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INTRODUCTION |
Integrase catalyzes two consecutive transesterification reactions
during its in vivo function (1, 2). In the "processing" reaction, the reverse transcriptase-generated DNA copy of the viral
genome is trimmed by the endonucleolytic removal of the 3'-dinucleotides from its ends. The two processed 3'-ends are then
inserted into opposing strands of the host DNA in the "joining" reaction via a concerted cleavage-ligation reaction (3-6). Purified integrase catalyzes both reactions on synthetic oligodeoxynucleotide substrates containing viral DNA end sequences in the presence of either
Mn2+ or Mg2+ as a cofactor (7-10). In
vitro, integrase also catalyzes the apparent reversal of the
joining reaction, the "disintegration" activity, on Y-shaped
oligodeoxynucleotide substrates (11) as illustrated in Fig. 1. These
Y-shaped substrates resemble products of integrase-catalyzed joining
and contain a nick immediately 5' of the joining site. The
disintegration reaction effectively reverses joining by resealing the
nick while concurrently displacing the inserted viral sequence. This
reaction is routinely used to assay integrase in vitro (8,
12-14).
Numerous structures of integrase catalytic core-containing fragments
determined from a variety of retroviral sources have all been dimeric
(15-21). However, the two active sites of the subunits in these
structures are outwardly oriented on opposite sides of the
crystallographic dimers, too far apart (>50 Å) to be spanned by the
requisite 5-6 bp stagger separating the two sites of concerted
integration on the host DNA (20). Although several tetrameric models
have been hypothesized based on comparisons with the structure of the
homologous bacterial Tn5 transposase (20-22), the structure for
neither a full-length integrase nor an integrase-DNA complex has been
solved, and the quaternary structure of the catalytically active
integrase enzyme remains unknown.
We have previously elucidated some mechanistic aspects of substrate
specificity for the processing reaction of the avian sarcoma virus
(ASV)1 integrase by
presteady-state kinetics (9, 10); however, we were unable to determine
the reaction stoichiometry using a synapsed processing substrate due to
substrate-induced aggregation, a problem common to retroviral
integrases (23). Here, we report the use of a presteady-state
disintegration assay that mitigated these aggregation problems,
allowing us to determine the reaction stoichiometry by active site
titrations. Volumetric analysis of integrase particles imaged by atomic
force microscopy during the first turnover additionally establishes the
quaternary structure of the functional unit. Our results will be
discussed in the context of the hypothesized tetramer models.
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EXPERIMENTAL PROCEDURES |
DNA and Proteins--
Oligodeoxyribonucleotides were synthesized
by the Center for Gene Research and Biotechnology Central Services
Laboratory (Oregon State University). Purification by denaturing PAGE,
spectrophotometric determination of concentrations, and
5'-radiolabeling were as described previously (9). Integrase was
overexpressed in Escherichia coli BL21(DE3), purified as
described (9), and stored at
80 °C in 50 mM HEPES, pH
7.5, 500 mM NaCl, and 40% glycerol.
Presteady-state Assays and Product Analysis by Denaturing
Acrylamide Gel Electrophoresis--
Standard reactions were carried
out at 37 °C in 20 mM Tris, pH 8.0, 10 mM
Na-HEPES, pH 7.5, 4% glycerol, 10 mM 2-mercaptoethanol, 0.050 mg ml
1 acetylated bovine serum albumin, 250 or 400 mM NaCl, and with or without 5 mM
MnCl2. Preincubations were carried out in the absence of
MnCl2 for 30 min and the reaction subsequently initiated by
the addition of 37 °C MnCl2 to 5 mM. A
complete range of preincubation times and temperatures were tested to
ensure that equilibrium had been achieved. Gel analysis, image
quantitation, and non-linear least squares fittings were performed
according to Bao et al. (9, 10).
