J. Mol.

Biol.

(1990)

214.

557-570

Complexes of RecA Protein in Solution A Study by Small Angle Neutron

Scattering

Elisabeth DiCapua’, Manfred Schnarti, Rob W. H. Ruigrok3 Peter Lindner’ and Peter A. Timmins’ ’ Institut

Laue-Langevin,

156X,

38042 Grenoble

Cedex, France

’ Institut de Biologic Moleculaire et Cellulaire, CNRS 15, rue Descartes, 67000 Strasbourg, France 3EMBL, (Received

156X,

38042 Grenoble

Cedex, France

9 October 1989; accepted 16 March

1990)

RecA complexes on DNA and self-polymers were analysed by small-angle neutron scattering in solution. By Guinier analysis at’ small angles and by model analysis of a subsidiary peak at wider angles, we find that the filaments fall into two groups: the DNA complex in the presence of ATPyS, an open helix with pitch 95 8, a cross-sectional radius of gyration of 33 A and a mass per length of about six RecA units per turn, which corresponds to the state of active enzyme; and the compact form (bound to single-stranded DNA in the absence of ATP. or binding ATPyS in the absence of DNA, or just the protein on its own), a helical structure with pitch 70 8, cross-sectional radius of gyration 40 .& and mass per length about five RecA units per turn, which corresponds to the conditions of inactive enzyme. The results are discussed in the perspective of unifying previous conflicting structural results obtained by electron microscopy.

1. Introduction Purified RecA protein from Escherichia coli (M, 37.8 kDa) has several activities in vitro that reflect it,s role in recombination (strand exchange between 2 DNA substrates: for a review, see Howard-Flanders et al., 1984; Radding, 1988; Griffith & Harris, 1988) and in the SOS response (proteolysis of the LexA repressor; for a review, see Little & Mount, 1982). Both reactions are DNAdependent and require the presence of ATP. RecA is also a DNA-dependent ATPase, with a turnover of t,he order of ten per minute (Weinstock et al., 1981). For these processes, the active species is the complex of RecA with DNA, single-stranded or double-stranded, in the presence of ATP. This complex has been studied extensively by biochemistry and electron microscopy (for a review, see Cox $ Lehman, 1987; Stasiak & Egelman, 1988), resulting in models where a helical sheath of protein

t Abbreviations used: dsDNA, double-stranded DNA; ssDru’A, single-stranded DNA; ATPyS, adenosine-5’-0($thiotriphosphate); SANS, small angle neutron radius of gyration; scattering; R,, cross-sectional M/L, mass per unit length; *H,O. deuterated water; Em. electron microscopy; bp, base-pair(s); nt. nncleot~ide. 0022-:!83fi/9oil405.57-14

$03.00/O

557

covers the DNA: six monomers of RecA form one turn of about 95 A (1 A = @1nm) pitch, covering 18 base-pairs of dsDNAT, or 18 to 36 nucleotides of ssDNA (the stoichiometry to ssDNA is controversial). In the absence of ATP, the complex with dsDNA is not formed, and the complex with ssDNA is inactive (for a review, seeStasiak & Egelman, 1988; Radding, 1988). In the absence of DNA, RecA selfpolymerizes into filaments (Flory & Radding, 1982; Williams & Spengler, 1986; Takahashi et al., 1986; Brenner et al., 1988). Electron microscopy suggests a more compact helix for both the ATP-free complex with ssDNA and the self-polymer, but the measured structural parameters vary strongly. It has been suggested (Chang et al., 1988) that the compact structure is very sensitive to mounting constraints (fixation, adsorption, stain), as samples in amorphous ice have a much higher helical pitch (75 A) than samples in negative stain (60 8). The method of small-angle scattering complements electron microscopy by analysing the structure in solution in the absence of these constraints. In the Guinier range (Guinier &. Fournet, 1955), the intensity of the signal is proportional to the mass of the object, while the slope of the intensity versus scattering angle is related to its overall mass distribution, i.e. its radius of gyration (for a review, see 0

1990 Academic

Press Limited

558

E. DiCapua

Jacrot’ & Zaccai’, 1981). This analysis has been extended to rod-like particles (Luzzati, 1960), when their length is more than about five times larger than the cross-sectional dimensions, and this approach has been successful in analysing, for example, DNA in solution by X-ray scattering (Luzzati, 1960), and filamentous phages by neutron scattering (Torbet et al.. 1981). At wider angles, the scattering reflects molecular details within the particle: in the caseof particles with helical symmetry, the internal regularit,y of the st*ructure may produce pronounced maxima and minima, Here we study by small-angle neutron scattering (SANS) all the filaments and complexes mentioned above in solution, in the absence of the constIraints of elect.ron microscopy, using the latter as a qualitative control.

et al p,,260mn

= 6.3 (Silver & Fersht. 1982). ins wril as pilo*: phatr analysis (Ames. 1966). Samples for neutron scattering wert’ prepared I)! mixing the protein (usually at a conrn of around 4 mg!/ml. i.e. 0.1 mM) with DN’B when occurring (d&N11 at 4.5 bp/Recg. ssDNA usually at 9 nt/Rec;1) in a bufiia~ containing 20 mM-pOtaSSiUm phosphate (pH 6.8). 3 m\rmercaptoethanol. .5 to 10~~0 plprerol. 0.1 rn~-EDT;\. Where appropriate, magnesium acetatr was added 10

2 mM, and STPyS (BoehringerMannheim) to 0.5 IIIM. .U samples were incubated at, 37°C for 30 to 60 min I)rior to filling into Hellma c&ells of I mm pathlrngth (for H,O samples) or 2 mm pathlength (for 2H20 samples). .lfttar that, they were at temperat,ures between 10 and 2o.Y fol, several hours (up to 48 h) except during data c~ollec+$m when they were thermost,atioally c~ontrolled. usually at 16°C. Data caollrction was in cyrles. so that all samples were measured at’ early times and at lat,rA times: 1%~ observed no significant cahanpes in the data betwren rarlh and late (*y&s.

