From the Department of Physical Chemistry, Chalmers
University of Technology, S-412 96 Gothenburg, Sweden, the
¶ Unité Mixte de Recherche 216, CNRS and Institut Curie,
F-91405 Orsay, France, and the ** Physical Department, Risø National
Laboratory, DK-4000 Roskilde, Denmark
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ABSTRACT |
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To gain insight into the mechanism of
pairing two complementary DNA strands by the RecA protein, we have
determined the nucleobase orientation of the first and the second bound
DNA strands in the RecA·DNA filament by combined measurements of
linear dichroism and small angle neutron scattering on flow-oriented
samples. An etheno-modified DNA, poly(dA) was adapted as the first
DNA and an oligo(dT) as the second DNA, making it possible to
distinguish between the linear dichroism signals of the two DNA
strands. The results indicate that binding of the second DNA does not
alter the nucleobase orientation of the first bound strand and that the
bases of the second DNA are almost coplanar to the bases of the first
strand although somewhat more tilted (60 degrees relative to the fiber
axis compared with 70 degrees for the first DNA strand). Similar
results were obtained for the RecA·DNA complex formed with unmodified
poly(dA) and oligo(dT). An almost coplanar orientation of nucleobases
of two DNA strands in a RecA-DNA filament would facilitate scanning
for, and recognition of, complementary base sequences. The slight
deviation from co-planarity could increase the free energy of the
duplex to facilitate dissociation in case of mismatching base
sequences.
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INTRODUCTION |
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The RecA protein plays a crucial role in homologous recombination in Escherichia coli: it regulates the synthesis of proteins involved in recombination reaction, including RecA itself, and catalyzes strand-exchange reaction (1). RecA can, in the presence of cofactor ATP, promote pairing of two complementary DNA strands (2) and also perform strand exchange between two homologous DNA molecules in vitro (3, 4). In these reactions, RecA first binds with high cooperativity to a single-stranded DNA (5) forming a nucleoprotein filament (6). In the complex, the protein monomers are arranged in a helical manner around the DNA, and the DNA is elongated about 50% in length. This RecA single-stranded DNA nucleofilament can then bind a second DNA (either single- or double-stranded DNA) and pair complementary parts.
From fluorescence measurements of various probes attached to the DNA bases, we have concluded that direct base-base interaction of the Watson-Crick type contributes to the sequence recognition by RecA (7, 8). Base-base interaction can occur between all three DNA strands in the RecA·DNA complex, supporting formation of a triplex DNA structure, as has been suggested by other studies (9, 10). Several models of different base-triads, involved in triplex-structure of recombination intermediates have been proposed (11, 12)
In order to investigate the base-base interactions and understand more about the mechanism of recognition between complementary DNA strands in RecA, we earlier assessed the orientation (roll and tilt angles) of DNA bases in RecA·DNA complexes by linear dichroism (LD)1 spectroscopy combined with small-angle neutron scattering (SANS) and chromophore-replacement studies (13-15). LD measures, on an aligned sample, the absorption difference of light polarized parallel and perpendicular to the sample orientation direction (16). The signal is related to both the local orientation of the chromophore in the macromolecule and to the degree of alignment of the whole molecule. LD can therefore be used to estimate the local orientation of the DNA bases in the RecA·DNA nucleofilament once the degree of alignment (the orientation factor) of the complex filaments under LD measurement condition is known. The orientation factor can be independently determined by SANS measurements on the same sample aligned under the identical conditions (13).
Chromophore-replacement analysis has been used to estimate the LD signal of a given chromophore in a complex which has more than two chromophores exhibiting overlapping signals. The analysis is based on the replacement of one of the components in the complex by an analog whose spectroscopic character differs from that of the original chromophore. Comparison of original and replaced spectra allows determination of the signal of a particular constituent (e.g. DNA) despite overlapping signals from other chromophores of the complex (e.g. ATP and RecA).