Imaging by Atomic Force Microscopy (AFM)--
Imaging was
performed with a Nanoscope IIIa instrument (Digital Instrument, Santa
Barbara, CA) using the tapping mode in air. Nanosensor
Pointprobe non-contact/tapping mode sensors with a nominal
spring constant of 48 newton/m and resonance frequencies of 190 kHz
were used for all images. The protein and DNA molecules were deposited
onto freshly cleaved mica (Spruce Pine Mica Co., Spruce Pine, NC),
immediately washed with deionized distilled water, and dried with a
stream of N2 (gas). Depositions of disintegration reactions
during the first catalytic turnover were obtained by preincubating 4 µM integrase with 5 µM substrate DNA in 20 mM Tris, pH 8.0, 10 mM HEPES, pH 7.5, and 250 mM NaCl for a minimum of 30 min on ice, diluting the
reaction to 64 nM integrase and 150 mM NaCl
immediately prior to initiating the reaction by the addition of
MnCl2 to 5 mM. Aliquots were then deposited
within 10 s of initiating the reaction. All images were collected
with a scan rate of 3.2 Hz, at 512 × 512 resolution, and a scan
size of 1 µm2. Volume analysis of AFM data was performed
with the freeware program ImageSXM (based on NIH Image developed
at the National Institutes of Health) using image plane fitting, image
analysis, volume calculations, and conversion to molecular weights
according to MWapp = (volume x nm
3 + 25)
1.31 kDa
1 as described (24, 25).
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RESULTS AND DISCUSSION |
Single-turnover Disintegration Assays--
For single-turnover
experiments, the substrate was 5'-radiolabeled on the 19-nt target
fragment, denoted by the asterisk in Fig.
1, which is converted to a 44-nt product
during disintegration (inset, Fig.
2). Substrate (0.25 µM) was
preincubated with excess ASV integrase (1 µM monomers)
for 30 min in the absence of Mn2+ to allow equilibrium
formation of productive complexes (9, 10). Reactions were initiated by
the addition of Mn2+ to 5 mM. Fig. 2
(closed circles) shows biphasic conversion of the
radiolabeled 19-nt substrate to the expected 44-nt product. Non-linear
least squares fit of the data to a double-exponential function yielded
observed rate constants of 0.05 and 0.002 s
1 with
respective amplitudes of 17 and 60%. When the reaction was initiated
by mixing integrase and substrate without preincubation but in the
presence of Mn2+, we observed a single 0.002 s
1 exponential phase (Fig. 2, open circles)
with an amplitude (75 ± 0.9%) that was, within error, equal to
the sum of the amplitudes (77 ± 1.4%) observed with
preincubation. The fast phase reflected 17% productive
integrase-substrate complexes formed during the preincubation period in
the absence of Mn2+ that was rapidly converted to product
at 0.05 s
1 upon addition of the metal cofactor. In
contrast, the 0.002 s
1 phase observed in both experiments
reflected slower assembly of productive complexes in the presence of
Mn2+. The significant amplitude of the slower exponential
phase in the preincubated experiment, therefore, indicated that 60%
more productive complexes were formed following the addition of the metal cofactor, suggesting that the formation of these complexes is
facilitated by the presence of the metal cofactor (26).

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Fig. 1.
Schematic depicts integrase-catalyzed
disintegration and joining reactions of the Y-substrate superimposed on
a generic tetramer model for integrase (left).
The sequences of the component single-stranded synthetic
oligonucleotides are: 5'-gcttgttgaataccatctaatcgtgtcgggtctcgtactgcggaa
(a), ttccgcagtacgagacccg (b),
aatgtagtcttatgcaatagc (c), and
gctattgcataagactacaacacgattagatggtattcaacagc (d). The
asterisk denotes the position of the
5'-32P-radiolabel.
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Fig. 2.
Single-turnover time course of the
disintegration reaction at 1 µM
integrase and 0.25 µM substrate DNA
was obtained by quantifying the amount of 44-mer formed following
separation by denaturing PAGE (inset).
Experiments were conducted with (closed circles)
and without (open circles) preincubating enzyme and
substrate prior to initiating the reaction. Lines
represent the best fit of the data to sum of exponential
terms with A1, preinc = 0.17 ± 0.01, A2, preinc = 0.60 ± 0.01, 1, preinc = 0.05 s 1 ± 0.006, 2, preinc = 0.002 s 1 ± 0.0001, Anonpreinc = 0.74 ± 0.009, and
nonpreinc = 0.002 s 1 ± 0.00004.
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Active Site Titrations--
When experiments were performed at
lower concentrations of integrase with preincubation, the amplitudes of
the two exponential phases became reduced and an additional linear
phase became apparent (Fig.
3A, inset). The
apparent "burst" kinetics behavior is characteristic of enzymatic
mechanisms where products are formed at the active site faster than
they dissociate, resulting in rapid turnover of the first equivalent of
substrates bound while subsequent steady-state turnovers are
rate-limited by slow product release to regenerate the free enzyme. As
in the single-turnover experiments, the first turnover burst consisted
of two exponential phases reflecting the rapid 0.05 s
1
conversion of productive complexes preformed in the absence of Mn2+ followed by a slower rate of assembling additional
complexes in the presence of the Mn2+.

