Visualization of two binding sites for the Escherichia coli UmuD′2C complex (DNA pol V) on RecA-ssDNA filaments1

doi:10.1006/jmbi.2000.3591 available online at http://www.idealibrary.com on

J. Mol. Biol. (2000) 297, 585±597

Visualization of Two Binding Sites for the Escherichia coli UmuD02C Complex (DNA pol V) on RecA-ssDNA Filaments Ekaterina G. Frank1{, Naiqian Cheng2{, Chat C. Do2, Mario E. Cerritelli2 Irina Bruck3, Myron F. Goodman3, Edward H. Egelman4 Roger Woodgate1* and Alasdair C. Steven2 1

4

The heterotrimeric UmuD02C complex of Escherichia coli has recently been shown to possess intrinsic DNA polymerase activity (DNA pol V) that facilitates error-prone translesion DNA synthesis (SOS mutagenesis). When overexpressed in vivo, UmuD02C also inhibits homologous recombination. In both activities, UmuD02C interacts with RecA nucleoprotein ®laments. To examine the biochemical and structural basis of these reactions, we have analyzed the ability of the UmuD02C complex to bind to RecA-ssDNA ®laments in vitro. As estimated by a gel retardation assay, binding saturates at a stoichiometry of approximately one complex per two RecA monomers. Visualized by cryo-electron microscopy under these conditions, UmuD02C is seen to bind uniformly along the ®laments, such that the complexes are completely submerged in the deep helical groove. This mode of binding would impede access to DNA in a RecA ®lament, thus explaining the ability of UmuD02C to inhibit homologous recombination. At sub-saturating binding, the distribution of UmuD02C complexes along RecA-ssDNA ®laments was characterized by immunogold labelling with anti-UmuC antibodies. These data revealed preferential binding at ®lament ends (most likely, at one end). End-speci®c binding is consistent with genetic models whereby such binding positions the UmuD02C complex (pol V) appropriately for its role in SOS mutagenesis.

*Corresponding author

Keywords: DNA repair; electron microscopy; homologous recombination; SOS mutagenesis

Section on DNA Replication Repair, and Mutagenesis National Institute of Child Health and Human Development, Bethesda, MD 20892-2725, USA 2

Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda MD 20892-2717, USA 3

Department of Biological Sciences, Hedco Molecular Biology Laboratories University of Southern California, Los Angeles, CA 90089-1340, USA Department of Biochemistry and Molecular Genetics University of Virginia Health Sciences Center, Charlottesville VA, 22908, USA

Introduction {These authors contributed equally to the work. Present address: I. Bruck, Laboratories of Molecular Biophysics, The Rockefeller University, New York, NY 10021, USA. Abbreviations used: ATPgS, adenosine-50 -(3thiotriphosphate); CSPD, disodium-3-(4-methoxyspiro [1,2-dioxetane-3,20 (50 -chloro) tricyclo (3.3.1.1.3,7) decan]4-yl) phenyl-phosphate; EM, electron microscopy; pol V, E. coli DNA polymerase V; PMSF, phenylmethylsulfonyl ¯uoride; ssDNA, single-stranded DNA; dsDNA, doublestranded DNA; SOS mutagenesis, error-prone translesion DNA synthesis. E-mail address of the corresponding author: [email protected] 0022-2836/00/030585±13 $35.00/0

The Escherichia coli recA locus was identi®ed in the early 1960s by Clark & Margulies (1965) who discovered that mutations in recA rendered the cell highly sensitive to UV-irradiation and unable to promote homologous recombination between F-plasmids and chromosomal DNA (Clark, 1996). Since that time, the RecA protein has been extensively characterized (Roca & Cox, 1990; West, 1992; Kowalczykowski et al., 1994; Roca & Cox, 1997). In addition to its role in promoting homologous recombination, RecA is known to positively regulate the damage-inducible SOS response by mediating the cleavage of the LexA transcriptional

