Cryo electron microscopy of unstained, unfixed RecA-cssDNA complexes

JOUAAL

OF ULTRASTRUCTURE

AND MOLECULAR

STRUCTURE RESEARCH 100,

166-172 (1988)

Cryo Electron M icroscopy of Unstained, Unfixed RecA-cssDNA Complexes CHUNG-FU

DEBBY A. RANKERT,’ TZYY-WEN JENG,~ DAVID G . MORGAN, MICHAEL F. SCHMID,~ AND WAH CHIU~,~ Department of Biochemistry, University of Arizona, Tucson, Arizona 85721 CHANG,’

Received October 23, 1987; and in revised form June 29, 1988 Complexes of RecA protein with $X174 circular single-stranded DNA (cssDNA) with and without ATP$S were rapidly frozen and embedded in a thin layer of vitreous ice. The electron micrographs of these frozen-hydrated complexes clearly show visible helicity. Quantitative image analyses of these micrographs reveal the helical pitch and the axial rise between DNA bases of these complexes. Both of these structural parameters of RecA-cssDNA complexes increase significantly when ATPyS is present. These observations agree qualitatively but not quantitatively with those from negative stained specimens and confii the general model that the interactions among RecA molecules and between RecA and DNA could change according to the functional states of the RecA-cssDNA complex. o 1988 ~detttk press, IW.

The RecA gene in Escherichia coli has been shown to be essential for homologous genetic recombination (Clark and Margulies, 1%5), DNA repair (Howard-Flanders and Theriot, 1966), and the expression of SOS functions (Radman, 1975). Biochemical studies showed that the RecA gene product (mol wt 37 842) possesses multiple enzymatic functions including (i) DNAdependent hydrolysis of nucleoside triphosphate (Weinstock et al., 1979), (ii) ATPdependent pairing of DNA strands (McEntee et al., 1979; Weinstock et al., 1979; Shibata et al., 1979), and (iii) inducing the autocleavage reaction of LexA in the presence of ssDNA (single-strand DNA) and ATP (or ATPyS) (Little, 1984). A complex of RecA and single-stranded DNA is the prerequisite for synapsis to occur during the recombination process (Kahn and Rad’ Present address: Loral Corp., Defense Systems Div., P.O. Box 85, Litchtield Park, AZ 85340. 2 Present address: University of Michigan, Department of Biophysics, Ann Arbor, MI 48109. 3 Present address: Abbott Laboratories, Abbott Park, IL 60064. 4 Present address: Baylor College of Medicine, Department of Biochemistry, Houston, TX 77030. ’ To whom correspondence should be addressed.

ding, 1984). Nucleoside triphosphate is not necessary for forming these complexes (Flory and Radding, 1982; Dunn et al., 1982; Koller et al., 1983). It has been shown that RecA will dissociate from the complex during ATP hydrolysis and subsequently reassociate with the complex at a slow rate (White and Strand, 1982). ATPyS [adenosine 5’-(y-thio)triphosphate] is a nonhydrolyzable analog of ATP and the RecA-ssDNA-ATPyS complex may represent an intermediate conformation of the RecA-ssDNA-ATP complex prior to the ATP hydrolysis (McEntee et al., 1981). A reliable structural model of RecA-ssDNA with and without ATPyS would be helpful for understanding the mechanism of this complex process. Electron microscopic studies of complexes of RecA and single-stranded DNA formed in the absence of ATP or ATPyS have been carried out under a number of mixing and chemical conditions (Flory and Radding, 1982; Dunn et al., 1982; Koller et al., 1983). In those reports, specimens were prepared either by the metal shadowing technique or by the negative staining method. It is possible that the negative staining could limit the structural resolution

166 0889-16OY88$3.00 Copyr&ht 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

CRY0 EM OF RecA-cssDNA COMPLEXES IN VITREOUS ICE

or alter the structural features. Therefore, we have undertaken a study of RecAcssDNA (circular single-stranded) complexes formed with and without ATPyS which were preserved in a frozen, hydrated state in vitreous ice, without fixative or stain. In order to evaluate the difference in results, between the negative stain and the ice embedding techniques for these specimens, we performed the same experiment with negative stained samples. We have followed the same m ixing and buffer conditions as those of Williams and Spengler (1986). The helical pitch and the axial separation between DNA bases were calculated to characterize these RecA-cssDNA complexes with and without ATPyS embedded in vitreous ice and in negative stain. MATERIALS (a) Incubation

