The Escherichia Coli Ruvb Branch Migration Protein Forms Double

Home , Branch migration

Proc. Natl. Acad. Sci. USA Vol. 91, pp. 7618-7622, August 1994 Biophysics The Escherichia coli RuvB branch migration protein forms double hexameric rings around DNA (DNA helicase/three-dmensonal reconsuctlon/dectron microscopy) ANDRZEJ STASIAK*, IRINA R. TSANEVAt, STEPHEN C. WESTt, CATHERINE J. B. BENSON*, XIONG Yul, AND EDWARD H. EGELMANO§ *Laboratory of Ultrastructural Analysis, University of Lausanne, CH-1015 Lausanne, Switzerland; tClare Hall Laboratories, Imperial Cancer Research Fund, South Mimms, Herts. EN6 3LD, United Kingdom; and *Department of Cell Biology and Neuroanatomy, University of Minnesota Medical School, Minneapolis, MN 55455 Communicated by Philip C. Hanawalt, April 22, 1994 (receivedfor review February 25, 1994)

ABSTRACT The RuvB protein is induced in Escherichia MATERIALS AND METHODS coft as part of the SOS response to DNA damage. It is required for genetic recombination and the postreplication repair of Preparation of RuvB-DNA Complexes. The RuvB protein DNA. In vitro, the RuvB protein promotes the branch migra- was purified as described (18) and incubated (at 150 pg/ml) tion of Holliday junctions and has a DNA helicase activt in for 5 min at 370C with relaxed 4X174 DNA (10 pLg/ml), 15 mM reactions that require ATP hydrolysis. We have used electron MgAc, and 1 mM adenosine [y-thio]triphosphate (ATP[y-S]) microscopy, image analysis, and three-dimensional reconstruc- in a 20 mM triethanolamine acetate buffer, pH 7.5. tion to show that the RuvB protein, in the presence of ATP, Eleron Miroscopy. The samples (either RuvB-ATP(y'SJ- forms a dodecamer on double-stranded DNA in which two DNA complexes or RuvB-ATP[(-S]) were stained with 2% stacked hexameric rings encircle the DNA and are oriented in uranyl acetate, and images were recorded under minimal-dose opposite directions with D6 symmetry. Although h a are conditions at either x45,000 on a Phillips CM12 electron mi- ubiquitous and essential for many aspects of DNA repair, croscope or at x30,000 on a JEOL 1200EXII electron micro- replication, and transcription, three-dimensional reconstruc- scope. For scanning transmission electron microscopy tion of a helicase has not yet been reported, to our knowledge. (STEM), suspensions of RuvB-DNA complexes were applied The structural arrangement that is seen may be common to to thin carbon films prepared by the wet-film technique (19). other helicases, such as the shiian virus 40 large tumor antigen. The grids were washed and wicked extensively, blotted to a thin layer of liquid, plunged into liquid nitrogen slush, freeze-dried The ruv locus on the Escherichia coli chromosome contains overnight, and transferred under vacuum to the microscope. three genes (ruvA, ruvB, and ruvC) that are important for Image Analysis. A reference-free algorithm (20) was used genetic recombination and DNA repair (1). The ruvC gene for determining the translations and rotations needed to bring encodes RuvC protein, an endonuclease that catalyzes the individual images of the RuvB complex into a common resolution of Holliday junctions (2-4). The ruvA and ruvB alignment. A hierarchical clustering algorithm (21) was used genes form part of the SOS response to DNA damage and for decomposing global averages into subsets based upon encode the RuvA and RuvB proteins. Together, RuvA and similarity, and a filtered back-projection algorithm was used RuvB promote the branch migration of Holliday junctions in for the three-dimensional reconstruction. All of these proce- a reaction that requires ATP hydrolysis (5-7). Each protein dures were implemented within the SPIDER software package plays a defined role, with RuvA responsible for DNA binding (22). (and, in particular, junction recognition), whereas the RuvB ATPase provides the motor for branch migration (8). Under RESULTS certain in vitro conditions, RuvB can promote branch migration without the need for RuvA (9). To visualize the binding of RuvB to DNA we used in vitro Sequence analysis (10) has identified RuvB as a member of conditions, including the use ofthe slowly hydrolyzable ATP a superfamily of helicases (11), and experimentally it has analog ATP[y-S], that favor the stable association of the been shown that RuvB, in the presence of RuvA, acts as an protein with double-stranded DNA (13). When purified RuvB ATP-dependent helicase in a standard assay where a sub- protein was incubated with covalently closed, relaxed dou- strate consists of a short single-stranded DNA fragment ble-stranded DNA under these conditions, double-ringed annealed to its complementary sequence in a long single- structures were seen on the DNA in the electron microscope stranded DNA molecule (12). The role of RuvA in this (Fig. 1). The DNA must be passing through the center of helicase assay may be to target RuvB to single-stranded DNA these rings, because the rings are always aligned along a because in the absence of RuvA, the affinity of RuvB for common axis. If the DNA were binding to the sides, one single-stranded DNA is very low (13). The apparent require- would expect to see a stagger ofthe rings about the axis ofthe ment by RuvB of RuvA for helicase activity may be similar DNA. We can also exclude any wrapping ofthe DNA, either to the accessory proteins required for certain other helicases: about or within these rings, because contour-length measure- eukaryotic translation initiation factor eIF4A requires eIF4B ments (Fig. 2) indicate that the double-stranded DNA has its (14), E. coli UvrB requires UvrA (15), herpes simplex virus normal, fully extended length, with a 3.4-A rise per base pair. UL5 requires UL52 (16), and herpes simplex virus UL9 Identical structures were observed in cryo-electron micro- requires ICP8 (17). The helicase activity of RuvB may be scopic images of unstained, frozen-hydrated specimens (J. directly involved in the mechanics of branch migration (12). Bednar and A.S., unpublished work), showing that these