586 repressor (Little & Mount, 1982; Friedberg et al., 1995; Koch & Woodgate, 1998). RecA also plays at least two roles in damage-induced error-prone translesion DNA synthesis (SOS mutagenesis) (Blanco et al., 1982; Nohmi et al., 1988; Sweasy et al., 1990) - a ``last resort'' pathway that is mobilized by the cell to allow DNA synthesis across otherwise replication-blocking lesions, albeit at a cost of relatively low ®delity. The ®rst such role appears to be indirect in that, like LexA, RecA mediates the cleavage of UmuD-like proteins to generate the mutagenically active UmuD0 -like proteins (Shinagawa et al., 1988; Burckhardt et al., 1988; Nohmi et al., 1988; Woodgate & Levine, 1996; McDonald et al., 1998). The second role imputed to RecA is direct (Sweasy et al., 1990; Bailone et al., 1991), whereby it targets functionally active UmuD02C complexes to DNA lesions where they can interact with b-subunits of DNA polymerase III (Sweasy et al., 1990; Bailone et al., 1991; Frank et al., 1993; Tang et al., 1998, 1999, 2000; Sutton et al., 1999). Such an interaction forms a so-called ``mutasome'' that is able to promote error-prone translesion DNA synthesis (Woodgate et al., 1989; Echols & Goodman, 1990). The requirement for RecA in translesion synthesis is most evident in the reconstituted system (Tang et al., 1998, 1999, 2000): wildtype RecA promotes ef®cient translesion synthesis, whereas a speci®c mutant of RecA (RecA1730) that has a reduced capacity to interact with the Umu proteins shows no bypass activity (Tang et al., 1998). Although at ®rst glance, these activities appear quite disparate, it is likely that they both originate in formation of a RecA nucleoprotein ®lament (Takahashi et al., 1996). For example, after DNA damage, single-stranded DNA (ssDNA) gaps are generated as DNA polymerase II reinitiates downstream from a replication-inhibiting lesion (Rupp & Howard-Flanders, 1968; Rangarajan et al., 1999). Indeed, it is thought that the regions of ssDNA which are generated during the cell's attempts to replicate damaged DNA constitute the SOS-inducing signal (Sassanfar & Roberts, 1990). Upon binding to DNA (in the presence of ATP), RecA achieves its ``activated'' state (DiCapua et al., 1992; Hewat et al., 1991) and becomes pro®cient for all of its known functions (Roca & Cox, 1997). Since RecA nucleates more avidly on ssDNA than on dsDNA (Lu & Echols, 1987), it seems likely that the LexA and Umu-like proteins have evolved to recognize such nucleoprotein structures, thus serving as biosensors of chromosomal damage (Woodgate & Sedgwick, 1992). During homologous recombination, dsDNA resides within the deep helical groove of a RecA ®lament (Egelman & Yu, 1989). LexA also binds in this groove (Yu & Egelman, 1993), and its binding can inhibit homologous recombination (Harmon et al., 1996; Rehrauer et al., 1996). A similar inhibition of recombination is seen when the UmuD02C complex is overexpressed and RecA is maintained at its normal repressed levels (Boudsocq et al.,

Binding of the UmuD 02C Complex to RecA

1997). Such a phenotype could potentially be explained by UmuD02C binding to the same site as dsDNA and LexA protein (Woodgate & Levine, 1996) or alternatively, by binding to the tips of RecA ®laments so as to impede their elongation (Boudsocq et al., 1997; Sommer et al., 1998). Recently, Rehrauer et al. (1998) have used surface plasmon resonance spectroscopy to show that maximal binding of UmuD02C appears to occur at a stoichiometry of one UmuD02C complex per two RecA monomers. They also demonstrated that upon binding UmuD02C, RecA's ability to mediate LexA cleavage and strand-exchange was impaired, leading them to propose that the UmuD02C binding site overlaps that of LexA and dsDNA in the RecA ®lament's deep helical groove. Here, we have attempted a more direct characterization of the binding of UmuD02C complexes to RecA ®laments. After establishing the threshold of saturating binding by a gel mobility shift assay, we visualized them by cryo-electron microscopy. Under these conditions of observation, specimens are preserved in their native states (Dubochet et al., 1988). To characterize the distribution of UmuD02C complexes along ®laments at subsaturating binding, we performed immuno-electron microscopy by negative staining. These observations are discussed in the context of the roles attributed to the UmuD02C complex in homologous recombination and SOS mutagenesis.

Results Stoichiometry of UmuD02C-RecA binding at saturation Previous studies have demonstrated that the UmuD02C complex binds to RecA-coated DNA (Bruck et al., 1996). Such binding can be directly visualized by staining DNA-agarose gels with ethidium bromide (Figure 1(a)). Binding of RecA to ssDNA results in greatly reduced mobility compared to free DNA, and the further addition of UmuD02C produces high molecular weight structures that hardly enter the gel. To determine the stoichiometry of the UmuD02C-RecA-ssDNA interaction, we utilized a gel mobility shift assay, with immuno-detection of the Umu proteins (Frank et al., 1993). Control reactions, in which ssDNA, RecA, or both reagents, were omitted allow us to identify the electrophoretic mobility of free UmuD02C, an ssDNA-UmuD02C complex or a RecA-UmuD02C complex (Figure 1(b)). In the experimental reactions, the amounts of ssDNA (20 mM) and RecA (6.7 mM) were kept constant at levels chosen to maximize ®lament formation (which occurs at a ratio of one RecA monomer per three nucleotides ssDNA). The amount of UmuD02C subsequently added was varied from 0.3 mM to 6 mM (Figure 1(b)). At lower concentrations, most of the UmuD02C complexes, as detected by anti-UmuD0 (Figure 1(c)) and anti-UmuC antibodies (data not shown), migrated at a position that is consistent

