J. Mol. Biol. (1990) 216, 223-228

Structure of a R e c A - D N A Complex from Linear Dichroism and Small-angle Neutron-scattering in Flow-oriented Solution B. Nord6n, C. Elvingson, T. Eriksson, M. Kubista Department of Physical Chemistry, Chalmers University of Technology S-412 96 Gothenburg, Sweden

B. SjOberg Department of Medical Biochemistry University of Gothenburg, Box 33031, S-400 33 Gothenburg, Sweden

M. Takahashi Groupe de Cancdrogdnese et Mutagdnese Moldculaire et Structurale Institut de Biologic Moldculaire et Cellulaire du CNRS, 15 rue Rdne Descartes F-67084 Strasbourg Cedex, France

and K. Mortensen Physics Department, Riso National Laboratory, Box 49 DK-4000 Roskilde, Denmark

(Received 10 April 1990; accepted 16 July 1990) Small-angle neutron-scattering (SANS) and ultraviolet linear dichroism (I.d.) were measured on identical samples of a RecA-double-stranded (ds) DNA complex, including cofactor adenosine 5'-O-thiotriphosphate, which were aligned by flow in two equikalent Couette devices made of niobium and silica, transparent to neutrons and to ull~r~violet light, respectively. The SANS anisotropy indicates a modest orientation of the RecA-dsDNA fiber with the helix axis parallel to the flow field. By correlation with the corresponding I.d. of the DNA at the same orientation conditions, it is inferred that the DNA bases have a local orientation that is approximately perpendicular to the helix axis. By comparison with the worse orientation in single-stranded DNA-RecA, this conclusion suggests that the dsDNA in its complex with RecA is not strand separated, and may be accommodated as an essentially unperturbed, straight double helix running along the RecA polymer fiber. The SANS anisotropy is also found to support the assignment of a subsidiary intensity maximum as originating from the pitch of a helical fiber.

RecA is a key protein in the DNA repair system of Escheriehia coli. It promotes general genetic recombination via exchange between two homologous DNA strands and promotes the induction of the SOS system (for reviews, see Howard-Flanders et al., 1984; DiCapua & Koller, 1987; Cox &

Lehman, 1987; Griffith & Harris, 1988; Radding, 1989). The strand exchange reaction can be observed in vitro in the presence of ATP and great efforts have been made to gain insight into these mechanisms by studying complexes between ReeA and DNA using various techniques (McEntee et al., 1981; Williams & Spengler, 1986; Heuser & Griffith, 1989; DiCapua et al., 1982, 1989). RecA interacts with single-stranded (sst) DNA, and in the presence of ATP (or the non-hydrolyzable analog adenosine 5'-O-thiotriphosphate,

Abbreviations used: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; I.d., linear dichroism; SANS, small-angle neutron-scattering; SAXS, smallangle X-ray scattering. 0022-2836/90/220223-06 $03.00/0