2. Materials and Methods RecA protein (M, = 37.8x 103) was extracted from strain pDR, 1453/KM 4104 according to Weinstock et al. (1979). The purity after phosphocellulose column chromatography was considered sufficient: the A,,,/A,,, r&o was between 1.6 and I.7 (Craig & Roberts, 1981). suggesting no major contaminant: no change in this ratio nor in specific ATPase activity could be obtained bJ either DEAE-cellulose, Sephacryl 300 or single-stranded DFA-cellulose chromatography. suggesting that what may be won in chemical purity is concomitantly lost by inactivation of the product. Heavily overloaded. Coomassie-stained SDSlpolyacrylamide gels reveal contaminating bands. none of which exceeded 1 o/6 of the main band intensity upon scanning on a Shimadzu CS 930; evaluation of the RecA band at a peak threshold led to a figure of 82y0 RecA. of 0.5 7; (of the main band) As staining of gels is not quantitative over a broad range of concentrations or between different protein species. this value is at best indicative. Elect’ron microscopy also gives an idea about the purity of the protein: quite clear backgrounds were obtained when full binding to DNA was observed. An interesting particle was sometimes observed in the presence of ATPyS: the star-shaped rings seen in the background of Figs 7 and 9. They are strongly reminiscent of the particles with 7-fold symmetry formed by GroE protein of E. co&i (Hendrix. 1979;Hohn et a.1.. 1979). Bands around 65 kDa are present on gels from our preparations (at, intensities lower than lo/b of the protein content), compatible with a contamination with GroEL. The presence of contaminants leads to an overestimation of the RecA concentration. The protein concentration was determined using an extinction coefficient E”0*‘77’m of 6.33 (Tsang et al.. 1985): using (Craig & Roberts, 1981) increases the E’ 0”,2*0”m =5.9 value by 5%. Plasmid DNA was extracted by a cleared lysate with Triton X-100; calf thymus DNA was from Sigma: singlestranded phage MI3 DNA was from phage mpll banded in CsCl (d = 1.3). All DNAs were re-purified by protease and RNAase treatment, extractions with phenol and precipitation with ethanol. Etheno DNA was prepared from melted calf thymus DNA as described by Silver & Fersht (1982), and purified from the reagent by extensive dialysis and repeated precipitation with ethanol. Its concentration was determined b.v absorbance using

SANS data were c*ollected on the instrument, I)1 1 (Ibrl. 1976) at the Tnstitut Laue-Langevin. (irenoble. Samplrs were contained in quartz cauvettes (Hellma) of pathlength 1 or 2 mm and thermostatically controlled. lnridrnt neutrons of wavelength J = 10 .A were selected by means of a helical slot velocity selector (A;./A = 8”,,). Th(s 2-dimensional multidetect,or was placed at a tlixt,ancahereforr. some rxprrirnents were performed also in 2H20 (yielding /L’,): the low intensity peak at wide angles c~~ulti IJC measured reliably only in ‘H,O. Second, the concentration of our samples \vas always close to 4 mg/ml. At’ this conc~cntration. the calculated interparticle distanchefor i\. hypot hrtic*al monomer of RecA (Mr 38 x 103) would be 250 ,A: for the gia,nt complex particle rnade of a rnolec*ulr of plasmid p1’(‘8 DSA (2700 bp) and 1 Il,~~l tinit, I1t.r’ 3 bp (t’otal MT= 10 x 106), there i:, an il\~ailabl~~ space of about 12 x IO9 A$3 per partic~lc~ and. assuming parallel rods of length 14 ,LL,a. 10(H)4 interparticle distance. Of course, the actual solution is lessideal. Test cbxprriment,s were performed in the range of c~oncentra~tionsacacessiblcto satnpltb prc’paration. i.e. up to 6 mg/ml. and to rc~asonabl~~ neutron data c*ollec4ion,i.r. above I rng/‘rnl: thcJrc% is no sign of partial aggregation. whi& ~voultl iw recognized us a strong rise in intensit)y at vtlry IOM angles. and no cahangrsin &her Et, valur or the cxtrapolatIed intensit,y at zero angltl (Pig. 2), l’h~ lack of conczentration dependence was c*hrckcd fi,r t.he self-polymer. the caomplex with sinplr-stranded Ml3 phage I)?;A and the complex with donblrstranded DSA. in H ,O as well as in ‘H,O. In aI1 cases, the shapt~of the>plot did not (+hiltlgStb. nor (lid the paratnet~rrs rxt~ra~ctrd from it vary bt~~~ontlt,l~ statistical error. and we c~onclutlethat int~~rpartic~lc~ interference was Iqligiblc undt>r our rxperimrntnl conditions.

