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Physical interactions between DinI and RecA nucleoprotein filament for the regulation of SOS mutagenesis

2001; Springer Nature; Volume: 20; Issue: 5 Linguagem: Inglês

10.1093/emboj/20.5.1192

1460-2075

Takeshi Yasuda,

CRISPR and Genetic Engineering

Article1 March 2001free access Physical interactions between DinI and RecA nucleoprotein filament for the regulation of SOS mutagenesis Takeshi Yasuda Takeshi Yasuda Institute for Virus Research, Kyoto University, Japan Present address: Cellular Physiology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan Search for more papers by this author Katsumi Morimatsu Katsumi Morimatsu Institut Curie and Centre National de la Recherche Scientifique, France Present address: Division of Biological Sciences, Sections of Microbiology and of Molecular and Cellular Biology, University of California, Davis, CA, 95616-8665 USA Search for more papers by this author Ryuichi Kato Ryuichi Kato Department of Biology, Graduate School of Science, Osaka University, Japan Search for more papers by this author Jiro Usukura Jiro Usukura Nagoya University Postgraduate School of Medicine, Japan Search for more papers by this author Masayuki Takahashi Masayuki Takahashi Institut Curie and Centre National de la Recherche Scientifique, France Present address: FRE 2230, CNRS and Universite de Nantes, F44322 Nantes, France Search for more papers by this author Haruo Ohmori Corresponding Author Haruo Ohmori Institute for Virus Research, Kyoto University, Japan Search for more papers by this author Takeshi Yasuda Takeshi Yasuda Institute for Virus Research, Kyoto University, Japan Present address: Cellular Physiology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan Search for more papers by this author Katsumi Morimatsu Katsumi Morimatsu Institut Curie and Centre National de la Recherche Scientifique, France Present address: Division of Biological Sciences, Sections of Microbiology and of Molecular and Cellular Biology, University of California, Davis, CA, 95616-8665 USA Search for more papers by this author Ryuichi Kato Ryuichi Kato Department of Biology, Graduate School of Science, Osaka University, Japan Search for more papers by this author Jiro Usukura Jiro Usukura Nagoya University Postgraduate School of Medicine, Japan Search for more papers by this author Masayuki Takahashi Masayuki Takahashi Institut Curie and Centre National de la Recherche Scientifique, France Present address: FRE 2230, CNRS and Universite de Nantes, F44322 Nantes, France Search for more papers by this author Haruo Ohmori Corresponding Author Haruo Ohmori Institute for Virus Research, Kyoto University, Japan Search for more papers by this author Author Information Takeshi Yasuda1,2, Katsumi Morimatsu3,4, Ryuichi Kato5, Jiro Usukura6, Masayuki Takahashi3,7 and Haruo Ohmori 1 1Institute for Virus Research, Kyoto University, Japan 2Present address: Cellular Physiology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan 3Institut Curie and Centre National de la Recherche Scientifique, France 4Present address: Division of Biological Sciences, Sections of Microbiology and of Molecular and Cellular Biology, University of California, Davis, CA, 95616-8665 USA 5Department of Biology, Graduate School of Science, Osaka University, Japan 6Nagoya University Postgraduate School of Medicine, Japan 7Present address: FRE 2230, CNRS and Universite de Nantes, F44322 Nantes, France *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:1192-1202https://doi.org/10.1093/emboj/20.5.1192 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Escherichia coli dinI gene is one of the LexA-regulated genes, which are induced upon DNA damage. Its overexpression conferred severe UV sensitivity on wild-type cells and resulted in the inhibition of LexA and UmuD processing, reactions that are normally dependent on activated RecA in a complex with single-stranded (ss)DNA. Here, we study the mechanism by which DinI inhibits the activities of RecA. While DinI neither binds to ssDNA nor prevents the formation of RecA nucleoprotein filament, it binds to active RecA filament, thereby inhibiting its coprotease activity but not the ATPase