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ATP~S) also with double-stranded (ds) DNA (McEntee et al., 1981). From electron microscopy studies (Williams & Spengler, 1986; Heuser & Griffith, 1989) it has been inferred that RecA itself forms a homopolymer in the shape of a compact spiral with a pitch of about 7nm. Also the RecA-DNA complexes virtually consist of RecA helices, with about six RecA molecules per turn (DiCapua et al., 1982), that accommodate DNA in the interior (DiCapua et al., 1989). For the RecA complex with ssDNA the pitch has been estimated to be 9"5 nm when formed in the presence of ATP~S and 6 nm when formed in the absence of this cofactor. With dsDNA (in the presence of ATP~S) a rigid, helical fiber is formed (pitch 9-5nm: see Williams & Spengler, 1986; Heuser & Griffith, 1989; Egetman & Stasiak, 1986). From fluorescence and flow linear dichroism (l.d.) studies information about stoichiometry and organization of chromophores in protein (tryptophan residues) and nucleic acid (DNA bases) in the RecA-DNA complexes has been obtained (Takahashi et al., 1987, 1989a,b). Owing to the great length, of the order of micrometers as judged from electron microscopy studies (Egelman & Stasiak, 1986), of the RecA--dsDNA filaments, efficient orientation could be anticipated by applying a shear gradient in a Couette flow cell (Takahashi et al., 1987, 1989a,b). We here report measurements of anisotropic small-angle neutronscattering (SANS) and ultraviolet I.d. on identical RecA--dsDNA samples, flow-oriented in two equivalent Couette cells one made of niobium (transparent to neutrons) and one of silica (transparent to ultraviolet light). While the I.d. provides information about the average orientations of the lightabsorbing chromophoric groups of the nucleic acids and protein (Nord6n & Kubista, 1988), the SANS anisotropy reflects the overall degree of fiber orientation. Correlation of SANS and 1.d. at the same flow rate, therefore, should permit conclusions about chromophore orientations in a local, fiberfixed framework. Moreover, as we shall see, the anisotropic SANS allows discrimination between structural repeat lengths along and across the filament, i.e. the pitch and the width of the fiber. RecA was prepared as described (Takahashi et al., 1989a) but in order to improve the purification, high-pressure DEAE 5PW (Tosoh) chromatography was used instead of the conventional DEAEcellulose chromatography. For dsDNA, freshly dissolved calf thymus DNA (Sigma, type I) was used. The complex was formed by mixing RecA and DNA (3 base-pairs/monomer) in a buffer containing I4 mM-NaH2POa, 6 mM-Na2HPO4, 50 mM-NaCl, 1 mM-ATP~S (Boehringer, Mannheim), 1 mM-MgC12, 1 mM-dithiothreitol. After one hour of incubation at room temperature, the mixture was dialyzed at 4 °C against a 50 times larger volume of the same buffer in 99% 2HzO (Sigma). The reason why RecA was not added in excess, as is often done in electron microscopy studies, is that free RecA would contribute to the SANS signal. As shown in direct titrations (Takahashi et al., 1989a,b) addition of

stoichiometric amounts of RecA is sufficient for obtaining the spectral properties of a fully saturated RecA-dsDNA complex. The dialysis buffer was changed three times and the total dialysis time was 18 hours. Longer incubation (up to 5h) of the P~ecA-DNA mixture did not significantly affect the SANS pattern, as expected from the kinetics (Takahashi et al., 1989b). The ultraviolet Couette cell was of the Wada-Kozawa type, i.e. with an inner cylindrical solid rotor with a diameter of 29 mm and an outer stator cylinder both made of birefringence-free silica (Suprasil) (Nord~n & Kubista, 1988). The sample to be studied is subjected to shear flow in the annular gap (AR=0"500(_+0"005) mm) between the two cylinders. In the experiments with DNA solutions, and from comparison with a corresponding celt having an outer rotating cylinder, neither ordinary turbulence nor centrifugal (Taylor) instability could be detected for dilute aqueous solutions for gradients G < 2000 s- 1. Several neutron Couette cells were constructed of aluminium and of niobium, both materials having high transparency and low scattering for neutrons (Q>_0"007 A -1 (1 A=0"l nm)). Niobium was preferred owing to its chemical inertness. Both inner and outer cylinders were manufactured of 1 mm thick metal plates (98O/o Nb) that were bent and laser welded into crude cylinders. These were mounted on solid polyethylene cylinders and ground to a cylindrical shape. A final grinding was performed on the surfaces facing the solution to ensure perfect centricity. The outer, static cylinder was mounted (epoxied) in a stainless steel holder with an approximately 10mm circular opening for the neutron beam; the welding seam was turned away from the opening in order not to risk its perfbration when under vaccuum. The apparatus is contained in a larger steel cylinder (diameter 200 ram) allowing removal of the driving motor and inner Couette cylinder from the top through the open shaft. Conical contact surfaces guarantee that the inner cylinder always is in perfect centricity with the outer cylinder. This design permits easy inspection of the sample cavity and filling and removal of the sample. The outer cylinder is mounted vaccuum tight to the bottom of the steel cylinder with a 120° V-shaped inside bottom to prevent air bubbles being caught when the cell is spun. The dimensions of the cylinders are essentially the same as those of the silica Couette cylinders, i.e. the diameter of inner cylinder is 29 mm but the annular gap can be varied between 0-5, 1 and 2 ram. The sample volume is 1"3 to 2"6 cm a depending on the gap chosen. The apparatus is placed in the horizontal neutron beam so that the neutrons pass radially through the center of the cylinders, and the total neutron path length is thus twice the annular gap width. The SANS data were collected using the facility at Ris~ National Laboratory, Denmark (K. Mortensen, unpublished method). For the RecA-dsDNA sample the SANS in the absence of flow was essentially identical with that observed in