The publication costs ofthis article were defrayed in part by page charge Abbreviations: STEM, scanning transmission electron microscopy; payment. This article must therefore be hereby marked "advertisement" ATP[-S], adenosine ['y-thio]triphosphate. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom all correspondence should be addressed. 7618 Downloaded by guest on September 29, 2021 Biophysics: Stasiak et al. Proc. Natl. Acad. Sci. USA 91 (1994) 7619 dissociate from the ends of linear double-stranded DNA. A similar observation has been made for E. coli helicase 1 (23), although the structural organization of that helicase has not been determined. STEM is a very valuable tool for determining the mass of high-molecular-weight complexes (19). Fig. 3a shows a dark- field STEM image of unstained RuvB-DNA-ATP[y-S] complexes, prepared under similar conditions to those used for negative stain in Fig. 1. After a background subtraction, integration of the intensities in the image allows for mass determination because the intensity at any point in the image is directly proportional to the mass. The mean mass found for the doublets, 471,398 ± 3,674 (SEM) Da (Fig. 3b), can be corrected for the DNA included within the circular areas that were integrated. Because the average diameter of the circles integrated was 165 A, this would correspond to 49 bp ofDNA (32 kDa), on average, included in each double-ringed mass measurement. The corrected average mass, 439,330 Da, can be divided by the molecular weight of the RuvB monomer, 37,177 Da (24, 25), to yield an estimate of 11.8 ± 0.2 RuvB monomers per double-ringed complex, where the error in- cludes the uncertainty in the amount ofDNA included in the mass measurements. Images exist (see below) where the two rings are symmetrical in projection (Fig. 4f), indicating an even number, 12, of RuvB subunits in the complex.

FiG. 1. Double rings of the RuvB protein on double-stranded DNA. (Insets) Ten top views of the RuvB rings, which were randomly selected from the 1000 top views analyzed; the top views were obtained from samples prepared both with and without DNA, and no differences were seen. (Bar = 400 A.) structures are not artefacts of uranyl acetate staining, dehy- dration, or adsorption to a substrate. Although it has proven difficult to obtain images in the presence of ATP, similar double-ring structures have been observed after glutaraldehyde fixation of RuvB protein incubated with double-stranded DNA, RuvA, and ATP (data not shown). Under these conditions, however, extensive aggregation was seen, hindering further analysis. Our observations of a double-ring structure formed with ATP, as well as with ATP[y.S], lead us to suggest that this particular multisubunit complex represents the active form of RuvB branch migration motor. The proposal is consistent with the elevated ATPase activity observed with circular DNA com- pared with linear DNA (13), suggesting that the ring structure can continuously translocate around a circle, whereas it will b 15 - 80- C 60 a) C, 10- v 40 aDcL) ci) IZ 5- L 20 0 300 400 500 600