587

Binding of the UmuD 02C Complex to RecA

Figure 1. (a) Mobility of a ssfX174 DNA in the presence of RecA and UmuD02C. DNA nucleoprotein mixtures were prepared as described in Materials and Methods, separated in a 1 % agarose gel and the DNA stained with ethidium bromide. Under these conditions, free ssDNA migrates several centimeters into the agarose gel. In contrast, the RecAssDNA nucleoprotein ®laments have limited mobility and addition of saturating amounts of UmuD02C to the reaction results in nucleoprotein complexes with virtually no electrophoretic mobility. (b) Electrophoretic mobility of UmuD02C in the absence of ssDNA and/or RecA protein. DNA nucleoprotein mixtures were prepared as described in Materials and Methods and separated in a 1 % agarose gel. The relative location of UmuD02C was determined by transferring the nucleoprotein complexes to an Immobilon P membrane that was probed with antisera raised against UmuD0 and subsequently visualized using the CSPD-Western light chemiluminescent detection kit (Tropix, Bedford, MA). (c) Stoichiometry of UmuD02C binding to RecA-ssDNA ®laments. Each lane represents a reaction of pre-formed ssDNA-RecA ®laments, containing 20 mM DNA (per nucleotide), 6.7 mM RecA, and increasing amounts of UmuD02C (from 0.3 to 6.0 mM, as indicated). The position of UmuD02C-RecA-DNA, UmuD02C-RecA, free UmuD02C and free UmuD02 is indicated on the right of the Figure. The ratio of UmuD02C to RecA in each reaction is indicated below each track. Note the disappearance of one of the UmuD02C-RecA-DNA bands and the appearance of free UmuD02C when the concentration of UmuD02C exceeds a ratio of one UmuD02C complex per two RecA monomers, suggesting that all of the UmuD02C binding sites on the RecA-ssDNA ®laments are saturated.

with their binding to RecA-ssDNA ®laments (cf. Figure 1(a) and (c)). Under these conditions we also detected some higher mobility UmuD0 , which we consider to be free dimers that dissociated during electrophoresis, since no such band is observed when gels were probed with UmuC antibodies (data not shown). As the concentration of UmuD02C in the reaction increased, so did the intensity of the UmuD02CRecA-ssDNA band. In addition to the free UmuD02, we observed another band that has a slightly greater mobility than the higher molecular weight UmuD02C-RecA-ssDNA band. This band was also apparent when we visualized the reaction with UmuC antibodies (not shown) and is therefore unlikely to simply represent UmuD0 binding to RecA (Frank et al., 1993). The appearance of two bands can potentially be explained by our ®nding that the UmuD02C has two binding sites on RecA (see below). We therefore hypothesize that the slower migrating band corresponds to a fully saturated UmuD02C-RecA-(circular) ssDNA complex and that the faster migrating band contains variable amounts of the UmuD02C complex bound to a

small fraction of RecA-coated linear ssDNA. This trend was maintained as the amount of Umu complex added was increased to one copy per two RecA monomers (Figure 1(c)). Beyond this point, the slightly faster mobility UmuD02C-RecA-ssDNA band disappears and is replaced by a more intense, broader band of higher mobility, that corresponds to UmuD02C-RecA complexes lacking ssDNA, and/or free UmuD02C. (We cannot distinguish between these two possibilities as both migrate at approximately similar positions, cf. Figure 1(b)). These observations are readily explained by the hypothesis that all of the available UmuD02C binding sites on the RecA-ssDNA ®lament have been occupied. These data, therefore, are consistent with the report that binding of UmuD02C complexes to RecA saturates at a stoichiometry of approximately one complex per two RecA monomers (Rehrauer et al., 1998). Visualization of UmuD02C binding to RecA-ssDNA filaments To investigate this interaction further, we examined the ®laments by cryo-electron microscopy.

588 Micrographs of RecA-ssDNA ®laments decorated with saturating amounts of UmuD02C and of undecorated ®laments are shown in Figure 2(a) and (b), respectively. As in previous studies by cryo-EM (Chang et al., 1988; Yu & Egelman, 1992),