225

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Figure 1. Isotropic and anisotropic 2-dimensional S A N S intensity patterns of RecA-dsDNA complex in the presence of ATPTS in 2H20. Q / A - ' on Z and Y scales. (a) Measured isotropic SANS in a Couette cell at rest (path length 2 ram). (b) Measured anisotropic SANS (path length 2 ram) in Couette flow (gradient G = 8 0 0 s-l). Flow direction horizontal (Z axis). (c) Calculated SANS pattern for a helix, aligned parallel to the Z axis, containing 10 turns, each turn modeled by a row of.12 end to end connected cylinders, each with a radius of 2 nm. Diameter and pitch of helix was 8"0 and 9"5 nm, respectively• Wavelength of neutrons was 0"40 nm. (d) Calculated SANS pattern for a set of partially aligned helices (parametrized as in (c)) simulating the orientational distribution in Couette flow (Z=orientation direction) for an orientation factor S = 0-32. Notice the elliptical shape of the central intensity region and the subsidiary maxima centered on the Z axis at Q=0-07 A - i . (e) Same as in (d) for a flow ensemble with S=0-09.

a c o r r e s p o n d i n g q u a r t z ceil, t h u s showing no d i s t u r b i n g effects b y the split s a m p l e configuration or the n i o b i u m walls in the C o u e t t e cell. T h e I.d. w a s m e a s u r e d as described (Nord6n &

Seth, 1985) using a modified J a s c o 500 s p e c t r o p o l a r imeter. T h e r e d u c e d dichroism 1.d.r=l.d.]A~.so for D N A (with Aiso, t h e isotropic a b s o r b a n c e ) is r e l a t e d to the effective angle 0, b e t w e e m t h e D N A bases

226

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and the local fiber axis of the RecA-DNA complex according to (Nord6n & Kubista, 1988): l.d., = 3 / 2 S ( 3 ( c o s 2 0 ) - l ),

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where S, the orientation factor for the whole fiber, is estimated from the SANS data (see below). Owing to extensive overlap with the absorption of ATPTS, the Aiso of DNA was calculated from the DNA concentration asuming an extinction coefficient of 6600 mol- 1 cm- 1. On the basis of the fact that the negative I.d. band at 260nm has a shape very similar to uncomplexed DNA, it was further considered justified to ignore any I.d. contributions, at this wavelength, from l'¢ecA and any oriented ATPTS. Figure 1 shows the SANS intensity patterns at rest and during flow for the RecA-dsDNA complex in the presence of ATPTS. At rest (Fig. l(a)) the SANS pattern clearly shows the perfectly circular symmetry characteristic of an isotropic solution, whereas in flow (Fig. l(b)) the intensity profile is markedly deformed into an elliptical shape, indicating anisotropy. As expected for an elongated particle oriented parallel to the flow direction ( Z = horizontal) the SANS intensity pattern is elongated along the vertical direction (Y). A subsidiary intensity maximum that is also observed in small angle X-ray scattering, (SAXS) (results not shown) at approximately Q=0-07 A-l can be related to the helical pitch of the fiber (see below). Figure l(c) to (e) show theoretical scattering patterns, calculated for a helical model fiber consisting of small cylinders, representing the RecA helix, joined into a solenoid (the presence of DNA has been ignored). The dimensions (see legend to Fig. ]) were chosen on the basis of current models emerging from electron microscopy (Egelman & Stasiak, 1986), together with the SAXS results and the isotropic SANS profile. Figure l(c) shows the calculated SANS pattern for an ensemble of helical fibers perfectly aligned along the Z co-ordinate axis. Note the intensity maxima between the Z and Y axes which are characteristic for a helical structure. In Figure l(d) to (e) SANS patterns are calculated for partially oriented ensembles demonstrating the effect of imperfect alignment. Each ensemble was simulated by some 60 helices distributed according to the Peterlin-Stuart flow orientation model for rigid particles (Peterlin & Stuart, 1939). The degree of orientation of each ensemble is characterized by the orientation factor S = (cos2zZ) - (cos~z Y), with z Z denoting the angle between the fiber axis (z) and the flow direction (Z), and z Y the corresponding angle to the Y axis (the brackets ( ) denote ensemble average). Flow linear dichroism spectra recorded on the same sample as studied by SANS are shown in Figure 2(a). The "isosbestic points" observed close to the baseline in the I.d. spectra recorded at varying gradients indicate orientational homogeneity, i.e. that the DNA (with I.d. at 260 nm) and the RecA protein (with l.d. at 290 nm Takahashi et