Here we have determined selectively the orientation of the second DNA
strand bound in a RecA·DNA filament using this approach. Poly(dA),
absorbing at higher wavelengths than normal DNA (around 320 nm), was
used as the first DNA strand in the RecA·DNA filament to be able to
distinguish its LD signal from that of the second DNA (oligo(dT)) in
the complex. The nucleobases of the second DNA are found to be rather
immobile and oriented less perpendicular to the axis of the protein
filament but still rather similar to the bases of the first DNA strand.
An almost coplanar base arrangement could favor base-base contacts
between two DNA strands in the RecA filament and may facilitate the
search for complementary sequences within the DNA molecules.
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EXPERIMENTAL PROCEDURES |
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RecA and poly(dA) were prepared as described previously (17,
18). Poly(dA) and oligo(dT) were from Amersham Pharmacia Biotech. The
size of polynucleotides was about 350 bases and that of the
oligonucleotides about 20 bases. ATP
S was from Boehringer Mannheim
and freshly dissolved solution was used without further purification.
The concentrations were determined from UV absorption with
280 nm = 21700 M
1
cm
1 for RecA,
264 nm = 8520 M
1 cm
1 in base for oligo(dT),
260 nm = 8600 m
1 cm
1 in
base for poly(dA), and
257 nm = 3800 M
1 cm
1 in base for
poly(d
A).
RecA·DNA complexes were formed in 20 mM sodium phosphate,
pH 6.8, with 50 mM NaCl, 1 mM
MgCl2, and 400 µM ATPS, all in
D2O. Initially, RecA was dialyzed against the
D2O buffer mentioned above. A first RecA·DNA complex was
formed by mixing 50 µM RecA and 150 µM
poly(d
A) in the above buffer and by incubation at room temperature.
To form a complex with two DNA strands, oligo(dT) was added to the
preformed RecA·poly(d
A) complex and further incubated. In another
experiment, RecA·poly(dA) complex was first formed, and subsequently
oligo(dT) was added in stoichiometric amounts in order to form a RecA
complex with two DNA strands.
Absorbance, LD, and SANS were measured on each sample. Absorbance was
measured in a Cary 2000 spectrophotometer (Varian) with a bandwidth of
2 nm. Linear dichroism was measured in a modified Jasco J-500
spectropolarimeter (19) with a bandwidth of 2 nm. No smoothing
procedure was applied to any of the spectra. SANS data were collected
in the facility at Riso National Laboratory, Denmark. The sample to
detector distance was 3.86 m, and neutron wavelength was 0.723 nm,
as described in Nordén et al. (13). LD and anisotropic
SANS measurements were performed on the sample aligned by an inner
rotating Couette cell of the same dimension and with identical shear
gradients, 620 s1.
Assessment of nucleobase orientation of DNA strands in RecA filaments was made as follows. LD is defined as the differential absorption, LD = Aparallel - Aperpendicular, between orthogonal forms of plane polarized light. The reduced dichroism, LDr = LD/Aiso for DNA (with Aiso the normal, isotropic absorbance) at a given wavelength, is related to the angle between the light-absorbing transition moments in the DNA and orientation axis of the RecA·DNA complex (i.e. the fiber axis) (16),
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(Eq. 1) |
The SANS and LD samples referred to identical flow cell geometry and flow rates. But in order to not exceed a maximum absorbance of 2, some LD sample was diluted 1:5. Control experiments, in the absorption region where the absorbance did not exceed about 2 absorbance units, showed that the orientation of the RecA·DNA fibers was not significantly dependent on the concentration (14).
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RESULTS |
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We first investigated whether the orientation of the nucleobases
of a first bound DNA strand in a RecA·DNA filament is altered upon
binding of a second DNA strand. We measured LD of RecA·DNA complexes
with poly(dA) before and after addition of a second DNA, oligo(dT).
Poly(d
A) exhibits an extra absorption band above 320 nm in which
wavelength region the signals from normal DNA, ATP
S, and RecA are
negligible (21). The LD signal above 320 nm is, thus, only related to
the orientation of etheno-adenine, with no interference from other
components. As a second DNA, we used short oligonucleotides (oligo(dT)
around 20 bases long) instead of polynucleotides, to avoid formation of
network structures which may prevent orientation of the complexes in
the shear flow (22).