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Fig. 3.
A, disintegration reactions performed
using less than 4-fold molar excess of integrase over substrate DNA
showed burst kinetics as shown in inset for 0.25 µM DNA
and 0.5 µM integrase at 250 mM NaCl. Best fit
of the data to a double-exponential plus a line yielded a total burst
amplitude of 0.4. Active site titrations for 0.25 µM DNA
with 250 mM NaCl (closed circles) and 0.5 µM DNA with 400 mM NaCl (open
circles) plotting the total burst amplitude as a function of the
molar ratio of integrase monomer to substrate DNA show that saturation
is reached at a four to one reaction stoichiometry. Best fits of the
data (solids lines) to the following equation,
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(Eq. 1Eq. )
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using an arbitrarily fixed Kd < 1 nM yielded maximum amplitudes, Amax,
of 0.72 and 0.86 and reaction stoichiometries, , of 3.8 and 3.7 at
250 mM and 400 mM NaCl, respectively.
B, when the Y-substrate was radiolabeled to monitor the
release of the viral fragment (open circles), an additional
competing hydrolysis reaction was detected at 2 × 10 5 s 1, which quantitatively accounted for
the missing amplitude observed for the disintegration reaction
(closed circles). The solid lines represent the
predicted time courses of disintegration for 0.5 µM DNA
and 2.0 µM integrase at 250 mM NaCl with and
without an additional exponential phase at 2 × 10 5
s 1.
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To determine the number of integrase protomers required to catalyze the
disintegration of a single substrate, we performed active site
titrations by measuring the sum of the two exponential amplitudes at
fixed DNA concentrations as a function of increasing integrase to
substrate DNA ratio. Fig. 3A shows two such titrations carried out at 250 mM NaCl with 0.25 µM DNA
and at 400 mM NaCl with 0.5 µM DNA. In both
cases, the total first turnover amplitude increased linearly with added
integrase up to a ratio of four integrase protomers per substrate DNA.
At integrase:substrate ratios above 4:1, a plateau was reached with no
further apparent change in the burst amplitude, indicating that the
substrate binding capacity of the added integrase exceeded the
concentration of substrate. These results defined a reaction
stoichiometry of four protomers per substrate molecule. Additionally,
the same reaction stoichiometry was obtained at different DNA and NaCl
concentrations, and titrations performed using integrase purified by
different protocols also yielded identical results (data not shown).
The maximum burst amplitude observed at both salt concentrations
represented less than complete binding of substrate DNA added albeit to
different extents. If the missing 15-25% reflected a subpopulation of
substrate that could not be bound by integrase, then the measured
reaction stoichiometry of four should be divided by this plateau value
to obtain the true reaction stoichiometry. On the other hand, if the
missing fraction represented a subpopulation of integrase-substrate
complex that was silent in the disintegration assay, i.e.
did not generate the monitored product, then an adjustment may not be
necessary. When we monitored the release of the viral-end fragment from
the Y-shaped substrate rather than the regeneration of the 44-nt target
fragment under identical conditions, we observed an additional
exponential phase in which the missing 15-25% of the substrate was
slowly cleaved (2 × 10
5 s
1) to
completion at the viral-target joining junction (data shown in Fig.
3B for 250 mM NaCl). This reaction was
integrase-catalyzed yet could not have arisen from 3'-hydroxyl attack
by the target 19-nt fragment as the corresponding target 44-mer product
was not generated concurrently. We therefore concluded that the missing 15-25% amplitude represented a competing but silent subpopulation of
the integrase-substrate complex that underwent hydrolytic
attack at the joining junction instead of disintegration. Thus, the
amplitude observed in the active site titrations represented a fraction of integrase-substrate complex that underwent disintegration as opposed
to hydrolysis, leading us to conclude that the measured reaction
stoichiometry of four was accurately determined without further adjustments.
Atomic Force Microscopy Volume Determination--
The
reaction stoichiometry of four defined by the active site titrations,
while necessary, is not sufficient to establish a tetrameric functional
oligomeric state. To verify an active tetrameric structure, we
determined the molecular weight of integrase complexes in the presence
and absence of DNA by AFM volumetric analysis using the linear
relationship between measured AFM volumes and molecular weights
previously established for proteins ranging from 41 to 670 kDa (24,
25). To ensure the functional relevance of the particles imaged,
depositions were made during the first turnover within 10 s of
initiating the reaction.