activity. Furthermore, even under in vitro conditions where UmuD cleavage dependent on RecA–ssDNA–adeno sine-5′-(3-thiotriphosphate) is blocked in the presence of DinI, LexA is cleaved normally. This result, taken together with electron microscopy observations and linear dichroism measurements, indicates that the ternary complex remains intact in the presence of DinI, and that the affinity to the RecA filament decreases in the order LexA, DinI and UmuD. DinI is thus suited to modulating UmuD processing so as to limit SOS mutagenesis. Introduction The Escherichia coli RecA protein has pivotal roles in DNA recombination and repair, and it has been extensively studied with respect to its structure and function (Kowalczykowski et al., 1994; Friedberg et al., 1995; Roca and Cox, 1997; Kuzminov, 1999). RecA promotes strand pairing and exchange reactions between homologous DNA molecules, and as such is often called a recombinase. In addition, RecA promotes the cleavage of LexA, UmuD and prophage repressors such as λcI in DNA-damaged cells. Since LexA and other proteins are self-cleaved under certain conditions in vitro, even in the absence of RecA, the term coprotease was coined for the activity of RecA in promoting the cleavage of LexA and other proteins (Little, 1984). Binding of RecA to single-stranded (ss)DNA is a prerequisite for both the recombinase and coprotease activities, leading to the formation of a nucleoprotein filament or so-called activated RecA. In vitro studies suggest that ATP (or dATP) is required for filament formation. LexA is the repressor of many DNA damage-inducible genes, including lexA itself and recA, and it undergoes a self-cleavage reaction upon interaction with a RecA nucleoprotein filament, thereby resulting in induction of many LexA-regulated genes. This reaction to DNA damage is called the SOS response. Thus, RecA functions as a sensor of DNA damage by virtue of its ability to bind ssDNA generated as a result of interrupted DNA replication at sites of DNA damage. Such binding results in RecA activation, and consequent amplification of the inducing signal by promoting the processing of LexA and other proteins. Most of the induced gene products function in repairing DNA damage, but the product of the umuDC operon, which is most tightly regulated at the transcriptional level by the LexA repressor, functions in DNA damage-induced mutagenesis. The nascent UmuD protein is inactive for mutagenesis and needs to be converted to an active form (UmuD′) by an intermolecular self-cleavage reaction, which is promoted by interaction with a RecA nucleoprotein filament (McDonald et al., 1998). Two UmuD′ molecules interact with one UmuC molecule to form a UmuD′2C complex (Woodgate et al., 1989), which functions as DNA polymerase V to bypass DNA lesions in conjunction with RecA, at the risk of causing mutations (Reuven et al., 1999; Tang et al., 1999, 2000). Such DNA damage-inducible mutagenesis is called SOS mutagenesis. Interactions between a RecA nucleoprotein filament and homologous DNA, or proteins such as LexA and UmuD, are mutually competitive. For example, excess amounts of either ssDNA or double-stranded (ds)DNA inhibited RecA coprotease activity in vitro (Craig and Roberts, 1980, 1981; Takahashi and Schnarr, 1989; Rehrauer et al., 1996). Conversely, in vivo overproduction of both UmuD′ and UmuC resulted in the inhibition of RecA-dependent homologous DNA recombination (Sommer et al., 1993). An uncleavable form of LexA protein (LexA-S119A) also inhibited DNA strand-exchange activity of RecA in vitro (Harmon et al., 1996). Furthermore, UmuD′2C inhibited in vitro LexA cleavage mediated by a RecA filament (Rehrauer et al., 1998). It is believed that such inhibition is achieved by LexA and UmuD′2C binding to the deep helical groove of the RecA nucleoprotein filament (Yu and Egelman, 1993; Frank et al., 2000), to which homologous DNA also binds (Story et al., 1992). Recently, we reported that the LexA-regulated dinI gene encodes a small protein (81 amino acids) that is involved in regulating RecA functions. We first identified the dinI gene as a multicopy suppressor of the cold-sensitive phenotype caused by the dinD68 mutation (Yasuda et al., 1996). While dinI null