Binding of the UmuD 02C Complex to RecA

the RecA ®laments are variably curved and have a distinctive zig-zag appearance, re¯ecting the deep groove in their helical structure, as viewed in lateral projection (Figure 2(b)). Many of the control ®laments are closed loops, indicating that their circular ssDNA molecules remained intact, but others are linear. The decorated ®laments (e.g. Figure 2(a)) are generally linear and shorter, suggesting a higher incidence of nuclease activity in our UmuD02C preparation: more signi®cantly for present purposes, the zig-zag motif is only faintly visible, at greatly reduced contrast. This distinction was quite reproducible. Moreover, it was consistently observed in three independent preparations of both complex-bound and control ®laments, and in each case, in all parts of the cryo-EM grids that were sampled. On this basis, we infer that the observed diminution of contrast does not arise from an increase in solvent density from partial drying (Cyrklaff et al., 1990) which would not be expected to be uniform across the grid. Moreover, there is no reason to suppose that the latter effect, which is physical in origin, should correlate with the protein composition of the ®laments. Thus, we conclude that binding of UmuD02C complexes to RecA ®laments is accompanied by a marked diminution in the contrast with which their helical groove is visualized. Pitch, width, and radial density distribution of decorated and undecorated filaments

Figure 2. Cryo-EM. (a) Stoichiometric binding of UmuD02C to RecA ®lament. (b) Control (undecorated) RecA ®laments. The micrographs were recorded at similar values of defocus, their ®rst zeros of the contrast Ê )ÿ1 (microtransfer function being at spacings of (28.5 A ÿ1 Ê graph (a)), and (30 A) (micrograph (b)). Bar represents 50 nm. The inset is a composite diffraction pattern. The top half-plane represents the sum of many individual diffraction patterns calculated from images of straightened Umu-saturated RecA ®laments, (‡); and the bottom half-plane was obtained in the same way from images of control (Umu-free) ®laments, (ÿ). Both patterns show the same strong ®rst-order layer-line, and this re¯ection is shifted towards the meridian as a result of Umu binding (‡ pattern). Both patterns also show a weak second-order layer-line.

To characterize the structural basis of the interaction further, the micrographs were subjected to image analysis. After computational straightening, the diffraction patterns of many ®laments were calculated, and the individual patterns were averaged to improve the signal-to-noise ratio. Both kinds of ®laments show a strong re¯ection at an axial spaÊ (cf. the upper and lower halves of cing of 90 A the composite diffraction pattern shown in Figure 2). This re¯ection derives from the deep helical groove, and corresponds to a single-start helix with 6.2 RecA subunits per turn (Egelman & Stasiak, 1989). That only one strong re¯ection is observed (the undecorated ®lament pattern also shows a weak second layer line at a spacing of Ê ) re¯ects local variability in the ®lament struc45 A ture, i.e. the absence of long-range order. Nevertheless, the average pitch of the helix is well de®ned, i.e. the re¯ection is quite sharp in the meridional direction. Its spacing is the same for decorated and undecorated ®laments, implying that, on average, the pitch of the RecA helix is not altered by UmuD02C binding. However, the re¯ection is shifted closer to the meridian in the case of decorated ®laments (cf. Figure 2), re¯ecting a change in their radial distribution of density. To examine this property, the pro®les of projected density across decorated and undecorated ®laments were calculated by axial averaging of the straightened images (Figure 3(a)). These traces were then scaled relative to each other to account

Binding of the UmuD 02C Complex to RecA

589 observation, we conclude that the UmuD02C complexes are submerged within the deep helical groove. Preferential binding of UmuD02C at the ends of RecA-ssDNA filaments

Figure 3. (a) Transverse scans of projected density across cryo-electron micrographs of RecA ®laments decorated (continuous line) with UmuD02C complexes and undecorated (broken line). The difference curve is shown below (dotted line). (b) Average radial density pro®les of decorated (continuous line) and undecorated (broken line) RecA ®lament, and the corresponding difference curve (dotted line). The values obtained at Ê ) are prone to even small very low radii (i.e. < 10 A amounts of residual noise and are not shown for that reason.

for the additional mass on the decorated ®laments. They show that the outer edges of decorated and undecorated ®laments coincide, indicating that the ®lament diameter is effectively unchanged upon binding the complexes. From these projection curves, the ®laments' average radial density pro®les were calculated (Figure 3(b)). Beyond a radius Ê , the pro®les are superimposable whereas of 45 A Ê in to at least 15 A Ê , additional density is from 45 A associated with the decorated ®laments. From this