First, all samples contained between 5 and IOO:, glycerol; glycerol interferes with the hydration layer of macromolecules, and thus introduces a complication in the interpretation of the scattering patt’ern. Glycerol, however, was a necessary component in the buffer to ensure stability of the structures as measurements extended over long periods (up to 2 days); early on, we found that in the absence of glycerol, samples could be degraded, which was sometimes recognized by the fall-off of the Guinier plot at very low angles (Fig. l(a)); however, the size of the degraded particles was such as to suggest an apparent cross-sectional radius of gyration, R, = 35 A. Such samples, when inspected by electron microscopy, contained particles of 100 to 200 a as a dominant species (Fig. l(b)); the self-polymer was lost altogether, and complexes with DNA were partially disintegrated. The binding activity. as assessedby etheno DNA fluorescence enhancement. was markedly decreased (not shown); however, the protein was not degraded when inspected by SDS/

Third. fi)r solution t~xperimt~ntIs.it is itnpurt~a~lf to avoid it mixture of bound and frtacbRec*,4. In thts presence c,f ATF’yS. the cbomplrxchsapptaar to fi)rm quantitatively and virtually irreversibly (rat her than to be in t’hermodynamic binding t~quilibriunr). This makes it easy to assesscsomplesformation t)?, DXasr protec+iorl assays. ATI’@ ticc%J-s sponta neously with time. and may btl slowly hydrolyscd by the ATPase act.ivit,y of fhe protein. This would lead to dissociation of t,he complex and possibly t h(a appearance of degraded particles similar to those> discuss4 aho\-P. However. controls with I)Nast~ resistanct>at thth rnd of’t,hP data c*ollec*tionas well ah by electron micaroscopysuggest that this was tlot a major problem in our experimentas: a triinor degr>1dation ma.y account, for some of t)he \rariat ion of t htt values we measured with different’ samplcls. In t)hr a.bsencbe of nurleot)ide co-facator. however. the complex with single-stranded DNA is in therm0 dynamic eyuilibriurn. This binding (Latlnot t)r, assayed absolutely by DNase resistanc~r~.as onI). t’he rate of DNA hydrolysis is decreased (Kryant ~1

Mass Distribution

z i; s

A

A

32 e

v

561

in RecA Complexes

x

v

$

30

I

I

I

I

I

I

I

I

I

2

3

4

5

6

7

0

9

I

0

I

I

I

5

ICI

15

I

I

I

I

1

20

25

30

35

40

Temperature

0

0

I

I

I

I

1

2

3 4 Concentration

I

I

,

5

6 (q/ml)

I 7

I 0

I 9

Figure 2. The parameters deduced at low angles are not dependent on concentration in the range used for analysis. The values of R, and [I(&)&]a-o/C were determined for a series of samples that were diluted sequentially from one original mixture (to avoid quality differences). The different symbols indicate different series. (A, V, v) complexes with dsDNA and ATPyS; (0) self-polymer without ATP; (0) self-polymer with ATP. The values on t’he scale Mass/len th are only indicative as most data were collected in QH,O (0, 0, V), where an accurate calculation is not possible (see the text), but the scale expansion is accurat’e (according to relative [Z(Q)]Qa&’ of each sample). The error in the R, value is about kO5 !L The error due to plot statistics in M/L is negligible (- $001 RecA/lOO A) as compared to the variation due t,o sample.

(1,1.. 19%). The binding to etheno l>XA can be measured by fluorescence enhancement, but the binding constant to etheno DNA is much higher than to unmodified ssDNA (Menetski 62 Kowalczykowski, 1985). We used a novel approach to measure the extent of binding: the contour length of complexes with circular ssDNA as determined by electron microscopy (to be published elsewhere). Briefly, the contour length varies systematically with protein concentration until it reaches a plateau above O-5 mg/ml; it varies with DNA/protein stoichiometry, again up to a plateau at 6 nt/RecA; and it varies considerably with temperature (Fig. 3), approaching saturation binding only above 25°C. Both t,he high concentration of samples for SANS and the use of excess DNA allow us to extrapolate that most RecA was bound under the conditions used for data collection. One control experiment was performed with a sample at 9 nt/RecA; both the Guinier plot and the subsidiary maximum remained unchanged at temperatures of 16”C, 25°C and 37°C.

("C)

Figure 3. The temperature dependence of RecA binding to ssDNA in the absence of ATPyS, as determined by the contour length of complexes with phage Ml3 DNA (7237 nt). Complexes were formed at 150 p-n (0) or 15 PM (0) RecA protein (6 or @6 mg/ml) and 6 nt/RecA in the buffer used for the SANS experiments. They were incubated for 10 min at the given temperature and fixed with @l y0 glutaraldehyde at the same temperature (for IO min (35 to 4O”C), 30 min (20 to 30X”), 4 h (5 to 15°C)). Sample (*) was formed at 37°C and then left to re-equilibrate at 22°C overnight; it was found to have the same contour length as on the curve above, suggesting that the effect is not due to the kinetics of binding. The error bars indicate the standard deviation of the average from about, 30 molecules.

As evaporation/condensation and stability of protein at temperatures above room temperature were a potential problem, routine measurements were performed at’ 16°C. Finally, all solution scattering experiments had to be performed at low Mg*+ concentrations (1 to 2 mM). Upon addition of 10 mM-Mg*+, the plots become non-linear, reflecting aggregat’ion. and this unfortunately precludes analysis of the structure under conditions of the strand exchange reaction (where aggregation is thought to be part of the reaction mechanism; for a review. see Cox & Lehman. 1987; Radding, 1988). (b) Self-polymers

of RecA protein

At the concentrations used for SANS, RecA forms rod-shaped self-polymers as illustrated in Figure 4(c) and (d), obtained by fixation of the SANS sample at high concentration, prior to dilution for electron microscopy. On the other hand, the scattering curve shows a slight drop in intensity at very low angles, indicating that the average particle is not very long (Fig. 4(a)); it is, however, at, least five times longer than it is wide, as model calculations suggest (not shown). The Guinier plot gives a crosssectional radius of gyration R, of 40 A and a mass per unit length corresponding to six to seven RecA units per 100 A (Table 1). These values and the shape of the plot were indistinguishable for the selfpolymer in 20 mM-phosphate alone, or in the presence of 2 mlvr-magnesium acetate, or in the presence of 2 mM-magnesium acetate and @5 mM-ATPyS, as

562

I3:. l)iC’apua

et al.

