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The Escherichia Coli Ruvb Branch Migration Protein Forms Double

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The Escherichia Coli Ruvb Branch Migration Protein Forms Double 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 migra- tion 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] com- plexes, 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 in- cubated with double-stranded DNA, RuvA, and ATP (data not shown). Under these conditions, however, extensive aggregation was seen, hindering further analysis. Our obser- vations 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 com- plexes, 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.
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