Do UmuD02C complexes bind randomly along a given ®lament? To address this question by electron microscopy, ®laments should be prepared under conditions of sub-saturating binding. However, individual complexes do not have enough mass for it to be possible to identify their locations on ®laments unambiguously in cryo-micrographs, particularly against a background of unbound molecules. Accordingly, we labelled the ®laments with gold particles coupled to anti-UmuC antibodies and visualized them by negative staining (Figure 4(a)). These experiments were performed under two conditions, corresponding to 10 % and 50 % of saturation, respectively. As expected, some gold particles were randomly scattered over the grid. However, the gold particles were preferentially associated with the ®laments, as attested by their average density in the immediate proximity of the ®lament, operationally de®ned as being Ê of the center of the ®lament, being within 250 A higher than the background density by factors of 2.4 and 3.4, respectively, in these two experiments. The higher ratio in the second experiment is in keeping with the larger amount of Umu complexes added. Many micrographs were recorded, and for each ®lament with at least one antibody bound, we measured its length and the positions of all bound antibodies along it relative to the closest end. The resulting distributions are plotted in Figure 4(b) as functions of both coordinates, i.e. ®lament length and gold particle position. They document the preponderance of shorter ®laments noted above, but also reveal enhanced binding of antibodies at ®lament ends. In a control experiment in which the anti-UmuC antibody was replaced with another antibody that is not speci®c for any of these components (RecA, UmuC, UmuD0 ), the average ®lament labelling density fell by a factor of eight compared with the ®rst experiment (UmuD02C added at 10 % of the saturating value), and no preference for end-labelling was observed for the few gold particles that appeared to be associated with ®laments. To combine data from ®laments of different lengths without introducing bias, the score of each 20-nm bin was divided by the number of half-®laments that were long enough to have such a bin, i.e. the scores were expressed as probabilities of occupancy. (Each ®lament was treated as two half®laments, since we were unable to consistently determine the relative polarities of all the RecA ®laments under these conditions of observation.) For instance, in experiment II (Figure 4(c), right panel), 38 % of the ®lament ends were labelled, and the average labelling density at interior sites

590

Binding of the UmuD 02C Complex to RecA

Figure 4 (legend opposite)

was approximately ®vefold lower, corresponding to an average probability of 0.08 per 20-nm bin. Under these conditions, there should theoretically

be about six UmuD02C complexes per bin, so the percentage of complexes actually labelled is rather low. This situation is not unusual in a secondary

591

Binding of the UmuD 02C Complex to RecA

labelling experiment, in which the ef®ciencies of both the primary and secondary labellings must be considered. Nevertheless, in both experiments, ®lament labelling was systematically higher than (random) background labelling (see above) and end-labelling was systematically higher than internal labelling (Figure 4). Accordingly, these data provide evidence for two distinct classes of binding sites. To con®rm our inferences from the above experiment, we performed a similar experiment, but with linear ssDNA of de®ned length. In this case, the length distribution of the RecA ®laments exhibited a cutoff at the expected value Figure 5(b) (we attribute the shorter molecules observed to premature termination of synthesis or residual exonuclease activity in the UmuD02C preparation). Some examples of labelled ®laments are shown in Figure 5(a), and the distribution of gold particles along these ®laments (Figure 5(c)) clearly con®rms a preference for end-labelling. Interestingly, these data imply that one end of the polar RecA ®lament is preferred (we cannot yet tell which), because of the observed dearth of ®laments with both ends labelled. If both ends were equally accessible to labelling, we would have been expected 16(4) of the 255 ®laments measured to have both ends labelled. In fact, no such ®laments were observed. This suggests that one end of the ®lament is preferentially labelled and the data (Figure 5(c)) imply 80 % of these sites were labelled.

Discussion Our cryo-EM analysis shows that UmuD02C binds deep in the helical groove of RecA-ssDNA ®laments, where its presence may impede homologous recombination. Sequestration in this site may also protect these proteins from proteolysis, explaining their increased stability (Frank et al., 1996) in certain E. coli recA strains that have an enhanced propensity to form ®laments (Lavery & Kowalczykowski, 1992). Our immuno-labelling data indicate that the complex has a substantially higher af®nity for ®laments ends (Figures 4 and 5). As discussed further below, the terminal high af®nity site may serve to target UmuD02C com-

plexes to DNA lesions to ful®l their role in SOS mutagenesis. Getting in the groove: basis for competitive inhibition We do not yet know how the UmuD0 dimer is oriented as the complex binds in the groove nor how UmuC is positioned, beyond the observation that it is accessible to antibodies when the complex is bound to ®laments, which suggests that UmuC may be on the outside with UmuD0 underneath. Nevertheless, the mutagenically active UmuD0 Ê across, as measured perpendicular dimer is 60 A to the dimer axis (Peat et al., 1996; Ferentz et al., 1997; McLenigan et al., 1998), which compares with Ê for two RecA subunit a contour length of 70 A steps along a helical path in the groove at a radius Ê , where the peak Umu-associated density is of 30 A observed (Figure 3(b)). These dimensions ®t readily with the observed saturating stoichiometry of one UmuD02C complex per two RecA subunits whereby the complex has a ``footprint'' that covers two RecA subunits. If both UmuD0 subunits in the dimer bind to RecA subunits, these interactions are necessarily non-equivalent (assuming that UmuD02 retains the same structure upon binding UmuC), since successive RecA subunits are oriented in parallel and the two UmuD0 subunits per dimer are antiparallel. This proposition is consistent with cross-linking experiments that suggest that UmuD0 makes spatially distinct interactions with RecA (Lee & Walker, 1996). Bearing in mind the caveats noted above, we docked the known structure of the UmuD0 dimer (Peat et al., 1996; Ferentz et al., 1997; McLenigan et al., 1998; Ohta et al., 1999) into an EM-derived density map of the RecA ®lament (Yu & Egelman, 1993) (Figure 6). Although we do not yet have enough information to specify a precise mode of binding nor the placement of the UmuC component, this modelling experiment con®rms that there is plenty of room within the groove to accommodate binding of the kind indicated by our cryo-electron microscopy data. Like dsDNA, both UmuD02C (this paper) and LexA (Yu & Egelman, 1993; Harmon et al., 1996; Rehrauer et al., 1996) bind in the deep helical groove of RecA ®laments, and it has been demonstrated that under saturating conditions, binding of