AND METHODS

of RecA and cssDNA Complexes

RecA was purchased from U.S. Biochemical Corp. Circular single-stranded DNA purified from +X174 virion (5386 bases) was purchased from New England BioLabs. The incubation of RecA and cssDNA was carried out at 37°C for 30 min with a RecA to DNA ratio of 100:1 or 4OO:l (w/w). The buffer used was 20-25 m M triethanolamine (TEA), pH 7.4-7.6, and 1 mMMg2+. !b) Electron Microscopy

The frozen-hydrated specimens were prepared by the perforated film method previously described (Adrian et al., 1984). Holey grids were prepared according to Fukami and Adachi (1%5). A layer of carbon film was vacuum deposited on the support net of the holey grid immediately prior to use for specimen preparation. The specimen was applied to the grid and blotted off with filter paper. Using a guillotine device the grid was immersed rapidly in liquid ethane cooled by liquid nitrogen. The frozen specimen was transferred to a cold stage via a specially designed airlock in a JEOL 1OOCXelectron microscope equipped with a top entry configuration (Taylor and Glaeser, 1975). Our cold stage (Jeng and Chiu, 1987) was a modification of that originally designed by Hayward and Glaeser (1980). The temperature of the stage was maintained at - 148°C during the experiment. Images were recorded at a magnification of x 26 000 or x 33 000 with a dose of less than 10 electrons/A’. TMV particles were mixed with the specimens. The position of the characteristic “23-A layer line” in the optical or computed Fourier transform of TMV images was used as an internal calibration of magnification.

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The objective lens was underfocused by approximateiy 1.5-2.0 pm so that a high value of contrast transfer function was produced at the resolution of interest. Uranyl formate, used as the staining reagent, was prepared at a concentration sufficient to give an absorbance value of 0.09 at 450 nm (Williams, 1981). Micrographs of negatively stained complexes were also recorded with the same electron microscope at room temperature. TMV particles were included for an internal calibration. (c) Computer Image Processing

Images of RecA-cssDNA complexes were digitized with a Perk&Elmer PDS microdensitometer. A spot size (and sampling distance) of 25 +m was used corresponding to 9.6 or 7.6 A at the specimen, depending upon the magnification of the micrograph. Computer image processing was performed on a VAX-11/750 computer system. The digitized image was displayed on a raster-graphics display system (AED 512, AED Inc., CA). Portions of RecA-cssDNA complexes which showed obvious zigzag patterns were chosen for further computer analysis. The curving of the helical axes of the complexes makes direct computer image processing with conventional Fourier analysis impractical (DeRosier and Moore, 1970). These curved fibers were straightened with an algorithm similar to that described by Steven et al. (1985). The helical pitch of the fibers was determined by the position of the major layer line in the computed Fourier transform of the straightened fiber. The contour lengths were measured by a simple algorithm interfacing a digitizer board (COMPLOT 7000) to the VAX system. The micrographs were first enlarged three times into a print so that the error of the measurement due to tracing the curves was less than 3%. RESULTS

It has been suggested that RecA alone can form fdamentous aggregates (McEntee et al., 1981; Flory and Radding, 1982; Cotterill and Fersht, 1983; Morrical and Cox, 1985) with a helical nature (Stasiak and DiCapua, 1982). We used +X174 circular single-stranded DNA to complex with RecA in order to ensure that the compiexes contain the DNA. We have employed the incubation conditions used by Williams and Spengler (1986). The helical pitch for negative stained RecA-+X174 DNA complexes was 59 ? 4 A and the contour length was 0.96 + 0.07 pm. Upon addition of ATP$S, these parameters became 94 + 3 A and 1.83

