INTRODUCTION

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When an organism's DNA replication machinery encounters a lesion in the DNA that, for a variety of reasons, was not repaired by accurate repair pathways, it stalls, leading to one of two possible outcomes: (i) damage avoidance, a poorly understood set of processes, including daughter strand gap repair, that appear to utilize the information in the newly synthesized daughter strand of the DNA duplex to somehow bypass the lesion (17) or (ii) translesion synthesis (TLS), in which a specialized DNA polymerase is recruited for bypassing the damaged site (17, 21, 67, 69). The latter pathway is potentially mutagenic due to the miscoding or noncoding nature of the DNA lesion (17).

TLS in Escherichia coli is dependent on the umuDC and recA gene products (17, 23, 61). The umuDC operon encodes a DNA polymerase, DNA polymerase V, with the unique ability to replicate over particular types of DNA lesions, including abasic sites and thymine-thymine cyclobutane dimers (55, 64, 65). Recent work indicates that UmuC is the founding member of a diverse and ubiquitous family of DNA polymerases capable of copying imperfect templates (16, 18, 21, 69). In addition to their role in TLS, the umuDC gene products also participate in cell cycle checkpoint control (41, 46). In response to DNA damage, RecA protein nucleates on single-stranded DNA (ssDNA) that is generated by the cell's failed attempts to bypass lesions in its genome (17, 57). These RecA-ssDNA nucleoprotein filaments act to mediate the cleavage of the LexA repressor (33, 34). Cleavage of LexA inactivates it as a repressor, leading to the expression of the SOS regulon, a collection of ~30 unlinked genes whose expression is coordinately regulated (11, 17, 25). The umuDC operon is among these ~30 LexA-regulated genes (9, 17). Importantly, UmuD also undergoes RecA-ssDNA-mediated cleavage. This cleavage serves to remove its amino-terminal 24 residues to produce UmuD' (5, 43, 58). It has been proposed that cleavage of UmuD serves to regulate the two physiological roles of the umuDC gene products so that they act in a temporally ordered fashion (41, 46), first by participating in a DNA damage checkpoint control system and second by participating in TLS.

UmuD is related to three distinct classes of proteins: (i) other UmuD-like proteins involved in mutagenesis, which also undergo RecA-ssDNA-facilitated cleavage (17, 29, 30, 52), (ii) transcriptional repressors belonging to the LexA-like family, which undergo RecA-ssDNA-facilitated cleavage (17, 52), and (iii) signal peptidases (49). Interestingly, despite the fact that UmuD' and E. coli signal peptidase have little sequence identity apart from their lysine-serine dyad, they have a remarkable degree of structural identity. Comparison of their crystal structures revealed that 69 C- atoms of UmuD' are superimposable (with a root mean squared of 1.6 Å) on the E. coli signal peptidase crystal structure (49).

The E. coli UmuD and UmuD' proteins interact with each other to form homo- and heterodimers (1, 5, 22) and also interact with UmuC (4, 22, 70), RecA-ssDNA nucleoprotein filaments (5, 32), and three components of the replicative DNA polymerase (62). Given the rather small sizes of UmuD and UmuD', an important question is which part(s) of UmuD and UmuD' is involved in interaction with each of these other proteins? A detailed understanding of these interactions will be required for a complete understanding of the molecular mechanisms underlying the roles of the umuDC gene products in checkpoint control and TLS. Determination of the crystal (10, 50) and solution (A. E. Ferentz, G. C. Walker, and G. Wagner, unpublished data) structures of the UmuD'₂ homodimer and genetic studies (39, 45) have identified the residues involved in its dimerization interface. Furthermore, biochemical characterizations of single-cysteine derivatives of UmuD and UmuD' by analyzing homo- and heterodimer cross-linking efficiency using thiol-specific cross-linking reagents and by studies of spin-labeled derivatives by electron spin resonance have identified important components of the dimerization interfaces of the UmuD-UmuD' heterodimer and the UmuD₂ homodimer (unpublished data). Finally, these single-cysteine derivatives of UmuD have also been used, in conjunction with the thiol-specific, photoactivatable, heterobifunctional cross-linking agent p-azidoiodoacetanilide (71), to identify residues of UmuD able to cross-link efficiently to RecA-ssDNA nucleoprotein filaments (32) (Fig. 1). Taken together, these studies have begun to provide a detailed molecular understanding of the roles of the umuD gene products in regulation of the cell cycle and TLS.

View larger version (38K): [in this window] [in a new window]   FIG. 1.   Side (A) and top (B) views of the UmuD'2 homodimer crystal structure reported by Peat et al. (50) with the homodimer interface reported by Ferentz et al. (10). Leu101 and Arg102 are shown in red. Val34, Ser57, Ser67, Ser81, and Ser112 are shown in green. These positions of UmuD2, when changed to cysteine and conjugated to the thiol-specific photoactivatable cross-linker p-azidoiodoacetanilide (71), were cross-linked efficiently to RecA-ssDNA in vitro (32).