(~ 8) no ATPyS

(0)

&=41.3(&0.6)j h’10],+o=O~02113~0 0 Y G

5 ,c

‘ ,

-5,5

(A) + ATPyS

-

,. 2s 0

R, =40,2

(_tO,S)

a

,‘.

[~~~~~lp~o

0 n

0~00039

C =6 mg/ml

0 a

O0

0

I

O-0005

0

.?

=0~02113~

0

0

‘1.

O

1 a00 &. 0

1

0~0010

‘-

o&z-

0.08

Q2(a-2,

0

(a-’

Figure 4. Analysis of self-polymers of RecA. (a) Solution scattering in the Guinier range for self-polymers in thtl absence (0) and in the presence (A) of ATPyS. Note that’ the intensities for the 2 samples are identical: the plot (A) has been displaced by 025 unit for ease of visualization: the 2 lines a.re virtually indistinguishable. The parameters deducaed from these typical plots are shown as insets. (b) Subsidiary maximum at wider angles for the same samples (measured in ‘H,O). The scale of lnI(&) is in arbitrary units but has t,he same expansion throughout Figs 3. 6 and 9. (c) and (d) Illustrative electron microscopy of t’he very samples of RecA self-polymers used for SANS data collection. fixed and adsorbed at the end of the experiment. demonstrating the presence of rods of similar appearance (c) in the absence and (d) presence of ATPyS. while the optical diffraetion suggests that the structure is helical. well as in the presence of Mg2+ and 1 OI 10 mM-ADP, suggesting that no strong change in conformation and state of aggregation take place with these ligands under these conditions. ATPyS is bound ate a stoichiometry of about one per RecA unit. as seen after equilibrium dialysis by absorbance (Fig. 5) (where, however. possible hyper-

Synoptic

Table 1 table of the results obtained Self-polymer

A. Cross-sectional ATPyS B. Mass~length

ATPyS (‘. Position ATPyR

3*1* 397+

Complex

radius 1.1(17) 1.7(10)

(RecA

64 & 0.6(5) 6.2kOqlO)

of dub&diary 2n/63 2n/63

with

by SAN8

ss Complex

with

ds

of gyration

R, (A) 393+ 1,4(15) 33?5& 1.3(21)

units/100

33.3 + 1.5(9)

if)

63 &O%(6) 58i:O3(12) maximum

(A ‘) 2x/63 21~175

*55_fO.3(9)

2X/75

The values shown are the average from several experiments (number shown in parentheses) and the error is the standard deviation of the mean (as opposed to the statistical error of a single experiment as in Figs 4, 6 and 9).

chromicity effects do not allow us to deduce t,hr precise stoichiometry) and by the counts of radioact’ive ATPyS (where the value is imprecise due to degradation of ATPyS; results not shown). The same experiment performed with ,4I)P. i.e. equilibrium dialysis of 0.1 rnM-RecA against 1 mar-ADP. showed that under these conditions less than 0.1 equivalent of ,4DP is bound (not shown). At wider scattering angles, we observe a peak with a maximum at & = 0097 = 2x/63 (A ‘) (Fig. 4(b)). In the presence of ADP, the subsidiar! maximum is superimposable with that of a RNA self-polymer in the absence of nucleotide (not shown). Optical diffraction from elect’ron micrographs was not very st,rong (example in Fig. 4(c). inset) reflecting a low degree of order, but inspection of 16 such diffract,ograms (each imperfect ppr SP) suggests a helical structure similar to that of t)hr compact complex with ssDNA (discussed below). (c) (‘otnplexeS There exist, conditions of complex in the complex in the

with single-stranded

IlsL4

under the two types of complexes our experiments: the “cornpart ‘I absence of ATP$3, and the “open“ presence of ATPyS.

Mass Distribution

Wavelength

result mainly emphasizes that a mixture of selfpolymers and complexes shows the same structural parameters, hence the two structures are very similar at this resolution. Electron microscopy suggests that whatever the stoichiometry, the local structure (width and helical repeat) does not change on the complexes (not shown). At wider scattering angles, we observe the peak of Figure 6(b) at Q = 2x163 (A-‘). The parameters of the compact complex (R, = 405( +05) 8, M/L = 6 to 7 RecA/lOO A, subsidiary peak at 2711638-l) are also found in the presence of ADP (and Mg), both at 1 mM and 10 mM-ADP (not shown). However, ADP is presumed to lead to dissociation of the protein from the DNA; indeed. electron microscopic studies of these samples showed that at 10 mM-ADP, no cirrular complexes could be found on Ml3 phage DNA: instead, the protein was present as self-polymer rods (not shown). The self-polymer having the same compact structure, this effect could not be detected by SANS. Electron microscopy was performed to control the extent of binding (Fig. 7(a)). It also illustrates the appearance of the compact structure. However, an electron microscopy artefact becomes evident here (and was demonstrated by Chang et al., 1988): adsorption under negative staining conditions usually leads to shrinkage of the structure. Figure 7(b) (a micrograph taken of an unfixed sample) illustrates this phenomenon: the particles have a mixed appearance, a mixed contour length, and two different optical diffractions; the dark molecules are short and display a helical pitch of 59( )2) A (n = 16); the lighter circles have a contour length longer by a factor of 1.2, and their helical pitch is 75( +4) a (n = 23). Samples in amorphous ice (Chang et al., 1988) have a pitch of 75A. Thus. negatively stained specimens of this type of complex should be considered with caution.

(nm)

Figure 5. ATF’yS is bound to the protein under the conditions of SANS data collection. Samples (at about 2 mg/ml; 50 ELM) of RecA were dialyzed to equilibrium and their absorpt.ion spectrum recorded against the dialysis buffer in a 2 mm pathlength cell (normalized to 10 mm for t,he absorbance in the Figure). (-) RecA in the absence of ATPyS; ( - - -) RecA in the presence of 0.5 mMATPyS: (. ) the difference spectrum is compatible with I mol of ATPyB hound per mol RecA.