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al., 1989a)) belong to the same orientable species.

This observation is consistent with a well-defined RecA-dsDNA complex. Moreover, neither 1.d. (Fig. 2(a), signal above 320nm) nor absorption spectrum (not shown) displayed any significant scattering feature that could indicate the presence of aggregates. The shape of the l.d. spectrum was close to that observed previously at lower concentrations (Takahashi et al., 1989a,b), so the structure of the complex does not seem to be affected by the large concentrations necessary for the SANS measurements. In contrast to the elongated SANS pattern corresponding to a perfectly aligned RecA-dsDNA helix (Fig. l(c)), a partially aligned ensemble of helices is anticipated to display an anisotropic SANS pattern with a more symmetric and only slightly elliptical shape, as illustrated by the calculated patterns in Figure l(d) to (e). Further, the subsidiary maxima

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characteristic of a helical scatterer, which are found on the Y Z diagonals for a single oriented fiber (Fig. ! (c)), are blurred into weak, elongated maxima centered at the Z axis (Fig. l(d)). By comparing the experimentally observed SANS pattern of the oriented RecA-dsDNA complex (Fig. l(b)) with those calculated for partially oriented ensembles of helical scatterers, we find that the fiber orientation in the SANS experiment is far fi'om perfect; a semi-quantitative agreement with the expected SANS pattern for a flow oriented ensemble with an orientation parameter S=0"09 is observed. No major changes in the anisotropy of the SANS pattern could be observed at gradients above 200 s -~, although the flow linear dichroism signal clearly increases without any tendency to reach saturation level (Fig. 2(b)). This is most likely owing to the much higher sensitivity of the I.d. technique, compared to SANS, to monitor orientation, and is not caused by changes in the internal structure of the RecA-dsDNA complex, since the overall spectra recorded at the various gradients display the same intensity distributions (Fig. 2(a)). Since the same flow gradient was applied in the I.d. experiment as in the SANS study, the information about overall fiber orientation deduced from the SANS experiment can be used to scale I.d. signals using equation (1), which allows us to estimate the tilt of the nucleotide bases relative to the fiber axis of the RecA-dsDNA filament. From the measured 1.d. and estimated Ai~o a rough value of ].d.~= -0.15( +0.05) is obtained, which can be inserted into equation (1). With S=0"09, obtained from the SANS study, we end up with a value of the optical factor (3(cos~0) - 1)/2 between -0"37 and -0"5, which corresponds to 0 = 7 3 to 90 °. i.e. the nucleic acid bases in the complex between dsDNA and RecA should be organized so that their planes are approximately perpendicular to the RecA-dsDNA fiber axis. A perpendicular base orientation is also consistent with the observation (results not shown) of approximately constant reduced 1.d. over the 260 nm DNA absorption band, after correction Ibr absorption of other chromophores (see Matsuoka & Nord~n, 1983). Such a structure would be anticipated if the dsDNA were running unperturbed (unstretched) and completely straight along the RecA fiber. Also with a stretched conformation of DNA, for which there has been much evidence (DiCapua et al., 1982; Williams & Spengler, 1986; Heuser & Griffith, 1989; Egeiman & Stasiak, 1986), the DNA base-pairs can display a perpendicular orientation by a ladder arrangement, although this seems to require some steric support from RecA in order to compensate for the absence of base stacking in an extended ladder form. Also the circular dichroism spectrum (Takahashi et al., 1989b) is in favour of a not too strongly perturbed B-form DNA. The present findings of a high local orientational order of the dsDNA bases perpendicular to the axis of the RecA helix, in conjunction with evidence for