mutants showed no difference in UV sensitivity from the parental strain, dinI overexpression conferred severe UV sensitivity on wild-type cells (Yasuda et al., 1998). In dinI-overexpressed cells, processing of LexA and UmuD after DNA damage was blocked, and homologous DNA recombination, as measured by P1 phage transduction, was also suppressed. Conversely, processing of UmuD occurred more rapidly and extensively in dinI null mutants than in the wild type, thus conferring dinI mutants with a mutator phenotype. However, no discernible change in the regulation of LexA after DNA-damaging treatment was observed between isogenic dinI and wild-type strains (our unpublished result). Furthermore, in an in vitro system with purified DinI, RecA and UmuD proteins, DinI inhibited UmuD processing even after a stable RecA–ssDNA–adenosine-5′-(3-thiotriphosphate) (ATPγS) complex was formed. In comparison, DinI did not inhibit the cleavage of LexA under the same conditions. These results suggested that DinI might directly interact with the RecA nucleoprotein filament, thereby inhibiting the UmuD processing. How ever, the question as to why DinI differentially affected LexA and UmuD processing remained unanswered. In this study, we investigated the interaction between DinI and RecA nucleoprotein filaments using various biochemical and physicochemical methods. Moreover, we compared DinI, LexA-S119A and UmuD-K97A for their ability to inhibit RecA coprotease activity, which enabled us to clarify why DinI inhibits UmuD processing more efficiently than LexA cleavage both in vivo and in vitro. Results DinI does not inhibit the formation of an active RecA–ssDNA complex In vivo overexpression of dinI resulted in the inhibition of both coprotease and recombinase activities of RecA (Yasuda et al., 1998). There are at least three different explanations for the inhibitory mechanism: (i) DinI may interact with either ssDNA or free RecA not bound to ssDNA, so as to prevent the formation of an active RecA–ssDNA complex; (ii) DinI may interact with the active RecA nucleoprotein filament to prevent it from interacting with LexA, UmuD or homologous DNA; (iii) DinI may interact with the RecA nucleoprotein filament in such a way as to dissociate the ternary complex necessary for activation. We have examined all of these possibilities. We first tested binding of DinI to an ssDNA–cellulose column. As expected, RecA bound to the column and was eluted with higher concentrations of NaCl (Figure 1A). In contrast, DinI was recovered in the flow-through and wash fractions (Figure 1B), indicating that DinI does not bind to the ssDNA–cellulose. We then examined the effect of DinI on the ssDNA-dependent ATPase activity of RecA. DinI itself showed no ATPase activity in the absence or presence of ssDNA (data not shown). As shown in Figure 2, DinI did not affect the ATPase activity of RecA, even in a 30-fold excess over RecA, when it was mixed with poly(dT) either before or after the addition of RecA to the reaction mixture, while the E.coli ssDNA-binding protein (SSB) very effectively inhibited the ATPase activity when premixed with poly(dT). No inhibition of the ATPase activity was observed when DinI and RecA were premixed and ssDNA then added to the mixture. These results, therefore, exclude the possibility that DinI inhibits the interaction between RecA and ssDNA or the access of ATP to the RecA–ssDNA complex. Figure 1.DinI protein does not bind to ssDNA. ssDNA binding assays for RecA (A) and DinI (B) were performed as described in Materials and methods. FT and W represent flow-throw and wash fraction, respectively. In the case of RecA, the wash was performed twice (W1 and W2). In lane RecA or DinI, purified RecA or DinI protein, respectively, was applied to the gel. Download figure Download PowerPoint Figure 2.DinI does not inhibit the ssDNA-dependent ATP hydrolysis promoted by RecA protein. DinI, SSB, RecA (all at 1 μM) and poly(dT) (3 μM) were added in different orders. DinI was added to the reaction mixture after RecA and poly(dT) were pre-incubated at 37°C for 5 min (open circles). RecA was added after DinI (open squares) or SSB (closed triangles) was pre-incubated with poly(dT). Poly(dT) was added to the