The LD signal of the ATPS·RecA·poly(d
A) complex above 320 nm
was negative (Fig. 1), as observed
previously (23), in quantitative agreement with a rather perpendicular
base orientation relative to the protein-filament axis. Upon addition
of oligo(dT) to saturate the second DNA-binding site in RecA, the
intensity of the negative signal around 260 nm increased, however,
without modifying the LD signal in the region above 320 nm (Fig. 1).
Binding of a second DNA to the RecA·DNA complex apparently does not
affect the average orientation of the nucleobases of poly(d
A) bound
in the first DNA-binding site of RecA.
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Thus, assuming an essentially unchanged base orientation of the first
bound DNA strand, the base orientation of the second DNA strand in the
complex could be assessed from the difference (LD spectrum of
ATPS·RecA·poly(d
A)·oligo(dT) complex) - (LD spectrum of
ATP
S·RecA·poly(d
A) complex). This difference spectrum had a
maximum centered around 260 nm and had a shape similar to that of the
oligo(dT) absorption spectrum which is centered at 264 nm (Fig. 1).
This observation of similarity between spectral shapes supports the
idea that the increase in negative LD around 260 nm upon oligo(dT)
binding to the RecA·poly(d
A) complex is indeed due to the base
orientation of the second DNA. Since the LD is negative, the
nucleobases of the second DNA too are oriented rather perpendicular to
the filament axis.
As mentioned under "Experimental Procedures," one can
quantitatively determine the effective angle of the nucleobases
relative to the orientation axis of the RecA·DNA filament from the LD
signal, provided information about the degree of alignment of the
overall fiber is available (see Fig. 3). From SANS anisotropy measured under identical flow conditions, the second-moment orientation function
can be estimated from a comparison of the experimental SANS data with
the anisotropy calculated for flow model orientation distributions
(13). The SANS pattern of RecA·poly(dA) as well as of
RecA·poly(d
A)·oligo(dT) complexes exhibited a certain anisotropy in the presence of flow (Fig. 2). As a
control that non-homogeneous detector sensibility is not the cause of
virtual anisotropy, the difference in SANS pattern (differential SANS)
between flow-oriented and randomly oriented sample was recorded,
showing a systematic difference (Fig. 2B). The measurements,
in fact, show that the degrees of flow alignment of the
RecA·poly(d
A) and RecA·poly(d
A)·oligo(dT) complexes were
similar, corresponding to an orientation factor S in
Equation 1 equal to about 0.12 (± 0.02).
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Using this value, we could compute the orientation angles of the first
and second DNA from the LD data (Fig. 3).
At a given degree of alignment of molecules (S), a reduced
LD (LD/normal UV absorption) corresponds to an orientation degree of
the chromophore to the alignment axis (angle in Equation 1). The
nucleobase orientation of first DNA (poly(d
A)) was estimated from
reduced LD at 320 nm and found to be approximately 70 degrees as
observed previously (13). Because of uncertainty on the S
value (0.12 ± 0.02), the orientation could be between 68 and 78 degrees (Fig. 3). Since we found that the base orientation of the first
DNA strand (poly(d
A)) was not altered upon the binding of the second DNA, it is reasonable to assume that the spectral difference (LD of
RecA·poly(d
A)·oligo(dT) complex - LD of RecA·poly(d
A)
complex) reflects directly the signal from oligo(dT) at the same
S value. We thus estimated the reduced LD signal (at 260 nm)
of oligo(dT) bound to the second site. The value of the reduced LD for
oligo(dT) was significantly smaller than that for poly(d
A). This is
not an effect of partial binding of oligo(dT) to the RecA·poly(d
A) filament because the titration controls showed that the second site was
saturated at the addition of 3 bases/RecA of oligo(dT) (not shown), as
expected for almost complete binding of oligo(dT). We therefore
conclude that the bases of the second strand are on average more tilted
than those of the first bound DNA strands. An angle of about 60 degrees
was estimated relative to the long axis of the RecA filament (Fig. 3).