(i) Pompacf complex

The compact complex has an R, value of about 40 14 and a mass per length between six and seven RecA/lOO A. These parameters do not change between the complex in buffer alone or in the presentaeof 2 rn~-Mg’+ (not shown) and they are identical within error to the values of the self-polymer (Table 1 and Fig. 6(a)). No difference could be detected between complexes with Ml3 phage DNA or etheno DNA (not shown). The same plots were obtained also with “complexes” at lower DNA/RecA ratios (Bnt/RecA, Snt/RecA, not shown). As, under these conditions, not all RecA is expected to be bound (see section (a), above), this

(0)

no ATPyS

R, q 39,5 ;,

563

in RecA Complexes

(20.5)

8

-4.5

o.ooo39

0 x .-;; 5

(4) -5.O-

+ ATPyS I

R,=33.6(?06)8 [IlO)

Ql,,,

= 0.02306

+ 0*00032

: ’

C=6mg/ml

a

t

L 0

0~0005

0~0010 0’

t8-‘1 (0)

Figure

0.0015

(b)

6. Analysis of complexes with single-stranded DNA. Solution scattering in (a) the (iuinier range and (b) subsidiary peak at wider angles for complexes with phage Ml3 DNA (9 nt/RecA) in the absence (0) and in the presence (A) of ATPyS. The plot (A) has been displaced by @25 unit for ease of visualization. Inl(Q) in the wide-angle plot is in arbit,ra.ry units (cf. Fig. 4).

564

E. DiCapua

et

al

(b)

Figure 7. Electron microscopy of complexes with single-stranded DKA. absence of ATPyS which illustrates the extensive binding of the protein experiment, (@7 nt/RecA; 4 mg/ml). although some self-polymers are also

(a) Electron micrograph of thr complex in the in this sample fixed at the end of the SANS present. (b) The shrinkage usually observed in

Mass Distribution

565

in RecA Complexes

(ii) Open comp1e.x The open complex is obtained in the presence of ATPyS. SANS measures an II, value of about 33 A and a mass per length of five to six RecA units/ 100 A (Table 1 and Fig. 6(a)). No significant difference in these values was detected when different single&randed DNAs were used for complex formation: freshly melted calf thymus DNA (2 experiments), Ml3 phage DNA (6 experiments), and etheno derivat’ized calf thymus DNA (6 experiments). In order to assessthe significance of the difference of the t,wo radii of gyration between the compact and the open struct.ure, we performed a titration of RecA with ssDNA in the presence of ATP#; we expected the R, value to go from the self-polymer value (40 A) to the open complex value (33 A) at the titration

point,

with

a linear

combination

excess Dh’A

is not’ detrimental

’ ’ ’ 0123456709 Input

’

’

’

!

’

stoichiometry(nt/RecA

’ (O),

’

II 12

bp/RecA

(0)

Figure 8. The compactform changesinto the openform upon titration with DPjA in the presence of ATPyS. (0) Etheno calf thymus DNA, (0) pUC8 &DNA.

of the two

numbers at intermediate points. Figure 8 shows the result. The experiment was performed with etheno DNA because this allowed us to follow the extent of binding by fluorescence measurements; one experiment with ssM13 phage DNA gave the same result (not’ shown). A titration point is observed at about three to four nucleotides per RecA unit, compatible with some published stoichiometry values in the presenceof ATPyS. This experiment also shows that to the structure

for example; leading to very

321

by,

short clusters of

protein.

At wider angles. the scattering from the open complex is characterized by a peak at Q = 2x175 (A ’ ) (Fig. 6(b)). Electron microscopy of this type of complex reproducibly gives an optical diffraction for a helix with 95( +5) A pitch, and visualizes the open structure as a clear zigzag (Fig. 7(e)); its local structure does not appear t,o vary with the stoichiometry

(not, shown). (c) The complex with double-stranded DNA in the presenceof A T PyS Only c!omplexes stabilized with ATPyS have been observed with dsDNA by electron microscopy, even after fixation. Neutron scattering is not able to distinguish the self-polymer from the compact complex; hence, we cannot contribute to t,he analysis of hinding to the double strand in the absenceof ATP?/S. Indeed, we observe the parameters of the self-polymer in a control experiment with dsDNA in the absence of ATPyS. The complex with dsDNA in the presence of ATPyS appears identical with the open filaments ohserved with ssDNA in the presence of ATPyS: R,

is about 33 A and the massper length is five to six RecA/lOO A (Table 1 and Fig. 9(a)). A detailed report on the Guinier analysis of this complex, in particular the location of the DNA in the axis of the filament, has been described (DiCapua et al., 1989). At higher angles, the typical peak at & = 2~175 (A-‘) is found, indistinguishable from the one with ssDNA complexes, paralleled by the diffraction from electron micrographs (Fig. 9(c)). These complexes are very reproducible and stable and were therefore used for attempts at. orientation. The purpose of these experiments was to try to relate the pronounced subsidiary maximum found in solution scattering to particular features of the RecA filament structure. Figure 10 shows the scattering

pattern

obtained

from

a sample

flow-oriented

in a rotating Couette cell (Lindner & Oberthiir, 1984) in a shear gradient of 500 s- ‘. The scattering pattern was unchanged at shear gradients from 50 to 5oOOs-‘, indicating that maximal orientation had been obtained