reaction mixture after RecA and DinI were pre-incubated (closed circles). After adding the components, the mixtures were incubated at 37°C for a further 5 min, and ATP hydrolysis reactions were started with the addition of ATP (1 mM). Download figure Download PowerPoint Interaction between DinI and activated RecA in vivo Next, we examined whether DinI could interact with free RecA monomer or activated RecA nucleoprotein filament in vivo using a cross-linker, dithiobis(succinimidylpropionate) (DSP). The reagent has a disulfide bond in the center, which can be cleaved by the addition of dithiothreitol (DTT) (see Figure 3A for the experimental scheme). We used two E.coli strains, DE192 (lexA51) and DE667 (lexA51 recA730), which were transformed with a plasmid overproducing a recombinant N-terminal His-tagged DinI. The His6-DinI protein behaved exactly as the normal DinI protein in vivo and in vitro: overproducing the His6-DinI protein in the cells blocked SOS induction after UV irradiation, and the purified His6-DinI protein inhibited RecA-dependent UmuD processing in vitro (data not shown). The above two E.coli strains carry the lexA51 (Def) mutation, which inactivates LexA repressor activity. One strain, DE192, overproduces the wild-type RecA protein, while the other, DE667, overproduces the coprotease-constitutive RecA730 protein. The RecA730 protein is believed to be constitutively activated by binding to ssDNA regions that are transiently generated during DNA replication in the absence of DNA damage because of a higher DNA-binding affinity (Lavery and Kowalczykowski, 1992). Figure 3.Cross-linking between DinI and activated RecA. (A) A schematic presentation of the procedure used. (B) DinI binds to activated RecA. The experiments were carried out with DE667 (lexA51 recA730) or DE192 (lexA51), both carrying pHR255 (lacIq). In lanes 1, 2, 4 and 5, the cells carried pYP92 (His6-DinI). In lanes 3 and 6, the cells carried pQE9 (vector). In lanes 2, 3, 5 and 6, samples were treated with DSP. In lanes C, purified RecA protein was loaded. Download figure Download PowerPoint After overproduction of the His6-DinI protein, the cells from both strains were converted to spheroplasts, to which DSP was added to cross-link any interacting proteins. The cell extracts were prepared and mixed with Ni-NTA–agarose, which binds His6-DinI together with any protein(s) cross-linked to it. The His6-DinI protein was eluted from the Ni-NTA–agarose. After treatment with DTT to cleave the disulfide bond present in DSP, the eluted samples were subjected to SDS–PAGE followed by western blot analysis with anti-RecA antibodies as the probe. As seen in Figure 3B, the RecA band was observed in the sample from DE667 with the coprotease-constitutive RecA730, but not from DE192 with the wild-type RecA. Because we detected no or little difference in the total amounts of RecA and His6-DinI proteins between DE667 and DE192 (data not shown), the above result should imply that DinI interacts in vivo with activated RecA but not with free RecA monomers. Interaction between DinI and a RecA nucleoprotein filament We verified an interaction between DinI and RecA nucleoprotein filament by spectroscopic measurements. First, we used circular dichroism (CD), which relates to the secondary structure of proteins. The CD spectrum of DinI (not shown) indicated that the protein contains a large proportion (70%) of α-helix, in good agreement with the prediction from its amino acid sequence. DinI was heat denatured at ∼58°C (Figure 4A), and RecA was also unfolded at ∼58°C when it formed a complex with poly(dT) and ATPγS (Figure 4B). As shown in Figure 4B, when DinI was added to the RecA–poly(dT)–ATPγS ternary complex at a molar ratio to RecA monomer of 1:1, two different melting temperatures of 58 and 65°C were observed. In contrast, the addition of DinI to RecA in the absence of poly(dT) generated only a slight increase in the melting temperature of RecA (Figure 4C). This slight difference (∼0.5°C) is of limited significance, although it could indicate a very weak interaction between DinI and free RecA under the in vitro conditions used. Our CD measurements, therefore, clearly indicate that DinI interacts with the