A typical 1-µm2 AFM image of integrase alone showed
primarily particles with volumes consistent with monomers and dimers
(Fig. 4A, top). By
comparison, increased numbers of tetramer-sized particles were observed
in images deposited in the presence of the Y-shaped substrate (Fig.
4A, bottom). Analysis of the integrated volumes for all particles imaged confirmed that in the absence of DNA, integrase appeared predominantly as monomers and dimers (Fig. 4B, top). In contrast, a new peak with a mean
molecular volume of 154 ± 7.5 nm3, corresponding to a
calculated molecular weight of 138 kDa, became apparent and accounted
for ~20% of the particles in the presence of DNA (Fig.
4B, bottom). When these population distributions were adjusted to more accurately reflect the number of integrase protomers subsumed within each volumetric subpopulation by taking into
account the mass of each particle (Fig. 4C), we observed that the presence of substrate DNA induced nearly half of the integrase
protomers analyzed to assemble into tetramers. Aggregates larger than
tetramers, though visible, did not accumulate in sufficient numbers to
segregate into discrete populations of a defined size, e.g.
an octamer, and were therefore excluded from the statistical analysis.
Additionally, the small number of these larger aggregates is
inconsistent with the magnitude of productive complexes observed in the
presteady-state burst.

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Fig. 4.
A, AFM images of integrase deposited at
64 nM integrase in 150 mM NaCl showed mostly
monomers and dimers with an occasional tetramer (top). This
population was shifted in favor of dimers and tetramers in the presence
of 80 nM substrate DNA (bottom). Excess DNA was
used to disfavor the formation of tetramers. Images at higher integrase
and NaCl concentrations also showed similar substrate-enhanced
tetramerization. Images were created with the freeware program WSxM
(www.nanotec.es). B, volume histograms of 405 and 381 particles analyzed in the absence (top) and presence
(bottom) of substrate, respectively, showed a statistically
significant increase in the tetramer population upon the addition of
DNA. The inset axis represents the molecular volume to
molecular weight conversion. C, when the histogram from
B with substrate present was adjusted to reflect the mass of
each particle, nearly half of the total integrase molecules were found
in the tetrameric state.
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The 138-kDa apparent mass observed was larger than expected for a
tetramer of four 32-kDa subunits presumably due to the additional presence of the bound DNA. However, the apparent difference of 10 kDa
underestimated the actual mass of the DNA. It is plausible that part of
the volume contributed by the DNA may be topologically obscured within
the concavity of the DNA binding site. Alternatively, the linear
calibration curve determined for globular proteins is unlikely to yield
accurate volume to mass conversion for DNA whose partial specific
volume in aqueous solution can be considerably smaller than for proteins.
Hypothetical Structural Models of Integrase Tetramers--
The
structure of the full-length 32-kDa integrase protein is unknown.
However, the central catalytic core domain (15-17) and all two-domain
fragments consisting of the core plus either the C (18-20) or N
terminus (21), from a variety of retroviral sources, form dimers in the
crystal structures. Based on these structures, several dimer-of-dimers
models have been proposed (20-22). Fig. 5A shows a recent model proposed based on
the structure of the core plus N-terminal two-domain structure of HIV-1
integrase (21). In this model, a pair of inward-facing functional
active sites is contributed by the two inner, or "proximal,"
protomers at the dimer-dimer interface. The two remaining
outward-facing active sites of the two "distal" protomers are not
used for catalysis (20-22). Coordinated processing of both ends of the
viral DNA would therefore require concurrent binding of the two viral
ends in active sites on both sides of the dimer-dimer interface (Fig. 5A).

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Fig. 5.
A, the dimer-of-dimer tetramer
proposed by Wang et al. (21) based on the structure of HIV-1
integrase1-212 (Protein Data Bank code 1K6Y) is schematically
represented as a pair of crystallographic dimers
(green-yellow and blue-red) using large and small
ellipsoids to represent the core and N terminus domains, respectively.
Viral DNA ends (white ribbons) are superimposed in the
active sites of the two proximal protomers (yellow
and blue). The dashed line indicates the plane of
the dimer-dimer interface. B, the molecular surface of the
tetramer is shown with positive and negative electrostatic potentials
shown in blue and red, respectively. The image
was created with DeepView/Swiss-PdbViewer (30). Products of the
disintegration reaction from a Y-shaped substrate are superimposed on
the tetramer guided by an active site and the positively charged
central cleft, which is canted ~40° from the plane of the
dimer-dimer interface. Black and white ribbons
represent target and viral DNA, respectively. The neutralization of the
positive charge at the dimer-dimer interface by the target DNA may
provide the molecular basis for the observed DNA-induced tetramer
assembly.