_ I.I.I.. I .- . ., 0...... I I dmmmmm.m.=.I, Molecular weight x 10-3 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 Contour length, ,um FIG. 3. (a) Image of unstained, freeze-dried RuvB-DNA complexes, recorded by STEM (Brookhaven) under dark-field conditions FiG. 2. The contour lengths of 4X174 double-stranded DNA (19). Image intensity is proportional to the number of electrons (5386 bp) circles that were nearly completely covered by RuvB were scattered, and this is directly related to the mass of the specimen. measured, and the maximum contours were -1.83 ,um, correspond- Tobacco mosaic virus particles (TMV) are used as an internal mass ing to a 3.4-A rise per bp. This result indicates that no stretching from standard. (Bar = 400 A.) (b) Histogram ofmass measurements for 192 B-form DNA occurs. Similarly, the contour-length measurements double rings. The mean mass was 471,398 ± 3,674 (SEM). After preclude any wrapping ofthe DNA about or within the RuvB double correction for the DNA included in the measurements (see text), this rings. Approximately 90-95 doublets could be counted on each ofthe indicates that there are 11.8 ± 0.2 RuvB subunits per double ring, fully covered circles. establishing that the double-ringed structure is a dodecamer ofRuvB. Downloaded by guest on September 29, 2021 7620 Biophysics: Stasiak et al. Proc. Natl. Acad. Sci. USA 91 (1994) a ___*~~~~~~~ b c