RecA–poly(dT)–ATPγS complex, stabilizing it against thermal unfolding. Figure 4.Effect of DinI on the thermal stability of RecA. Thermal unfolding of DinI and RecA proteins was detected by CD change upon temperature elevation, and presented by its first derivative, d(CD)/dT. (A) DinI alone (8.3 μM). (B) RecA–poly(dT)–ATPγS complex (thin line); mixture of DinI and RecA–poly(dT)–ATPγS complex (thick line); expected theoretical curve of the mixture when there is no interaction between DinI and RecA–poly(dT)–ATPγS complex (broken line). RecA, 8.3 μM; DinI, 8.3 μM; poly(dT), 24.9 μM. (C) RecA with ATPγS (solid line) in the absence of ssDNA; expected theoretical curve of the mixture when there is no interaction between DinI and RecA with ATPγS (broken line). RecA, 4 μM; DinI, 4 μM. Download figure Download PowerPoint We then studied the interaction of DinI with a RecA nucleoprotein filament by flow linear dichroism (LD) spectroscopy. RecA nucleoprotein filaments can easily be flow-oriented and provide large LD signals (Norden et al., 1990, 1992). By contrast, small proteins, like DinI, do not provide any LD signal because they can not be oriented by flow. However, if a protein binds to a RecA nucleoprotein filament and is flow-oriented together with the filament, the addition of such a protein to the RecA filament should generate a change in the LD signal. To examine the validity of this argument, we used lysozyme as a negative control and LexA-S119A (an uncleavable mutant form of LexA) as a positive control. When RecA (4 μM) and poly(dT) (12 μM in nucleotides) were mixed in the presence of 50 μM ATPγS and placed for >3 h at room temperature, an LD signal was observed. The addition of lysozyme to the RecA–poly(dT)–ATPγS complex did not generate any change in the LD signal (not shown), but the addition of LexA-S119A to the ternary complex caused an increase in intensity of the positive LD signal at ∼196 nm, without significant change in the signal at ∼260 nm (Figure 5A), indicating an additional positive signal from LexA. We applied this method to study the interaction of UmuD-K97A, a non-cleavable mutant form of UmuD, with the RecA nucleoprotein filament. In contrast to LexA-S119A, the addition of UmuD-K97A to the RecA–poly(dT)–ATPγS complex caused a decrease in the LD signal at ∼196 nm (Figure 5B), implying that UmuD-K97A bound to the filament and generated a negative LD signal. In the inset of Figure 5A and B, the changes in the LD value at 196 nm were plotted against the amount of LexA-S119A or UmuD-K97A added to the reaction mixture. The results suggest that LexA-S119A and UmuD-K97A bind to the RecA–poly(dT)–ATPγS complex at the molar ratio to RecA monomer of 1:2, although more experiments are necessary to determine the stoichiometry definitely by LD measurements. LD can thus detect the interaction between a RecA nucleoprotein filament and proteins that bind to it. Figure 5.Effect of LexA-S119A and UmuD-K97A on the LD signal of RecA–poly(dT)–ATPγS complex. RecA (4 μM) and poly(dT) (12 μM in nucleotides) were used. LD was measured with a shear force of 20/s at 25°C. (A) LexA-S119A was added at a molar ratio to RecA of 0.23, 0.45, 067, 0.89 or 1.35. Only the spectra for RecA alone or with LexA-S119A added at 0.45 or 1.35 molar ratio are shown. (B) UmuD-K97A was added at a molar ratio to RecA of 0.16, 0.32, 0.47, 0.63 or 0.95. Only the spectra for RecA alone or with UmuD-K97A added at 0.32 or 0.95 molar ratio are shown. In the insets of (A) and (B), the change in the LD value at 196 nm was plotted against the molar ratio of LexA-S119A or UmuD-K97A to RecA. Download figure Download PowerPoint To investigate the interaction between DinI and RecA nucleoprotein filament by the LD method, we used either poly(dϵA) or poly(dT) as ssDNA, and native calf thymus DNA as dsDNA. The LD signal of free poly(dϵA) was too small to distinguish from the baseline. When RecA (4 μM) and poly(dϵA) (12 μM) were mixed in the presence of 50 μM ATPγS, a significant LD signal was observed. No LD signal appeared upon the addition of DinI alone to poly(dϵA) (Figure 6A), supporting the previous result that DinI does not bind directly to ssDNA. However, the addition of DinI (4 μM) to RecA–poly(dϵA)–ATPγS