(albeit,

not a perfect

alignment,

see Fig. 10). The broad maxima are clearly due to repeating features along the axial direction of the filaments; they do not in themselves show the existence of a helical structure. The presence of intensity on the meridian is, at first sight, difficult to explain, as optical diffraction from electron micrographs shows a helical pattern that is characterized by only off-meridional intensity. Such a pattern can only be found, however, with perfectly aligned filaments. Intensity on the meridian can be explained by disorder in the plane of rotation of the Couette cell. In our experimental set-up there is a significant disordering of the filaments in the plane of rotation due to the torque induced by the shear gradient and

negative stain: complex (i) “shrunk”, with optical diffraction (A) (ii) showing 60 A pitch, complex (iii) more native, with optical diffraction (B, 75 A pitch) and mixed appearanceof complex. Unlike all other micrographspresentedhere, this sample was prepared at O-4 mg/ml and 6 nt/RecA and not fixed for microscopy (because of dilution the self-polymers disaggregatcd). Samples that were fixed always showed the “shrunk” appearance as in (i) and (a). (c) Electron micrograph of the complex in the presence of ATPyS and its optical diffraction (C). The contour length of the filaments is not

bars

significant as there was DNA were obtained from catalase.

excess

present

(9 nt/RecA).

Also.

t’he circular

phage

DNA

was often

broken.

The

scale

E. DiCapua

566

et al

(bi .

- . . 5+5

. . l

.

. l *e.

.

. .

l * l

. .

.

. .

.

. . 6

,

Figure 9. Analysis of the complex with double-stranded subsidiary peak at wider angles obtained from a complex InI in the wide-angle plot is in arbitrary units (cf. Fig. diffraction.

.4

of ATPyS. (a) (:uillit~t~ plot and (l)J Dh’A in the presencr with linear pl~(“8 DNA at 4.5 hp/Rt~xA ( 1.,5 x 1)X:\ rscLt,ss). 4). (c) Elect.ron micarograph of thr sample atltl ((1) its opticaal

to the How being on a c*ylindrical straight’ path. Therefore. we

rather than a conclude t,hat the diffraction of the flow-orient,ed samplr is compatibk with a helical structure. but the disordering means that we are unable to derive accurately the helical parameters of the filaments as the t)rue position ot the layer-line and it,s radial maxima, clannot b( determined. The dat,a; however. do allow us to put limits on the helical pitch and in part,icular t,o shoM that it is consistent wit,h the parameters derived from electron micrographs. i.e. a pitch of 05( & 5) a. Moreover, it confirms that the subsidiary maximum observed in solution scattering arises from these helical features of the structure as no other significant intensity at, similar & values is found. for example. on the equator (where signals arising frotn cross-sectional features would be). 1 ots-‘,

0

0.02

0.04

..-...I 0.06

(d) 0.08

C’alaulation

of modA

scattcriug

curws

0.10

Figure 10. Neutron diffraction pattern of flow-orient,ed solutions of double-stranded complexes in ATP$3. The d-dimensional spectrum was recorded in laminar shear flow at a gradient of 500 s-l and is a computer-generated prey-scale representation of the scattered intensity. The broad meridional maxima are clearly visible. Because of the Couette cell geometry, the meridian is horizontal. The white square in t,he middle is the beam-stop.

The experimental result’s described abovr rprrai that RecA polymerizes into t)wo stSruc%urrs, the compact filament, in the absence of either DNA or ATP or both, and the open filament, upon binding to any DNA in the presence of ATPyS. Csing programs described in Materials and Methods, the theoretical scattering c~nrrea from arrays of spheres were computed and c*omparpd

Mass Distribution

567

in RecA Complexes

I.0 G < -1.0 r -3.0 . 0

004

0.08 Ot8)

0.12

(a)

-3a

-

0

0.04

0.08

0.12

O(8)

Compact

Open

5 RecA/turn 70 X pitch

6 RecA/turn 95 X pitch

Figure

12. Sketch of the result from the SANS study.

(b)

Figure 11. Comparison of curves calculated from models (smooth lines) to the scattering data points. (a) Compact form. The data points are from the complex with ssDNA in t’he absence of ATPyS in ‘Hz0 (analogous to Fig. 6(a) + (b)). The full line is the scattering curve calculated for a model with 5 RecA units at a radius 35 a per turn of a helix with 70 ,% pitch. The broken lines show the effect, of changing the pitch to 65 or 75 A. (b) Open form. The data points are from the complex with dsDXA in the presence of ATPyS in ‘Hz0 (analogousto Fig. 9(a) + (b)). The full line was calculated for a model with 6 KecA units at a radius 28 L%per turn of a helix with 95 A pitch. The broken lines show the effect of rhanging the pitch to 90 or 100 a.

with the experimental data, i.e. the combined spectra at low angles and at higher angles. (i) Co7npactstructure As the helical structure is apparent in the electron microscope, we decided t’o interpret the high angle scattering from RecA solutions as being due to that from helical rods and to determine the parameters of this helical structure. About 30 models were tried, varying the radius from 25 to 40 A, and the pitch from 60 to 80 A: the best fit to the observed data is shown in Figure 11(a). This fit comes from a model in which five RecA subunits are arranged on a helix of pitch 70 A. each subunit centre being located 35 A4from the helix axis. The lowest angle data is well-modelled as is the position of the maximum, although the intensity is not so well explained. (‘hanging t.he pitch value by 5 w up or down leads to

the two broken curves shown in Figure 1 I (a), giving an idea of how strong the contribution of pitch is to the scattering curve at those angles. The spheres themselves (of radius 22.5 8) and the distance between them (about 45 A) will not) lead to strong peaks until further out (Q values of - @14 A- ‘, not shown). (ii) Open structure Again, the curves were computed for many models, varying the pitch and the radius. The best fit was obtained (Fig. 11(b)) from a model having six subunits per turn of a helix of pitch 95 w with each subunit located at 28 a from the helical axis, confirming the existing models and image reconstructions from negatively stained electron micrographs (Egelman & Stasiak, 1986). Again, the lowest angle data tits perfectly. and the position of the peak is correct.