Estimation is more precise at this
value (60 ± 2 degrees).
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We also examined the complex formed between RecA and two complementary,
unmodified DNA strands, poly(dA) and oligo(dT). LD spectra of
ATPS· RecA·poly(dA) complex before and after addition of
oligo(dT) as well as the differential LD spectrum for the oligo(dT) are
shown in Fig. 4. For a quantitative
analysis, the orientation factors of each complex were also determined
by SANS measurements on the same samples under the same conditions as
for the LD measurements. In this case, however, we could not
experimentally distinguish the signal of oligo(dT) from that of
poly(dA). We must, therefore, assume that the binding of second DNA
does not alter the base orientation of first DNA, as was the case of
the Rec A·poly(d
A)·oligo(dT) complex, to be able to
estimate the base orientation of the second DNA.
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The results show that the average orientation of the first DNA bases
was about 68 degrees and that of the second DNA bases was 60 degrees
relative to the filament axis. The results were thus very similar to
those for the RecA-poly(dA)-oligo(dT) complex.
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DISCUSSION |
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To gain better insight into the pairing mechanism of complementary DNA strands by RecA and the base-base interactions occurring between the two DNA strands in the RecA filament, we have investigated the orientation of DNA bases in the complex. The base orientations were determined by combining LD and SANS data as described previously (13).
The results indicate for both of the types of DNAs investigated that the nucleobases of the second DNA are somewhat more tilted than those of the first DNA, but the difference is small so that the nucleobases of the two DNA strands are almost coplanar, although we cannot exclude the possibility that the base tilts of the two DNA strands are in opposite direction (Fig. 5). This is also the case when the two DNA molecules are not complementary in sequence. An almost coplanar structure is logical since it may facilitate effective base-base hydrogen bondings between the two DNA strands with the purpose of comparing sequences and of search for complementarity.
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The quantitative evaluation indicates that bases are neither perfectly perpendicular to the fiber axis nor coplanar to each other. This must be taken into account upon model building of the base-base interactions in the RecA·DNA complex filament. Most models of base triads of RecA-promoted triplexes are based on the assumption that all the nucleobases of the three DNA strands are perpendicular to the main DNA axis and are thus perfectly coplanar to each other, although the bases of conventional poly(dT):poly(dA):poly(dT) triplexes are generally not perfectly perpendicular to the helix axis (24, 25). Our observation that the bases of the second bound DNA strand are more tilted than those of the first DNA could indicate a mechanism by which the RecA filament acts as a scaffold preventing the formation of classical Watson-Crick-Hoogsteen hydrogen bonding, keeping the free energy of the complex higher than that of an ideal triplex. Such an arrangement would facilitate dissociation, in case of mismatch, and also facilitate longitudinal sliding of the DNA molecules relative to each other, which would be required for an efficient search-for-homology mechanism.
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FOOTNOTES |
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* This work has been supported by grants from the European Community (BioMed program) and French Ministry of Education (ACC-SDV) and by Institut Curie and the French and Swedish Research Councils (CNRS and NFR).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.
§ Present address: Beckman Institute, California Institute of Technology, Pasadena, 91125 CA.
Recipient of fellowships from the Association pour la
Recherche sur le Cancer and the French government.
To whom correspondence should be addressed: Institut Curie,
Bat. 110, Centre Universitaire Paris-Sud, F-91405 Orsay, France. Tel.,
and Fax: 33-1-69-86-30 11; E-mail:
Masa.Takahashi{at}curie.u-psud.fr.
1
The abbreviations used are: LD, linear
dichroism; ATPS, adenosine 5'-O-3-thiotriphosphate;
poly(d
A),
poly(1,N6-etheno-deoxyadenosine); SANS,
small angle neutron scattering.
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REFERENCES |
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