and RecA–poly(dT)–ATPγS complexes increased the LD signals (Figure 6A and C). In the inset of Figure 6C, the changes in the LD value at 196 nm were plotted against the amount of DinI added to the reaction mixture. The result suggests that DinI binds to the RecA nucleoprotein filament at a ratio to RecA monomer of 1:1, while the results shown in the inset of Figure 5A and B suggest that LexA and UmuD bind at a ratio of 1:2. In contrast to such results with RecA–ssDNA–ATPγS complexes, the addition of DinI caused little or no effect on the LD signal of the RecA–dsDNA–ATPγS complex (Figure 6B). These results indicate that DinI interacts with RecA bound to ssDNA, but not with that bound to dsDNA. Figure 6.Effect of DinI on the LD signal of RecA–DNA–ATPγS complexes. LD was measured with a shear force of 60/s (A and C) or 20/s (B) at 20°C. (A) poly(dϵA) (12 μM in nucleotides) was mixed with 4 μM RecA alone or plus DinI (4 or 8 μM). The curves of ssDNA alone or with DinI are almost at baseline. (B) RecA (4 μM) and calf thymus dsDNA (12 μM) were mixed with or without DinI (8 μM). All the curves are almost superimposed. (C) RecA (4 μM) and poly(dT) (12 μM) were mixed with 1, 2, 3, 4 or 8 μM DinI. Only the spectra for RecA alone or with DinI added at 0.5 or 2.0 molar ratio are shown. In the inset of (C), the LD value at 196 nm was plotted against the molar ratio of DinI added to RecA. Download figure Download PowerPoint DinI does not dissociate the RecA–ssDNA–ATPγS complex From the results described above, it is unlikely that DinI dissociates the RecA–ssDNA–ATPγS ternary complex. This was confirmed directly as described below. In our previous in vitro experiments with the purified proteins, activated RecA-dependent UmuD processing was effectively inhibited in the presence of DinI, but LexA processing was not (Yasuda et al., 1998). If DinI inhibited UmuD processing by dissociating the Rec–ssDNA–ATPγS complex, then the cleavage of LexA added later to the reaction mixture containing the ternary complex, DinI and UmuD should also be inhibited. However, it may be argued that even if RecA–ssDNA–ATPγS complex is disassembled by the addition of DinI, reassociation of RecA monomers with ssDNA might occur and thereby promote preferential LexA cleavage. Such reassociation can, however, be blocked in the presence of SSB, a potent competitor for binding to ssDNA (Figure 2). In fact, SSB completely prevented LexA processing when it was mixed with RecA, ssDNA and ATPγS prior to the addition of LexA (data not shown). As shown in Figure 7B (the experimental scheme is given in Figure 7A), UmuD and LexA were cleaved in the presence of RecA–poly(dT)–ATPγS (lanes 4–6) but not in its absence (lanes 1–3). In the presence of DinI, UmuD processing was inhibited (lanes 7–11), but LexA subsequently added to the same reaction mixture was cleaved normally in the presence or absence of SSB (lanes 9 and 11). This result indicates that the RecA–poly(dT)–ATPγS complex remains active for LexA cleavage in the presence of DinI, while being inactive for UmuD processing. We believe, therefore, that these findings eliminate the possibility that DinI dissociates the RecA–ssDNA–ATPγS ternary complex. Figure 7.DinI does not dissociate a RecA–ssDNA–ATPγS complex. (A) The experimental scheme. (B) SDS–PAGE analysis of the reaction products. The in vitro reactions were performed as described in Materials and methods. Proteins and ssDNA were added to the reaction mixture as indicated in (A). The lane number corresponds to the sampling time taken from the reaction mixture, as indicated by the number in (A). Download figure Download PowerPoint We visualized the RecA–ssDNA–ATPγS complex structure with or without DinI by electron microscopy (Figure 8). When RecA was mixed with poly(dT) (average length 400 nucleotides) in the presence of ATPγS and placed at 37°C, the intermolecular end-joining of RecA nucleoprotein filaments was observed (Figure 8A and B), as previously reported (Register and Griffith, 1986). We observed very similar structures when DinI was added after the RecA–poly(dT)–ATPγS complex was placed at 37°C for 1 h (Figure 8C and D), indicating that the ternary complex was maintained in the presence of DinI. Since