4. Discussion The results from the SANS study are summarized in the sketch of Figure 12: the self-polymer of RecA (with or without ATP@) and the inactive complex with single-stranded DNA form a compact structure with five RecA units per turn of a helix with 70 A pitch and a cross-sectional radius of gyration of 40 8. The complex formed with DNA (singlestranded or double-stranded) in the presence of ATPyS is an open helix with six RecA units per turn

568

E. DiCapua

of 95 d pitch and a cross-sectional radius of gyration of 33 A. Small-angle neutron scattering is not a very common method due to the scarcity of experimental facilities. Its power lies in the fact that samples are st,udied in aqueous solution with non-damaging radiation, thus the macromolecules and assemblies are as native as they can be. Data collection has been optimized on the instrument Dll of ILL: a relatively high neutron flux and a detector with many elements allow scattering curves with good statistics to be collected and the intensity curves are therefore quite reliable. One weakness is the rather long time involved in data collection, which may lea,d to changes in sample quality. Aware of this, we have monitored data at early versm late time: and biochemical quality before and after the experiments. and observed no systematic changes (discussed in Materials and Methods and Results). However, a considerable variation in the results exists beyond the counting statistics of the scattering experiments. This variation expresses itself in the statistical error of the average values (Table I). which amounts to a few per cent for t,he radius of gyration, and in the order of 109;, for the mass per lengt’h. The computation of the mass per length from the intensity relies as much on biochemical values (concentrations) as on scat,tering dat’a. The values given in Table 1 were collected over a period of about three years with several preparations of the protein and at varying glycerol concent,rations (between 5 and lOf/,). The purity of the protein and the level of binding activity may have varied within a range of 80 t,o 900/, active protein (not worse, as activity tests were performed routinely and showed plateau values). The protein concentration does not, show the concentration of “good” protein, i.e. protein involved in regular st,ruct)ures, but, includes inactive heterogeneous protein that should br subtracted as background for correct analysis. Furthermore, the concentration of glycerol goes into the mass per length calculation wit,h an effect, of several per cent compared to pure aqueous solvents; however. the concentration of glycerol at the surface of the prot,ein and its interaction with the hydration layer cannot be controlled. Taken together, these errors allow the mass per length to be up to 20% larger (mainly due t,o the error in active concentration) and 50/;, up or down due to salvation effects, allowing 52 to 7 RecA units pet 100 ‘4 for the ATP complex. and 5.9 to 7.8 RecA units per 100 A for the compact complex. We have changed t,he average values of M/L (Table 1) by about. I So/b t,o sketch the models in Figure 11. choosing to equate the result’ in solution to the results of electron microscopy for the open helix. This is a subjective decision that we defend bJ the fact that the stru&ural parameters of t’hat complex have not been found to vary. and six RecA units per turn of pitch 95 A have been found from several points of view: cont’our length and biochemical stoichiometry (DiCapua, 1986; DiCapua rt

et d. al., 1982), mass per lengt’h by STEM (DiC’apua r/ u/.. 1982), image reconstruction (Egelman & St,asiak. 1986) and (i-fold symmetry of sideways associat iota (Stasiak rf al., 19X3: Egelman &, Stasiak. 1988). Without, this correction. the JZ//i value would imply a reproducible shrinkage of the open complex by about 18Y,, in electron microscopy. A slight shrinkage by 6i”;, has been put) forward from c*r,vo electron-microscopy by Chang it nl. (19%) whet1 t’hey measured contour length and helicsal pitch. The peak at Q = 2475 (.A ‘) in neutron sc*att,ering is fitt)ed best by models with a pii.ch of 95 4, Ijut. ~ultl accommodate a few per cent change>. It c~)uld no1 accommodata> 1124 (if 18°d shrinka~gfb Ir>ads to 95 A). I ncidentally. model-fitting is inscnsitivv to changes in :W/I, in thfx order of No,, a,s far as t h(i position of the peak is concerned. The solut)ion result excludes the structure \vit lr two Re(aA units per 95 A proposed by (‘hrysogelos cjt ~1. (1983). It is also in contradiction with th~~~rnodrl of four base-pairs per RecA unit if WC’ a(bccpt that t,here a,re 1X ba,se-pairs per turn (as found by Stasiak & I>i(‘apua, 1982; Dombroski of ~1. 19x3: C’hysogelos of al.. 1983: Pugh et al.. 1988). sincrb ow RNA unit- per’ four basr-pairs will give- an ,V//, value of 4.5 HecA units per turn of 18 basC>-pairs. it value t)oo IOU- by, an extra IO?,, c~omparrd to tlic lowest c*alculation from neutron scattering. Neutrorl scatt,ering has the pnt,mtial to locate I)SA within a protein assembly. as the scattering densit>- of the t.wo compounds differ. However, in complexes with RecA, DNA rna,kes up only 1.50;, of t.hr mass in double strand complexes, and between 2.5 and 7.,5’“,, in single strand complexes depending orI ttw st,oichiometry (3 to 8 nucleotides per RecLA unit ). which precludes precise detection. I)onhle-st,rand~:d l)NA was found localized near the a,xis of t’hr* complex when using deut,rrat,cd DNA (lX(‘a,pua rf nl., 1989). which was confirmed 1)~ imagtt recoil struc+on (Egelman X- Yu, 1989). The mass per lcngt,h of the compact form is fi)untl to be greater by about II”;, t,han that of t.hc open form, Although t*his difference is not beyond thr variability of our measurements. it tn;iy br signiti-. cant as it was usually found when doitq ~1 svriva of samples u-it h one preparat,ion of RecA. Howrvc>r. ~1 difference of 12 OC,does not ac*count for “stret thing” of the one helical form into thca other upon binding of ,L\TP (or 1)X=\ and ATT’). as this would imply a differenc*cl of about 300/,, (‘&retching” t hc) mass from 70 A to 95 w). M’e must c~oncludt~ t,hai the transition of one form t,o t’he other involvcls t+thrr ?I concomit,ant rot,ation around the axis of the tiltsrnent (t,o incorporate protein from the ncxi turn) or a reshuffling of t’he structure involving t hr addition of more R,ecA: indeed. this is not surprising irr view of the different st,oic:hiomrt,rirs in t’he compac4 singl(a strand c*ornplex as compared with the, ATI’ form (Ruigrok & t)i(lapua, unpublished result~s: Mrnet,ski & Kowalczykowski. 1985: Bryant ~1 ~1.. 19X5: Takahashi B Schnarr. 1989: Takahashi 4 crl.. 1989). Xlternatively, the compact form reflect~s t.hr (son formation of RecA after ATP hydrolysis. 1 hts loss it)