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+- 0.15 pm, respectively. The pitch measurements were averaged from more than 200 turns of different filaments and the contour lengths were determined from over 10 circular complexes. These results are in good agreement with those of Williams and Spengler (1986). In order to ensure that the protein concentration used was sufficiently high to fully saturate the DNA, we have also tested the effect of increasing the protein:DNA ratio by a factor of 4. This increase did not significantly affect our results. Images of frozen-hydrated complexes of RecA and cssDNA without and with ATPyS are shown in Fig. 1. The images show good contrast between the proteinDNA complex and the background vitreous ice. The low-resolution features of the helical fiber are clearly discerned. The high contrast arises from the difference between the scattering density of the protein-DNA complexes and that of water and from the values of the defocus used. The absence of a layer of carbon support film under the specimen further enhances the image contrast. The helical pitch, contour length, and DNA axial rise of these complexes embedded in vitreous ice are shown in Table I. The contour length measurements were made from at least 30 RecA-cssDNA complexes. The axial rise between DNA bases is derived by dividing the contour length of the complex by the known number of DNA bases. An increase by a factor of 1.7 in axial rise between DNA bases is seen in the presence of ATPyS. The helical pitch measurements are derived from areas of circular complexes on different micrographs that had been straightened as previously de-

scribed. The total number of helical turns used in the pitch determination is indicated in Table I. A 27-A increase in helical pitch is seen when ATPyS was added after the complex was formed. Fourier transforms of the straightened fibers show only one consistently observed layer line with high amplitude relative to its neighboring background. The layer line corresponds to 102 and 75 A when complexes are formed with and without ATPyS, respectively (Table I). With the defocus used for recording images, the contrast transfer function reaches zero at a resolution around 30 A. We have not been able to detect any high-resolution structural information in these complexes partly, at least, because of their flexible nature. In our study, the resolution of image data is not sufficiently high to determine the number of DNA bases and of RecA monomers per helical turn. DISCUSSION

Negative stain electron microscopy has been employed by a number of investigators to study the RecA-cssDNA complex. We have confirmed that our sample preparations are the same as those of Williams and Spengler (1986) by repeating their experiments in negative stain. Our results agreed with theirs. The frozen-hydration specimen preparation technique provides an unique means of observing specimens in a defined chemical environment (Unwin and Ennis, 1984; Chiu, 1986).Observations derived from frozen specimensare more likely to reflect the true structure of specimensin solution. Our recent work on the 3D reconstruction of TMV embedded in vitreous ice in compar-

FIG. 1. Electron images of RecA-+X174 cssDNA complex in a buffer solution of 1 mkf w+, 20-25 mit4 TEA, pH 7.4-7.6 (A) in the absence of ATPyS and (B) in the presence of 0.4 mM ATPyS and 5 mM MgZf. Complexes were rapidly frozen in liquid ethane at a temperature of - 170°C. The frozen specimens were imaged at - 148°C with a total electron dose received by the specimen of less than 10 e/AZ. There is no fixative or stain present in the buffer solution. The contour length and the helical pitch of the complex thus formed were significantly larger than those of the complexes formed in the absence of ATPyS (see Table I). The scale bar represents 1000 A.

CRY0

EM OF RecA-cssDNA

COMPLEXES

IN VITREOUS

ICE

169

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CHANG ET AL. TABLE I

STRUCTURALPARAMETERSOF RecA-cssDNA COMPLEXES Axial rise Specimen RecA-+X174 RecA-+X174 w/ATPyS

Pitch (4

Contour length (pm)

75 f 2 1.21 2 0.06 (30) (356) 102 k 1 2.02 2 0.19 (39) (312)