Figure 4. (a) A negatively stained electron micrograph of RecA ®laments decorated with UmuD02C complexes under subsaturating conditions (nominally 20 % of saturation), with sites occupied by complexes marked by antiUmuC antibodies, detected by a secondary antibody labelled with 5-nm colloidal gold. Bar represents 100 nm. Below the micrograph is a schematic interpretation. Filled disks represent gold particles taken to be ®lament-associated, whereas the open disks represent gold particles that are not. (b) Two-dimensional distributions of the incidence of gold particles as a function of both ®lament length and position of particle from the ®lament end. In experiments I and II, the amount of complexes added was 10 % and 50 % of saturation, respectively. Measurements were made only on linear ®laments. The ®laments were, on average, shorter in the latter experiment, implying the possible presence of contaminating nuclease activity in the complex preparation. (c) Histograms combining the data shown in (b).

592

Binding of the UmuD 02C Complex to RecA

Figure 5. (a) Examples of negatively stained electron micrographs of RecA-ssDNA ®laments with linear, 788 bp-long DNA, decorated with UmuD02C complexes under subsaturating conditions (nominally 10 % of saturation), labelled with anti-UmuC antibodies, and detected by a secondary antibody labelled with 5-nm colloidal gold. Bar represents 100 nm. (b) Distributions of the ®lament length: the expected length of the ®laments is 390 nm (195 nm halflength arrow). (c) Distribution of gold particles along the ®laments. To within counting errors, the data indicate uniform density of labelling (broken line: arrow) away from the ends and substantially higher endlabelling. If in fact, only one end of the polar RecA ®lament is preferentially labelled (see Results), the probability of its occupancy is higher, i.e. 0.8.

one molecule competitively inhibits the binding of others (Rehrauer & Kowalczykowski, 1996; Harmon et al., 1996; Rehrauer et al., 1996, 1998). However, it should be stressed that competitive inhibition does not necessarily imply that they are competing for the same binding site nor even that their binding sites overlap: merely that one molecule, once bound, blocks access of the other molecule to its binding site. Regardless of the orientation of the bound UmuD02C complex relative to the RecA ®lament and whatever its contact point(s), once the complex is installed in the deep helical groove, it would block the subsequent binding of LexA or dsDNA. The latter, would, of course, result in an inhibition of homologous recombination (Figure 7). Therefore, such binding can explain the observed inhibition of recombination by UmuD02C when overproduced in vivo (Sommer et al., 1993, 1998; Boudsocq et al., 1997) and in vitro under similar conditions (Rehrauer et al., 1998). End-specific binding of UmuD02C to RecA filaments: identification of lesion sites for mutagenic repair The observed binding of the complex in the deep helical groove does not explain how the limited number (100 or so) of UmuD02C complexes normally present in a cell participate in SOS mutagen-

esis. The much greater abundance of RecA implies that, if the complexes were to bind randomly to RecA-DNA ®laments, they would be unlikely to be correctly positioned for their role in SOS mutagenesis (Friedberg et al., 1995; Woodgate & Levine, 1996; Tang et al., 1998; Reuven et al., 1998). However, in binding experiments in which the concentration of UmuD02C was much lower than that of RecA, and should perhaps be considered more physiological, we observed preferential binding of the complexes to tips of RecA ®laments (Figures 4 and 5), i.e. to sites within 20 nm of the end of a ®lament - the estimated resolution limit of our labelling experiment. This observation implies preferential binding of the UmuD02C complex to ®lament tips. While we cannot entirely rule out the possibility that these observations simply re¯ect greater accessibility of the antibody to the UmuC protein at the tips, this explanation of preferential end-labelling seems unlikely considering the paucity of ®laments with both ends labelled in the experiment documented in Figure 5. This mode of binding is, moreover, consistent with genetic analyses revealing that a preponderance of mutations that affect Umu-RecA interactions map to sites expected at the exposed end of a RecA ®lament (Sommer et al., 1998). Presumably, tip-speci®c binding correctly positions the Umu/pol V complex at sites containing DNA

593

Binding of the UmuD 02C Complex to RecA

bition of recombination requires much higher levels of MucA02B compared to UmuD02C (Venderbure et al., 1999). These observations are commensurate with MucA02B presumably having a higher af®nity for the tip of a RecA ®lament (thereby promoting SOS mutagenesis) than for the deep helical groove (leading to inhibition of recombination), than their chromosomally encoded E. coli counterparts, UmuD02C.