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Our results suggest that concerted integration may further
require binding of the target DNA bridging the dimer-dimer interface (Fig. 5B). Although we cannot be certain of the molecular
details of DNA binding in the absence of cocrystal structure, the
observation that the Y-substrate, with only a single viral-end mimic,
was able to induced active tetramer assembly would suggest significant contribution by the target DNA in mediating the dimer-dimer
interaction. The calculated electrostatic potential at the surface of
the proposed tetramer (Fig. 5B) shows that a ~30-Å wide,
positively charged groove extends along this interface, suggesting that
the assembly of the tetramer would require the juxtaposition of two
positively charged surfaces. The neutralization of this like-charge
repulsion by the binding of the negatively charged target DNA into this groove would greatly enhance the stability of the tetramer and may form
the molecular basis for the observed DNA-induced assembly.
Relevance of the Disintegration Reaction--
Despite being widely
employed for assaying the activity of integrase and integrase mutants
(8, 12-14), including that of the catalytic core domain constructs
used in crystallographic studies (15-17, 27, 28), concerns persist
that the disintegration reaction may not be mechanistically
relevant. The skepticism arises in part from the observation that the
catalytic core domain can catalyze the disintegration reaction but not
the forward joining reaction, leading to the notion that the
disintegration reaction might represent a less "stringent" measure
of activity. While it may be more compelling to determine the reaction
stoichiometry for the more obviously relevant processing or joining
reactions, the results reported here with the disintegration reaction
represent the first successful quantitative measure of a reaction
stoichiometry using any integrase assay, because the
propensity of the enzyme to aggregate has precluded the use of both the
processing and the joining reaction in active site titrations (9, 10).
Additionally, the reported weak disintegration activity of the ASV
catalytic core (16) is > 100-fold slower than the competing,
nonspecific hydrolysis reaction shown in Fig. 3B and at
least 5 orders of magnitude slower than the disintegration activity
reported here for the full-length enzyme (data not shown). This result,
therefore, suggests that the disintegration reaction may not be as
permissive as previously thought, at least for the ASV enzyme.
A more substantive concern regarding the use of the
disintegration assay stems from the inclusion of only one viral-end
mimic in the structural design of the Y-shaped substrate. As a result, our data do not directly rule out an octameric (dimer of tetramers) model (20-22) for the binding of two viral ends. However, as the minimum requisite set of active sites are present in a tetramer to bind
both viral ends plus the target DNA, the available data likewise do not
dictate any direct need to recruit additional active sites. In the
absence of direct evidence for a higher order aggregate, we therefore
favor the minimally sufficient tetrameric model. We note further that
while the tetramer with a single Y-shaped substrate bound can
accommodate the additional binding of a second viral end, the 30-Å
wide groove of the tetramer is too narrow to permit the binding of a
second Y-shaped substrate. Additionally, the complementary relationship
between the structure of this substrate and its ability to mediate
functional tetramer assembly lends compelling support for the
structural and functional relevance of the disintegration substrate in
modeling the active-site architecture.
Implications for Structural Studies and Antiviral Design--
Our
ability to complete active site titrations suggests that the presence
of target DNA in the disintegration substrate mitigated protein
aggregation problems observed with other DNA substrates. Alternatively,
the disintegration substrate conferred sufficient binding stability to
maintain active complexes at the higher NaCl concentrations required to
prevent aggregation. These results suggest potential benefits from
using disintegration-like substrates, containing both target and viral
DNA, in structural studies of the active integrase unit to promote
tetramer formation as well as to minimize non-productive aggregation.
The observed substrate DNA- and metal cofactor-dependent
oligomerization further suggests that the assembly of the active tetramer may be an integral and dynamic component of the catalytic pathway. The dynamic nature of the dimer-dimer interface should make it
an ideal target for inhibitor design. The diketo acid family of
inhibitors targeting the active site of integrase has recently been
shown to inhibit the V(D)J recombinase, RAG1/2 (29), important for T-
and B-cell development. Recently, we have independently characterized a
novel RAG1/2-like "splicing" activity of ASV integrase that further
suggests that any active-site directed inhibitor of integrase could
similarly interfere with V(D)J recombination (9). If the substrate
binding-induced assembly of an active tetramer lies along the kinetic
pathway of catalysis, then inhibitors that disrupt the dimer-dimer
interface may provide an enzyme-specific means of disrupting catalytic
activity while avoiding possible adverse effects resulting from
directly targeting the active site.