As part of our ongoing effort to better understand how UmuD and UmuD' interact with other proteins to enable checkpoint control and TLS, we have embarked on a site-directed mutational analysis of the umuD gene products (1, 45). Comparison of the deduced amino acid sequences of members of the UmuD-like mutagenesis proteins to those of the LexA-like transcriptional repressor family identified a small number of residues that were well conserved exclusively among the UmuD-like mutagenesis proteins (1) (see Fig. 2). This observation suggested that these might be residues that are important for the biological roles of the umuD gene products rather than for the RecA-ssDNA-facilitated cleavage of UmuD. Mutations affecting most of these highly conserved positions have already been characterized (19, 39, 45). Here we describe our construction and genetic and biochemical characterizations of a UmuD derivative in which two such highly conserved residues, leucine-101 (Leu101) and arginine-102 (Arg102), have been replaced with glycines. Our characterizations of this mutant UmuD protein, which we unexpectedly found to be deficient in RecA-ssDNA-facilitated cleavage, suggest a possible mechanism for how the interaction of UmuD₂ with the RecA-ssDNA nucleoprotein filament results in the cleavage of one UmuD molecule by its intradimer partner to yield UmuD' (10, 37).

	MATERIALS AND METHODS

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Bacteriological techniques. E. coli strains and plasmid DNAs used in this study are described in Table 1. Generalized transduction using P1vir was performed as described previously (40). E. coli was grown in either Luria-Bertani medium or M9 minimal medium (40) as indicated in Fig. 3 and 4. When required, the following antibiotics were used at the indicated concentrations: ampicillin, 150 µg/ml; kanamycin (KAN), 40 µg/ml; tetracycline, 20 µg/ml; and rifampin, 50 µg/ml. Bacterial transformation was by the calcium chloride technique (56). Plasmid DNAs were purified using the QIA-spin mini prep kit (Qiagen) per the manufacturer's recommendations. Missense mutations within the umuD and umuD' coding regions were generated using the Quickchange kit (Stratagene) per the manufacturer's recommendations, and the nucleotide sequences of all constructs were verified by automated DNA sequence analysis.

Proteins and reagents. The UmuD and UmuD1012 proteins were purified as described previously from 1-liter cultures of BL21(DE3) transformants except that Superose 12 chromatography was omitted (10). The chromatographic characteristics of UmuD1012 were essentially identical to those of the wild-type UmuD throughout its purification, suggesting that it had a native conformation and that the Leu101-to-glycine and Arg102-to-glycine substitutions had at most a minimal effect on the overall structure of the protein. RecA protein was purified as described elsewhere (15). M13mp18 ssDNA was from New England Biolabs, glutaraldehyde and adenosine 5'-O-(3-thiotriphosphate) were from Sigma, and the Western-Light chemiluminescence kit was from Tropix.

SOS mutagenesis assays and Western blot analysis. SOS mutagenesis activity was measured by either the argE3(Oc)Arg⁺ reversion assay as described elsewhere (9) or a rifampin resistance assay. For the rifampin resistance assay, 0.5 ml of cultures grown overnight at 42°C was used to inoculate 25 ml of M9 minimal medium supplemented with 0.4% Casamino Acids, 0.4% glucose, 100 µg of adenine per ml, and KAN. Cultures were grown at 42°C with shaking. We used these growth conditions because they have been previously demonstrated to enhance the coprotease activity of RecA protein (6). When cultures reached an optical density at 595 nm of ~0.6, 6-ml aliquots of each were removed and either irradiated with UV (20 J/m²) or mock irradiated in sterile Pyrex dishes (100 mm by 15 mm). Irradiated and mock-irradiated cultures were then returned to 42°C and grown with aeration to allow the expression of SOS-regulated functions. Forty-five minutes after irradiation, 4-ml aliquots were removed from each culture for determination of their optical density at 595 nm and for preparation of whole-cell lysates to monitor the relative abundance of UmuD (or UmuD1012) and UmuD' (or UmuD1012') by Western blotting as described previously (45, 46). The remaining 2 ml of each culture was allowed to grow for an additional 1 h 15 min at 42°C to fix any induced mutations, and then serial dilutions of each were plated onto supplemented M9 minimal media containing adenine and KAN, with and without rifampin, to measure SOS mutagenesis activity. Plates were incubated at 42°C for 2 days prior to their counting.