Mass Distribution mass per helical turn possibly due to release of some of the RecA concomitant with the release of ADP. The pitch of t,he two forms in solution is found to he 70 and 95 8, with an imprecision of about 5%. This allows for some spring-like flexibility of both structures, but excludes intermediate values. It also excludes the low values found for the compact form by negative stain electron microscopy. Apparently, the latter method leads to a shrinkage of the complex, as shown by Chang et al. (1988) by cryomicroscopy, and explains the wide variation of the pit,ch found so far by electron microscopy of negatively stained samples (64 a by Stasiak & Egelman (1986): 45 a by Dunn et al. (1982), 55 a as well as 75 A by Williams & Spengler (1986)). The self-polymer in the presence of ATPyS was found to be in the compact conformation (R, = 40 L% and pitch = 70 8). ATPyS is bound under these conditions (our results and Cotterill et al., 1982). We must therefore conclude that the binding of the cofactor alone does not induce the conformational change, which occurs only upon synergistic binding of both DNA and ATP. McEntee et al. (1981) have observed by electron microscopy long helical filaments of the same appearance as the ATPyS stabilized complex with DNA (the open helix); Register & Griffith (1985) have proposed these filaments to be due to contaminating polynucleot’ides. Williams Ik Spengler (1986) d rscribe the self-polymer in t’he absence and the presence of ATPyS as indistinguishable. with a helical repeat of about 75 A (in contrast to t’he helitaal repeat of 95 a in the complex wit’h DNA). Tn our SASS experiments, the El, value and the clear maximum at wider angles are of the compact type. However. closer inspection of the spectrum of t,hr self-polymer in the presence of ATPyS (Fig. 4(b)) shows that the minimum is less pronounced, indicating a partial structural change as compared to the self-polymer without ATP. Tndeed. prelitninar? results indicat,e that the SANS maximum typical for the compact form will move to the position for the open helix upon addition of salt)s. paralleling the induction of the DNXindependent, XTPasp activitv (Pugh OzCox, 198X). Tt is conceivable that other ions and pH may have similar effects that mimic the binding of DNA. leading to an activation of the protein. RrcA is perfectly active for strand exchange and LexA cbleavagein the 20 mlcl-potassium phosphate

(pH 6.X) buffer used for the neutron scattering experiments (E. DiC’apua, unpublished results, and evident from the literature). ATPyS is thought t’o be a faithful strucatural replacement for ATP (Flory et nl.. 1984: Sogo et (cl.. 1987; Pugh & Cox, 1988), suggesting that that open complex with DPU’A is indeed the presynaptic complex active in strand exchange (Radding, 1982). Our solution study suggests to DNA

that the whole population is in this conformation,

of RecA bound which may have

been quest>ionedon the basis of ele&on microscopy alone (since the technique itself may select molecules through e.g. preferential adsorpt,ion to the grid).

569

in RecA Complexes

In summary, solution studies by small-angle scattering have confirmed the model of an open helical structure for the complex of RecA with DNA in the presence of ATP, which is active as a strand transferase, an ATPase and in the induction of LexA cleavage. Under conditions where these activities are silent, RecA is found in a compact form with clearly different structural features. We thank Dr M. Takahashi who took part in the early experiments and Dr Andrzej Stasiakfor useful comments. We also thank Ed Egelman, Paul Langan, Trevor Forsyth, Watson Fuller, Jim Torbet. David Blow and Stephen &sack for helpful discussions on helical diffract,ion. This project was supported by EMBC) with short term and long term fellowships to E.D.C.. who also thanks Th. Koller and the Institute for Cell Biology. Ziirich, as well as D. Leach and t’he Department of Molecular Biology, Edinburgh. for support in early and late stages of the project.

References Ames, B. N. (1966). Methods Enzymol. 8. 155-118. Brenner, S. L., Zlotnick, A. & Grifit,h. J. D. (1988). J. Mol.

Biol.

204,

959-972.

Bryant,

F. R.. Taylor, A. R. & Lehman. I. R. (1985). J. Biol. Chem. 260, 1196-1202. Chang, C.-F., Rankert, 1). A., Jeng, T.-W.. Morgan, D. G.. Schmid. M. F. & Chiu, W. (1988). ,J. (~ltmstrwt. Mol. Strut.

Res.

Chrysogelos.

100, 166-172.

S., Register,

J. Biol.

Chem.

258,

,J. C. & Griffith.

,J. I). (1983).

12624-12631.

Cotterill, S. M., Satterthwait,. A. (‘. & Fersht. A. R. (1982). Biochemistry, 21, 4332-4337. VOX, M. M. & Lehman, I. R. (1987). Annu. Ku/:. Biochem. 56, 229-262. Craig, N. 1,. & Roberts,