DinI is so small, we did not expect any difference in the structure of the RecA filament in the presence or absence of DinI. We did, however, notice that the striated pattern of the RecA nucleoprotein filament seemed to be more condensed in the presence of DinI than in the absence of it (compare Figure 8C with A). Figure 8.Electron micrographs of RecA–poly(dT)–ATPγS complex without DinI (A and B) and with DinI (C and D). The bars indicate 200 nm (A and C) and 500 nm (B and D). Download figure Download PowerPoint Competitive binding of DinI, LexA and UmuD to a RecA nucleoprotein filament The results described above support the notion that DinI interacts with a RecA nucleoprotein filament so as to prevent it from interacting with UmuD. Why, then, does DinI inhibit UmuD processing but not LexA cleavage? We reasoned that such distinct effects could be caused by differences in the binding affinity of each protein to a RecA nucleoprotein filament. To address this question, we assayed the inhibitory effects of DinI, LexA-S119A and UmuD-K97A on RecA coprotease activities with either LexA or UmuD as the substrate. We first compared the effect of LexA-S119A and DinI on the inhibition of UmuD processing, using either poly(dT) or M13 DNA as ssDNA (Figure 9). In both cases, LexA-S119A inhibited UmuD processing more efficiently than did DinI, as shown in Figure 9C where the extent of inhibition is plotted against the amount of LexA-S119 or DinI added to the reaction. Next, we compared the effects of DinI and UmuD-K97A on inhibiting LexA cleavage. LexA cleavage occurred much faster than UmuD processing: LexA was completely cleaved within 15 min at 37°C, while complete UmuD processing took ∼3 h under the same conditions. When DinI or UmuD-K97A was added to the RecA–poly(dT)–ATPγS complex and then LexA added to the mixture, LexA was completely cleaved after a further 15 min incubation at 37°C (data not shown). We subsequently performed the reaction at 20°C instead of 37°C, in order to slow down the LexA cleavage reaction. Indeed, complete LexA cleavage took ∼30 min at 20°C (data not shown). Under these conditions, we could detect inhibitory effects of DinI and UmuD-K97A on LexA cleavage, although the extent of inhibition was much weaker than that of LexA-S119A or DinI on UmuD processing. The result summarized in Figure 10 demonstrates that DinI inhibited LexA cleavage more efficiently than UmuD-K97A. The above experiments to measure the RecA coprotease activity were carried out in the presence of ATPγS, which stabilizes the RecA nucleoprotein filament. However, when LexA cleavage was measured at 37°C in the presence of ATP instead of ATPγS, no inhibition was observed upon the addition of DinI (data not shown). This eliminates the possibility that the failure of DinI to inhibit LexA cleavage in vitro is an artifact caused by ATPγS. Figure 9.Inhibition of UmuD processing by DinI and LexA-S119A. (A and B) The in vitro reactions were performed as described in Materials and methods. Different amounts of DinI (A) or LexA-S119A (B) were added to the reaction mixtures to measure the inhibition of processing of UmuD (6 μM). The proteins were analyzed by 16.5% SDS–PAGE with a Tricine buffer system. (C) The densities of the UmuD and UmuD′ bands in the presence of poly(dT) (A and B) or M13 ssDNA (data not shown) were measured, and the relative amounts of UmuD′ against UmuD + UmuD′ were plotted against the amount of DinI or LexA-S119A added to the reaction. Download figure Download PowerPoint Figure 10.Inhibition of LexA processing by DinI and UmuD-K97A. The in vitro reactions in the presence of poly(dT) were performed as described in Materials and methods at 20°C. Different amounts of DinI or UmuD-K97A were added to the reaction mixtures to measure the inhibition of cleavage of LexA (6 μM). The densities of the LexA bands were measured, and the relative amounts of the cleaved LexA were plotted against the amount of DinI or UmuD-K97A added to the reaction. Download figure Download PowerPoint Discussion Direct binding of DinI to the RecA filament for the inhibition of RecA functions In this study, we have examined in detail how DinI inhibits RecA coprotease activities, by a variety of in vivo a