle.
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