g n

1800 aa1800 1800 -

15a150 a1500

Cl) C 1200 0) 1209_-12a200 9

I0-0 00

300 300 300

0 a. a a a . a . 0 a. a a a 1 O . a a a . a. . 0 20 406000 100120 140160 0 20 406000 100 120 140 160 0 20 406000 100120 140160 xA x, A x A FIG. 4. (a) Averaged image of 799 double rings, alligned using a reference-free search (20) for the translations and rotations needed to bring the different images into an optimal alignment. The path of the DNA would be vertical, running through the center of the structure from the top to bottom. The average shows a partial mirror symmetry, which arises from a 2-fold axis in the plane of the projection. (b) An average of 1000 top views of the double rings, aligned, and averaged with the same reference-free algorithm. No sigifcant rotational symmetry can be seen in this average. (c) The top-view projection ofthe reconstruction ofFig. 5 shows no 6-fold rotational symmetry and a weak 12-fold symmetry. This result is consistent with the failure to observe a 6-fold symmetry in the average ofb and the presence ofa weak 12-fold symmetry in averages of various subsets extracted from the total population. (d) Model for D6 symmetry in which the two hexameric rings are rotated so that there would be a pseudo 12-fold symmetry in a top-view projection, with no 6-fold symmetry seen at low resolution. The rod through the center of the rings indicates the location of the 6-fold axis, which is also the path of the DNA. The most dissimilar projections of the two rings would occur in the orientation shown, generating the three symmetrical peaks from one ring and the two peaks fr-om the other ring, as shown in g. (e andf) The global average of a has been decomposed into subsets based upon similarity. A threshold was chosen that yielded 12 subsets. Sqix of these subsets together contained only 106 imagesand weresbe'_tsnot simply related to the other subsets. The imkages ithin these six were not used further. The remaining six subsets (containing 693 images) fell into two groups. Three of these subsets (containing 434 images) had an asymmetry between the two rings, and the average of these 434 images is shown in e. Two of the subsets (containing 171 images) had the greatest symmetry between the two rings, and the average of these 171 images is shown inf. The remaining subset, containing 88 images, was in between the average of e andf The one-dimensional projections of e andfare shown in h and i, respectively, with the solid lines showing Downloaded by guest on September 29, 2021 Biophysics: Stasiak et al. Proc. Natl. Acad. Sci. USA 91 (1994) 7621 Because images of individual molecules or macromolecu- equivalent to a 2-fold rotation perpendicular to the plane of lar complexes obtained in the electron microscope suffer projection, with a reversal of the rings. Thus, the averaged from a very poor signal-to-noise ratio, image averaging was subset of Fig. 4e can be used to provide the two most used to generate a reliable picture. Approximately 800 double dissimilar projections ofthe rings. Three-dimensional recon- rings have been aligned and added together (Fig. 4a). This structions of macromolecular assemblies visualized in the average has an approximate mirror symmetry, with one electron microscope are traditionally made by either per- mirror plane running perpendicular to the path of the DNA, forming tilts to collect different projections ofthe structure or and a second mirror plane running along the path ofthe DNA. by examining helical assemblies, where a single image ofthe Each ring is polar, with the mirror plane relating one ring to structure contains many different projections ofthe repeating the other to form a bipolar structure. Deviations are seen subunit that are related by rotations about the helical axis. from perfect mirror symmetry, and we can show that the We have exploited the internal symmetry of the structure to deviations from the mirror symmetry perpendicular to the obtain the required projections. When the two different path of the DNA arise from different rotations of the rings, projections ofthe ring in Fig. 4e are combined with the 6-fold whereas the left-right deviations from the mirror symmetry rotational symmetry of the structure, 12 projections of the about the path of the DNA may arise from an intrinsic ...120-A diameter structure are obtained that can be used to asymmetry in the structure produced by the bound DNA. generate a three-dimensional reconstruction at --30-A reso- Because a biological structure cannot have mirror symme- lution (26). try, we know that the mirror symmetry in projection must A reconstruction generated in this manner is shown in Fig. arise from a 2-fold axis lying in the plane of the projection. 