Materials and Methods Reagents The UmuD02C complex was puri®ed as described previously (Bruck et al., 1996; Tang et al., 1999). Af®nity-puri®ed rabbit polyclonal antibodies against the UmuD0 and UmuC proteins have also been described by Frank et al. (1996). The remaining reagents were purchased: fX174 ssDNA and RecA protein (New England Biolabs, Beverly, MA); ATPgS and PMSF (Calbiochem, La Jolla, CA); Immobilon-P membrane (Millipore, Bedford, MA); 5 nm gold-labeled goat anti-rabbit sera (Goldmark, Phillipsburg, NJ) and the CSPD-chemiluminescent immunodetection kit (Tropix, Bedford, MA). DNA gel mobility shift assay

Figure 6. The surface of a three-dimensional reconstruction of a RecA-ATP-DNA ®lament (Yu & Egelman, 1993) is shown in blue, while two dimers of UmuD0 (Peat et al., 1996) are shown as space-®lling atomic models in the deep groove of the RecA ®lament. The two dimers are related by the RecA helical symmetry, and separated by ®ve RecA subunits. While the details of the orientation of UmuD0 in the RecA groove are not known, it can be seen that the groove is deep enough for a UmuD02C complex to be easily accommodated.

Binding was effected in two steps. First, RecA-ssDNA ®laments were formed by mixing 30 ng of fX174 ssDNA and 1.0-1.5 mg of RecA in a 6 ml reaction volume of 20 mM Hepes, 50 mM NaCl, 1 mM ATPgS, 1 mM MgCl2, 1 mM DTT, and incubating at 37  C for 20 minutes. Then, UmuD02C (0.25 mg to 3 mg in the same buffer as above, but containing 1 mM PMSF) was added to give a total reaction volume of 10 ml, and the incubation continued at room temperature for an additional 30 minutes, when the samples were lightly cross-linked by adding glutaraldehyde to a ®nal concentration of 0.01 %. The reactants were then separated in a 1 % (w/v) agarose gel in TAE (Tris-acetate-EDTA) buffer, transferred to an Immobilon-P membrane, and the Umu proteins detected using af®nity puri®ed polyclonal antibodies against UmuD0 (Frank et al., 1996) and the CSPD-Western light chemiluminescent detection kit as previously described (Frank et al., 1996). Electron microscopy

lesions, where they are able to promote translesion DNA synthesis (Sommer et al., 1993, 1998; Boudsocq et al., 1997) (Figure 7). The idea that the UmuD02C complex (and presumably, functionally related proteins) has two binding sites on RecA; at the tip of a ®lament and within the deep helical groove, may help explain the differences observed between Umu homologs and their respective abilities to inhibit recombination and promote SOS mutagenesis. For example, the plasmid-encoded MucA02B proteins are extremely active for SOS mutagenesis at much lower cellular concentrations than UmuD02C, yet inhi-

For cryo-electron microscopy, UmuD02C was mixed with RecA-DNA at a ratio of approximately one complex (Mr 72,000) per two molecules of RecA (Mr 38,000) per six nucleotides of ssDNA. Reaction mixtures containing 30 ng of ssDNA fX174, 1.16 mg RecA, 1.05 mg UmuD02C, 20 mM Hepes, 50 mM NaCl, 1 mM ATPgS, 1 mM MgCl2 and 1 mM DTT were prepared as described above for the gel assays, and 5-ml drops were applied to holey carbon ®lms mounted on copper grids. They were then blotted to thin ®lms, vitri®ed on a Reichert KF80 cryo-station, inserted into a Gatan 626 cryoholder, transferred into Philips CM120 or CM200-FEG electron microscopes operating at 120 keV, and observed under minimal electron dose conditions, essentially as described (Conway et al., 1998). To visualize substoichiometric binding of UmuD02C to RecA, samples were prepared as described above except that the amount of UmuD02C was reduced to 50 % and