In vitro cleavage of UmuD reconstituted with purified components. In vitro cleavage of UmuD was reconstituted with purified components essentially as described elsewhere (5). Briefly, reaction mixtures (20 µl) containing the indicated amounts (indicated in Fig. 5) of purified UmuD, UmuD1012, and RecA; 1.7 µM ssDNA 20-mer oligonucleotide; 2.3 mM adenosine 5'-O-(3-thiotriphosphate); 50 mM Tris-HCl (pH 7.5); 100 mM NaCl; 20 mM MgCl₂; 0.1 mM EDTA; 0.1 mM dithiothreitol, and 10% glycerol were assembled on ice. Reactions were initiated by incubation at 37°C and were quenched at the indicated times (indicated in Fig. 5) by addition of 0.25 volume of 4× sodium dodecyl sulfate (SDS) sample buffer (200 mM Tris-HCl [pH 6.8], 100 mM dithiothreitol, 8% SDS, 0.8% bromophenol blue, and 40% glycerol) followed by heating to 95°C for 5 min. Aliquots of each sample were then electrophoresed in SDS-14% polyacrylamide gels followed by staining with Coomassie blue R-250. Cleavage efficiency was quantitated by densitometric analysis of the stained gels relative to appropriate standard curves (data not shown) using the Molecular Analyst software package (Bio-Rad). For time course experiments, reaction mixtures (140 µl) containing 24.5 µg of RecA (4.6 µM as a monomer) and 14 µg of either UmuD or UmuD1012 (3.3 µM as a dimer) were assembled on ice and then transferred to 37°C. Twenty-microliter aliquots were removed after 0, 15, 30, 60, 90, and 120 min of incubation at 37°C and quenched as described above. For RecA-ssDNA titrations, the ssDNA concentration was maintained at 1.7 µM. Reaction mixtures (20 µl) contained 0, 0.44, 0.9, 1.75, 3.5, or 7 µg of RecA (0.58 to 9.2 µM as a monomer) and 2 µg of UmuD or UmuD1012 (3.3 µM as a dimer). Incubation was at 37°C for 30 min.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

umuD'1012, but not umuD1012, is active in SOS mutagenesis. We employed site-directed mutagenesis to construct a plasmid-carried umuDC operon that expresses a UmuD derivative having glycines in place of Leu101 and Arg102. These residues are highly conserved among the UmuD-like mutagenesis proteins but not among members of the structurally related LexA-like transcriptional repressor family (Fig. 2), a fact that initially suggested to us that these residues might be important for the specific biological roles of the umuD gene products rather than for the RecA-ssDNA-facilitated self-cleavage of UmuD. We chose to change Leu101 and Arg102 to glycines instead of alanines because, based on the crystal structure of the UmuD'₂ homodimer (10, 50) and the solution structure determined by nuclear magnetic resonance (Ferentz et al., unpublished data), they are located in a solvent-exposed loop of UmuD₂ (Fig. 1 and 2). We chose to analyze the double mutant in this study because the close proximity of Leu101 and Arg102 together with their high degree of conservation among the UmuD-like mutagenesis proteins suggests their mutual participation in a single function(s). Thus, we reasoned that the double mutant would exhibit a more pronounced phenotype than either of the single mutants. Finally, for the sake of simplicity, we will ascribe all phenotypes unique to UmuD1012 and/or UmuD'1012 to both substitutions throughout this report.

View larger version (63K): [in this window] [in a new window]   FIG. 2.   Partial amino acid alignment of proteins similar to E. coli UmuD. Shown is the region between amino acids 94 and 108 of UmuD. This figure is modified from references 1 and 51. UmuD-like mutagenesis proteins (I) and LexA-like transcriptional repressors (II) are grouped separately. The active-site lysine (Lys97) in E. coli UmuD is boxed. Residues 101 to 104 of E. coli UmuD, which are based on the crystal structure (10, 50) as well as the recently solved solution structure (Ferentz et al., unpublished data) form a solvent-exposed loop, as well as the corresponding residues in the related proteins, are indicated by the shaded box. Leu101 and Arg102 are represented as white letters. Ec, E. coli; St, Salmonella enterica serovar Typhimurium.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Effect of plasmids carrying either wild-type or mutant umuDC or umuD'C operons on UV-induced (20 J/m2) reversion of argE3(Oc)Arg+ in a umuDC E. coli strain (GW8023). UV mutability is expressed as the number of Arg+ revertants per 107 survivors (9). Plasmid genotypes are as follows: pBR322kan, umuDC; pSE117, umuD+C+; pSE117-1012, umuD1012C+; pGW3751, umuD'+C+; pGW3751-1012, umuD'1012C+.