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an inside location (DiCapua et al., 1989) and indications that the DNA orientation in the corresponding RecA complex with ssDNA is much less pronounced (Takahashi et al., 1987), supports our conclusion that the dsDNA in the dsDNA-RecA complex is straight and runs on the inside of the RecA-dsDNA filament, more or less parallel to the central axis of the fiber (Fig. 3). If the DNA, for example, followed

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a groove of the RecA fiber, a relatively low DNA base order would be expected and finally if it was running outside the RecA fiber it should most certainly not have shown resistance to nuclease digestion. Our conclusion agrees with recent electron microscopy image analysis and stereochemical considerations suggesting the DNA to be close to the center of the filament (Egehnan & Yu, 1989). DiCapua el al. (personal communication) have recently also measured SANS on flow oriented RecA-dsDNA solutions and obtained results very similar to ours, though their solutions, in addition to ATP~S and 2H20 buffer, contained 10% (v/v) glycerol with the intent of counteracting degradation. T h e y too observed a subsidiary intensity maximum t h a t they proposed be related to the pitch of a helical fiber. In our case the measurements were carried to completion within a few hours after preparing the solutions and no stabilizing additivies were found to be necessary. An i m p o r t a n t related conclusion of the anisotropic SANS follows from the observation t h a t the intensity of the subsidiary maximum is centered on the Z axis, i.e. the direction of preferred fiber alignment, and not on the Y axis. The consistent prediction of this behavior by the simulation for a partially aligned set of helical fibers (Fig. l(d)), supported by the fact t h a t a transverse repeat distance, such as for example the fiber width or a hole-diameter of a hollow cylindrical fiber, would instead have contributed to Y centered intensity, is conclusive for our assignment of the subsidiary maximum as indeed originating from the pitch of a helical fiber.

This work is supported by the Swedish Natural Science Research Council.

References Cox, M. M. & Lehman, I. R. (1987). Annu. Rev. Biochem. 56, 229-262. DiCapua, E. & Koller, Th. (1987). In Nucleic Acids and Molecular Biology (Eckstein, F. & Lilley, D. M. J., eds), vol. l, pp. 174-185, Springer-Verlag, Berlin, Heidelberg and New York. DiCapua, E., Engel, A., Stasiak, A. & Koller, Th. (1982). J. Mol. Biol. 157, 87-103. DiCapua, E., Schnarr, M. & Tirnmins, P. (1989). Biochemistry, 28, 3287-3292. Egelman, E. H. & Stasiak, A. (1986). J. Mot. Biol. 191, 677-697. Egehnan, E. H. & Yu, X. (1989). Science, 245,404~o407. Griffith, J. D. & Harris, L. (1988). CRC Cril. Rev. Biochem. 23, $43-$86. Heuser, J. & Griffith, J. (1989). J. Mol. Biol. 210, 473-484. Howard-Flanders, P., West, S. C. & Stasiak, A. (1984). Nature (London), 309, 215-220. Matsuoka, Y. & Nord~n, B. (1983). Biopolymer.~. 22. 1731-1746. MeEntee, K., Weinstoek, G. M. & Lehman, I. R. (1981). J. Biol. Chem. 256, 8835-8844. Nordc~n, B. & Kubista, M. (1988). In Polarized Spectroscopy of Ordered Syste~, SamorL B. & Thulstrup, E., eds), pp. 133-165, D. Reidel Publishing Company, Dordrecht. Nord~n, B. & Seth, S. (1985). Appl. Spectrosc. 39, 647-655. Peterlin, A. & Stuart, H. A. (1939). Z. Physiol. 112, 1-25. Radding, M. (1989). Biochim. Biophys. Acla. 1008. 131-145. Takahashi, M., Kubista. M. & Nord~n, B. (1987). J. Biol. Chem. 252, 8109-8111. Takahashi, M., Kubista, M. & Nord~n, B. (1989a). J. Mol. Biol. 205, 137-147. Takaha~hi, M., Kubista, M. & Nord~n, B. (1989b). J. Biol. Chem. 264, 8568-8574. Williams, g. C. & Spengler, S. T. (1986). J. Mol. Biol. 187, 109-118.

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