594

Binding of the UmuD 02C Complex to RecA

Figure 7. Cartoon of the two UmuD02C binding sites on RecA-ssDNA and their effect on SOS mutagenesis and homologous recombination, respectively. SOS mutagenesis: a RecA nucleoprotein ®lament has formed on a region of single stranded DNA generated during the cells attempt to replicate lesioned DNA (X). The cartoon shows the UmuD02C complex, which has a footprint of two RecA subunits, as binding to the extreme tip of the ®lament. This is its most likely site, although the limited resolution of the immuno-labelling data restricts the precision with which the high-af®nity site has been localized to the terminal 20 nm of the ®lament (11 RecA subunits). Binding to the tip of this ®lament does, however, provide a targeting mechanism that allows the normally limiting number of UmuD02C (pol V) molecules within the cell to be appropriately positioned at sites of DNA damage. The pol V together with RecA, the b-clamp and g-complex from DNA polymerase III, forms the so-called ``mutasome'' which is able to ef®ciently replicate damaged DNA, but with greatly reduced ®delity (Tang et al., 1999, 2000). The mutasome is non-processive and is likely to dissociate from the template shortly after translesion DNA synthesis to be replaced by pol III holoenzyme which completes genome duplication (Woodgate, 1999). Homologous recombination: RecA has formed a ®lament on ssDNA as it prepares to initiate recombination with an homologous dsDNA. At the low physiological concentrations of UmuD02C normally found in vivo, the Umu complex binds to the end of the ®lament, and does not interfere with recombination. In the presence of overproduced levels of UmuD02C, however, the Umu complex binds within the deep helical groove of RecA, thereby masking the binding site of dsDNA and as a consequence, competitively inhibits homologous recombination.

10 %, respectively. Where noted, the circular fX174 ssDNA was replaced with a linear 788 bp ssDNA fragment that was generated by heat denaturation of a 788 bp dsDNA PCR product. After the incubations, drops of sample were applied for 20 seconds. to carboncollodion substrates mounted on 400-mesh copper grids. Each grid was then ¯oated, specimen side down, on a 20 ml-droplet of blocking solution (I-block, Tropix, Bedford, MA) for 25 minutes, washed three times with TBST buffer, then ¯oated on a drop of anti-UmuC antiserum (diluted 1:50 in TBST) for 30 minutes, washed three times with TBST, and incubated with 5-nm goldconjugated goat anti-rabbit serum (Goldmark, Phillipsburg, NJ) diluted 1:20 in TBST. Finally, the specimen was stained with 1 % (w/v) uranyl acetate and visualized in a Philips CM12 electron microscope.

These segments were straightened by means of a cubic spline algorithm (Steven et al., 1991) implemented in the PIC-III image processing program (Trus et al., 1996), which was also used for the other operations described below. To calculate radial density pro®les, segments were axially averaged, and these averages combined and centro-symmetrized. For decorated ®laments, 18 segments, totaling 3.5 mm, were used, and for control ®laments, eight segments, totaling 1.19 mm. Finally, the pro®les were centro-symmetrized and band-limited to Ê )ÿ1, and radial density pro®les were calculated as (20 A described (Steven et al., 1984). Prior to calculating each diffraction pattern, the straightened ®lament segment was padded out to a length of 512 pixels with the requisite number of copies of its axially averaged transverse scan, and the background density was subtracted.

Radial reconstruction of decorated and undecorated filaments

Statistical analysis of gold particle distributions

Micrographs were digitized with a SCAI scanner (Zeiss Photogrammetrics, Fort Collins, CO) at a ®nal Ê per pixel. Fields of interest were sampling rate of 5.7 A displayed on a DEC Alpha workstation, and ®lament segments were designated interactively with a mouse.

Micrographs of the negatively stained preparations recorded at a magni®cation of 35,000 were digitized with a SCAI scanner (Zeiss Photogrametrics, Fort ColÊ per pixel lins, CO) at a ®nal sampling rate of 13.5 A Ê per pixel (experiment II and (experiment I) and 16.7 A controls). In some experiments, photographic prints were

Binding of the UmuD 02C Complex to RecA digitized on a ¯atbed scanner. The images were analyzed interactively in the NIH Image program (Rasband & Bright, 1995). running on a Apple Macintosh computer, using a mouse to measure contour lengths. Statistical analyses were performed with Mathlab software v.5.2.1 (MathWorks Inc., Natick, MA). In compiling data on gold particle distributions, considered as functions of the distance from an end, each ®lament was treated as two half-®laments because we cannot determine the relative polarities of the ®laments in these images. To combine the data on gold particle distributions from ®laments of different lengths, the score of each 20 nm bin was weighted by dividing by the total number of ®laments long enough to have such a bin, i.e. the scores were expressed as overall probabilities of occupancy.

Acknowledgments We thank M. Tang for providing the UmuD02C complex used in Figure 5; A. Rodriguez FernaÂndez de Henestrosa for providing the 788 bp PCR product also used in Figure 5; A. S. Levine for helpful suggestions during the course of this study; and J. Conway and B. Trus for provision of image processing resources. The work was supported in part by the NIH intramural research program and grants from NIH (GM35269) and the HFSP to E.H.E. #2000 US Government

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Edited by M. Gottesman (Received 18 June 1999; received in revised form 28 January 2000; accepted 3 February 2000)