5. An important test is that even though the D6 symmetry has Thus, the double rings are seen to be symmetrical and related been imposed on the reconstruction, the "top view" projec- by a 2-fold axis that is perpendicularto the DNA. This bipolar tion of the reconstruction (Fig. 4c) generates an image that symmetry would limit the rings to double rings, whereas an has no apparent 6-fold symmetry, and only a very weak arrangement where the rings are stacked in a polar fashion 12-fold symmetry, consistent with what is observed in the would allow for the infinite polymerization of rings. Based real "top views" (Fig. 4b), even though this data has not been upon the mass analysis, each ring is a hexamer ofRuvB, and used in the reconstruction. An additional test is that the the only reasonable symmetry for the structure is D6, a reconstruction can generate the mirror-symmetric projection dihedral symmetry relating two 6-fold symmetric structures seen in Fig. 4f, and this projection has not been used, either, by a 2-fold axis that is perpendicular to the 6-fold axis. In in the reconstruction. agreement with this, a gel-filtration analysis ofRuvB showed The reconstruction (Fig. 5) reveals a hollow core, suggest- structures consistent with both a hexameric and dodecameric ing that the DNA is binding stain positively and is thus not form (A. Mitchell and S.C.W., unpublished work). These visualized. The holes at the ends of the structure are =20-25 solution observations were made in the absence of DNA, A in diameter. Because the hollow core is significantly larger consistent with our observations in the electron microscope in diameter in the remainder of the structure, with the next that the double rings do not require DNA for formation. smallest diameter being -40 A at the center, this suggests One thousand "top" views of the double rings were also that the main RuvB-DNA contacts only occur at these ends, aligned and averaged (Fig. 4b). The projection of a structure where DNA enters and exits the rings. Alternatively, or in with D6 symmetry down the 6-fold axis must generate a addition, asymmetric contacts between individual RuvB sub- projection with a 6-fold symmetry; however, this symmetry units and the DNA would not be visualized in the recon- may not be visible at low resolution. Ifthe subunits in the two struction, given the imposition of 6-fold symmetry. It is thus rings were stacked on top of each other, we would expect to possible that contacts might exist between an individual see a strong 6-fold rotational symmetry in this low-resolution RuvB subunit and the DNA at locations other than the two average. However, no 6-fold rotational symmetry can be seen ends. This asymmetry could account for the deviations from in the average ofFig. 4b. In various subsets ofthe top views, left-right mirror symmetry observed in the averaged images. weak 11-, 12-, and 13-fold symmetries were seen (data not shown), suggesting that the two rings may be rotated such that the centers of mass of the six subunits in one ring are DISCUSSION approximately in between the centers of mass of the six This article reports the three-dimensional visualization of a subunits in the other ring; this would generate a pseudo helicase on DNA; some information exists about the orga- 12-fold rotational symmetry at low resolution. A model nization of other helicases. The simian virus 40 large tumor having this arrangement is shown in Fig. 4d. We will show antigen also forms a two-ringed dodecamer, consisting oftwo that a decomposition of the 799 side views into sets based hexameric rings, on DNA in the presence of ATP (27). It is upon similarity confirms this model. quite likely that simian virus 40 large tumor antigen will also Multivariate statistical analysis and hierarchical clustering have the D6 symmetry (with a 2-fold axis between the rings) (21, 22) were used to generate subsets of the average of Fig. that we find for RuvB, because a simple translation between 4a. Because the side views ofthe RuvB dodecamer will have the two rings would lead to the extended polymerization of random rotational orientations on the grid about their central many rings. The E. coli rho transcription termination protein axis (along which the DNA is located), it is expected that is a helicase that forms hexameric rings (28) as well as dodecamers with similar rotational orientations will be clus- dodecamers (29, 30). Interestingly, although the postulated tered into discrete subsets. The greatest asymmetry between D3 symmetry ofthe individual hexameric rho rings (31) differs the two rings should exist when the rings are oriented as from the polar rings that we observe for RuvB, the dodeca- shown for the model in Fig. 4d, with one ring generating three meric organization of rho is believed to occur only in the peaks in projection, and the other ring generating two peaks. presence of an oligonucleotide cofactor that binds to three of The subset of Fig. 4e, containing 434 images of the double the six RNA-binding sites (29, 30). Under these conditions, rings, has this character. A 300 rotation (about the central the individual hexameric rho rings might have apolarity (with axis) of the structure from this position will merely be RNA bound to one side), and the dodecameric form of rho