between DNA bases (A) 2.2 3.8

ison with that determined from X-ray fiber diffraction analysis has helped establish the reliability of this ice embedding procedure (Jeng et al., unpublished). According to our measurements, the helical pitches of frozen-hydrated complexes of RecA-$X174 cssDNA and of RecA-$X174 cssDNAATP-yS are larger than those of the corresponding negatively stained specimens by 8a (27%) and 9a (lo%), respectively. We have routinely mixed tobacco mosaic virus (TMV) particles with our specimens as an internal calibration of the magnification of the electron microscope. In addition, we have analyzed 15 separate images of TMV particles and found that the standard deviation of the 23-A layer line in the optical transforms is less than +0.5 A and the inferred microscope magnification differs from the microscope reading by no more than 3%. In our recent work on TMV embedded in vitreous ice, we have found that the TMV, used in our helical diffraction analysis, was tilted usually no more than a few degrees. Moreover, the morphology of the TMV was recognizably different when it was tilted to more than 0.5”. Therefore, the TMV particles used for calibration were not appreciably tilted. Consequently, any discrepancy due to significant error in magnification of the microscope can be ruled out. A plausible explanation for the finding that the negative stained complexes have smaller pitches is that the air-drying process or chemical alteration by stain caused complexes to shrink. However, the differ-

ence in the structural parameters between frozen-hydrated and negatively stained RecA-cssDNA-ATPyS complexes is less than the difference for the complex without ATPyS. This suggests that the extent of discrepancy between negative staining and frozen-hydration is specimen and specimen-state dependent. A potential error in the contour length measurement is the possibility of the nonplanar orientation of the specimen embedded in vitreous ice with respect to the electron beam. This may result in an underestimate of the contour length and, consequently, the axial base rise. We estimate that only 2% error would have been introduced in the contour length even with a specimen tilted by IO”. Another possible source of error in contour length measurement is the formation of secondary structure in the cssDNA (Koller et al., 1983; Williams and Spengler, 1986). Because the region of the secondary structure would not be complexed with RecA (Williams and Spengler, 1986) the measurement of the contour length will be smaller if such secondary structure regions are present. The extent of the formation of secondary structures in the complex can be controlled by manipulating the incubation conditions, such as Mg2+ concentration and temperature. The salt concentration used in our experiments is sufficiently low that the amount of secondary structure in the cssDNA should be minimal (Kowalczykowski and Krupp, 1987). The difference in sensitivity to negative stain between the two complexes and the pitch difference between the two complexes embedded in ice provides further evidence to support the belief that there is a difference in the protein packing in these two functional states (Dunn et al., 1982; Williams and Spengler, 1986). This change may reflect a change in the number of RecA proteins per helical turn. The resolution of our data is not sufftciently high to allow us to verify unambiguously this possibility.

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CRY0 EM OF RecA-cssDNA COMPLEXES IN VITREOUS ICE

This type of molecular rearrangement among the proteins may result from a slight conformational change of the RecA protein upon binding of its nucleotide ligand. It has been shown crystallographically that the quaternary structure of a protein can be altered upon its binding to a ligand (Baldwin and Chothia, 1979; Unwin and Ennis, 1984). Although the pitch distance is altered by as much as 27 A, the protein conformation or protein-protein interactions need not necessarily change a great deal. Moreover, the change in axial rise of DNA with and without ATPyS is seen in our ice embedded complexes. This confirms qualitatively but not quantitatively the observation from negative stained complexes that the DNA conformation is altered within the complex upon the binding of ATPyS.

We thank Drs. E. DiCapua, Robley Williams, Jack Grifftth, and Howard White for valuable suggestions and discussions. This research has been supported by NIH research Grants GM27061 and RR02250. REFERENCES ADRIAN, M., DUBOCHET, J., LEPAULT, J., AND McDOWALL, A. W. (1984) Nature (London) 308, 37-

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CONCLUSION

This study shows the feasibility of visualizing an unstained, unfixed RecA-cssDNA complex with and without ATPyS, embedded in vitreous ice. In the past few years, there have been a number of studies utilizing the vitreous ice embedding technique. In many instances, the use of this technique gives rise to no improvement in structural resolution, which is likely lim ited by the periodicity of the specimens. This is also true in our RecA-cssDNA complex. However, we have found that the structural parameters used to describe the complex can be different between the negative stain and the ice embedding preparative methods. This must be taken into account when evaluating the negative stained data even at low resolution. Our analysis confirms the general belief that this complex can assume different conformations dependent on its functional states. Furthermore, this study suggests the feasibility of examining RecA complexed with other macromolecular components, such as dsDNA or repressors, embedded in ice in order to determine their structural conformations.

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