the projection of the bottom ring and the dashed lines showing the projections of the top ring. The projection in h is very similar to that of the model (d and g) in its displayed orientation. The subset inf has a near mirror symmetry, which is due to the 2-fold axis lying in the plane of the projection. Symmetry suggests that this view is related by a 150 rotation from that in e. Downloaded by guest on September 29, 2021 7622 Biophysics: Stasiak et A Proc. Nati. Acad. Sci. USA 91 (1994)

FIG. 5. Three views ofthe three-dimensional reconstruction ofthe RuvB dodecamer. The structure in the center is ofhalfofthe dodecamer, cut along the plane in which the DNA runs. The hollow central channel ofthe dodecamer can be seen, suggesting that the DNA in the complex is staining positively and is not visualized in the reconstruction. The surface has been chosen so as to enclose the expected volume of the dodecamer, assuming a partial specific volume of protein of 0.75 cm3/g. The subset of Fig. 4e (434 images) generated the 12 views ofthe rings used for the reconstruction (with the image as shown corresponding to projections at 0°, 60°, 120", 1800, 2400, and 300° and with the image rotated by 1800 corresponding to the projections at 300, 900, 1500, 2100, 2700, and 3300). might be similar to that of RuvB, in that it would consist of 8. West, S. C. (1994) Cell 76, 9-15. 9. Mfliler, B., Tsaneva, I. R. & West, S. C. (1993) J. Biol. Chem. 268, two rings of oppositely oriented polarity. The E. coli DnaB 17179-17184. helicase also forms a hexamer (32), but little more is known 10. Lloyd, R. G. & Sharples, G. J. (1993) Nucleic Acids Res. 21, about its organization. 1719-1725. The bipolar symmetry ofthe RuvB dodecamer is surprising 11. Gorbalenya, A. E., Koonin, E. V., Donchenko, A. P. & Blinov, be which can V. M. (1989) Nucleic Acids Res. 17, 4713-4730. because it is to expected that branch migration, 12. Tsaneva, I. R., Miller, B. & West, S. C. (1993) Proc. Nat!. Acad. spontaneously proceed in either direction, would be cata- Sci. USA 90, 1315-1319. lyzed by a polar structure. Moreover, previous studies 13. M~ller, B., Tsaneva, I. R. & West, S. C. (1993) J. Biol. Chem. 268, showed that the DNA helicase activity catalyzed by the 17185-17189. RuvB ATPase occurs with a specific polarity (5'-3') relative 14. Rozen, F., Edery, I., Meerovitch, K., Dever, T. E., Merrick, W. C. & Sonenberg, N. (1990) Mol. Cell. Biol. 10, 1134-1144. to single-stranded DNA (12). Most likely, polarity is provided 15. Oh, E. Y. & Grossman, L. (1989) J. Biol. Chem. 264, 1336-1343. by the RuvA protein, which may bind asymmetrically to the 16. Dodson, M. S. & Lehman, I. R. (1991) Proc. Nat!. Acad. Sci. USA RuvB dodecamer. Alternatively, RuvA may nucleate or 88, 1105-1109. stabilize a hexameric form ofRuvB. During branch migration 17. Boehmer, P. E., Dodson, M. S. & Lehman, I. R. (1993) J. Biol. Chem. 268, 1220-1225. we propose that RuvA, which forms a stable RuvAB complex 18. Tsaneva, I. R., Illing, G. T., Lloyd, R. G. & West, S. C. (1992) with RuvB on a Hollidayjunction (33) plays a dominant role Mol. Gen. Genet. 235, 1-10. in defining biological polarity. It is interesting to speculate 19. Wall, J. S. & Hainfeld, J. F. (1986) Annu. Rev. Biophys. Biophys. that a 2-fold axis relating the two rings may be congruent with Chem. 15, 355-376. 20. Penczek, P., Radermacher, M. & Frank, J. (1992) Ultramicroscopy the 2-fold axis in double-stranded DNA, such that each 40, 33-53. strand ofthe DNA is bound symmetrically by each ring. The 21. Frank, J., Bretaudiere, J. P., Carazo, J. M., Verschoor, A. & reconstruction provides a starting point for understanding Wagenknecht, T. (1988) J. Microsc. 150, 99-115. why all DNA helicases thus far examined exist as multimeric 22. Frank, J., Shimkin, B. & Dowse, H. (1981) Ultramicroscopy 6, structures (34). 343-358. 23. Lahue, E. E. & Matson, S. W. (1988) J. Biol. Chem. 263, 3208- 3215. We thank Joe Wall and Martha Simon ofthe Brookhaven National 24. Benson, F. E., Illing, G. T., Sharples, G. J. & Lloyd, R. G. (1988) Laboratory STEM Facility (National Institutes of Health Biotech- Nucleic Acids Res. 16, 1541-1550. nology Resource) for their invaluable assistance. This work was 25. Shinagawa, H., Makino, K., Amemura, M., Kimura, S., Iwasaki, supported by National Institutes of Health GM35269 (E.H.E.), the H. & Nakata, A. (1988) J. Bacteriol. 170, 4322-4329. Imperial Cancer Research Fund (S.C.W.), and the Swiss National 26. Crowther, R. A., DeRosier, D. J. & Klug, A. (1970) Proc. R. Soc. Science Foundation (A.S.). London A 317, 319-340. 27. Mastrangelo, I. A., Hough, P. V. C., Wall, J. S., Dobson, M., 1. Sharples, G. J., Benson, F. E., flling, G. T. & Lloyd, R. G. (1990) Dean, F. B. & Hurwitz, J. (1989) Nature (London) 338, 658-662. Mol. Gen. Genet. 221, 219-226. 28. Gogol, E. P., Seifried, S. E. & von Hippel, P. H. (1991) J. Mol. 2. Dunderdale, H. J., Benson, F. E., Parsons, C. A., Sharples, G. J., Biol. 221, 1127-1138. Lloyd, R. G. & West, S. C. (1991) Nature (London) 354, 506-510. 29. Geiselmann, J., Yager, T. D., Gill, S. C., Calmettes, P. & von 3. Bennett, R. J., Dunderdale, H. J. & West, S. C. (1993) Cell 74, Hippel, P. H. (1992) Biochemistry 31, 111-121. 1021-1031. 30. Geiselmann, J., Yager, T. D. & von Hippel, P. H. (1992) Protein 4. Iwasaki, H., Takahagi, M., Shiba, T., Nakata, A. & Shinagawa, H. Sci. 1, 861-873. (1991) EMBO J. 10, 4381-4389. 31. Geiselmann, J., Seifried, S. E., Yager, T. D., Liang, C. & von 5. Shiba, T., Iwasaki, H., Nakata, A. & Shinagawa, H. (1991) Proc. Hippel, P. H. (1992) Biochemistry 31, 121-132. Nat!. Acad. Sci. USA 88, 8445-8449. 32. Reha-Krantz, L. J. & Hurwitz, J. (1978) J. Biol. Chem. 253, 6. Parsons, C. A., Tsaneva, I., Lloyd, R. G. & West, S. C. (1992) 4043-4050. Proc. Nat!. Acad. Sci. USA 89, 5452-5456. 33. Parsons, C. A. & West, S. C. (1993) J. Mol. Biol. 232, 397-405. 7. Tsaneva, I. R., Mfller, B. & West, S. C. (1992) Cell 69, 1171-1180. 34. Lohman, T. M. (1992) Mol. Microbiol. 6, 5-14. Downloaded by guest on September 29, 2021