umuD1012C⁺ confers a cold sensitivity for growth slightly greater than that conferred by wild-type umuDC. Another indication that the UmuD1012 protein was deficient in RecA-ssDNA-mediated cleavage came from our examination of the ability of the umuD1012C⁺ operon to cause umuD⁺C⁺-mediated cold sensitivity. This phenomenon refers to the inability of E. coli lexA(Def) strains that constitutively express a plasmid-carried umuDC operon to grow at 30°C; interestingly, these same strains exhibit no growth defects at 42°C (36, 47). A careful analysis of the genetic requirements for umuDC-mediated cold sensitivity has revealed that they are different from those for SOS mutagenesis in the sense that uncleaved UmuD, together with UmuC, confers a greater degree of cold sensitivity than does UmuD', together with UmuC (47). One of the clearest illustrations of this point was the finding that UmuD derivatives containing missense mutations rendering them noncleavable by RecA-ssDNA conferred a more severe cold sensitive phenotype than did the wild-type operon (between ~2- and ~60-fold more severe) (47). The ability of uncleaved UmuD together with UmuC to cause cold sensitivity for growth when overproduced, by virtue of an increased gene dosage, appears to be related to the proteins' ability to participate in a DNA damage checkpoint (41, 46). We observed that the plasmid-carried umuD1012C⁺ operon confers an approximately 20-fold more severe cold sensitive phenotype than did the wild-type umuDC operon (Table 2). This finding strongly parallels our earlier results with the noncleavable umuD alleles (47) and provides an additional suggestion that the UmuD1012 protein is refractory to RecA-ssDNA-mediated cleavage. Incidentally, whatever properties of UmuD are necessary for it to participate in umuDC-mediated cold sensitivity must remain largely intact in the UmuD1012 protein.

UmuD1012 is defective in RecA-ssDNA-facilitated self-cleavage in vivo. As a direct test of the possibility that the UmuD1012 protein was defective in RecA-ssDNA-facilitated cleavage, we measured its cleavage in vivo to yield UmuD'1012 following a UV dose of 20 J/m². Cleavage was monitored directly by Western blotting of whole-cell extracts using affinity-purified anti-UmuD-UmuD' antibodies as previously described (45, 46). Preliminary experiments indicated that whereas wild-type UmuD was efficiently cleaved to yield UmuD' after UV irradiation of a recA⁺ strain, UmuD1012 was not. We therefore investigated whether cleavage of UmuD1012 could be facilitated in vivo by RecA derivatives whose coprotease activity is altered. For these experiments we used culturing conditions previously demonstrated to enhance the coprotease activity of RecA protein (see Materials and Methods). Plasmids carrying umuD⁺C⁺ or umuD1012C⁺ were introduced into umuDC derivatives of recA⁺, recA430, recA441, or recA730 strains. The recA430 allele encodes a mutant RecA protein containing a glycine-204-to-serine (G204S) substitution that renders it deficient in facilitating self-cleavage of UmuD, LexA, and cI repressor while leaving it proficient in homologous recombination (24). The recA441 and recA730 alleles encode a common Q38K substitution, with the recA441 allele encoding a V298I substitution as well (26, 68). The recA441 mutant is coprotease constitutive at 42 but not 30°C, while the recA730 mutant is coprotease constitutive at both temperatures (68; reviewed in reference 28). Since the RecA441 mutant is coprotease constitutive at 42°C and since we wanted to maximize the coprotease activity of all RecA derivatives, we grew all cultures, regardless of their recA genotype, at the permissive temperature of 42°C (see Materials and Methods).

View larger version (31K): [in this window] [in a new window]   FIG. 4.   Relative in vivo cleavage efficiency and SOS mutator activity of umuD+C+ and umuD1012C+ as a function of the recA allele. (A) In vivo cleavage efficiency of UmuD and UmuD1012 was measured following UV irradiation (20 J/m2) or mock irradiation using four isogenic E. coli strains that differed in their recA allele as described in Materials and Methods [GW8023 differed additionally in that it was lexA+ and not lexA(Def); RecA430 protein cannot cleave LexA and therefore requires a lexA(Def) allele to express SOS-regulated functions (24)]. The RecA441 and RecA730 proteins are coprotease constitutive at 42°C and hence are phenotypically LexA(Def) regardless of the presence or absence of the lexA+ allele (reviewed in reference 28). All strains were grown at 42°C in M9 minimal medium supplemented with 0.4% Casamino Acids, 0.4% glucose, 100 µg of adenine per ml, and 40 µg of KAN per ml. These growth conditions have been previously reported as enhancing the coprotease activity of RecA protein (6). Note that UmuD1012 runs slightly ahead of UmuD+ in SDS-polyacrylamide gel electrophoresis. Aliquots of the same cultures that were UV irradiated and used for measuring cleavage efficiency were also used to measure SOS mutator activity (B to E). Thus, the results in panel A can be compared directly to those in panels B to E. Isogenic strains bearing either recA+ (GW8023 [B]), recA430 (GW8040 [C]), recA441 (GW8025 [D]), or recA730 (GW8110 [E]) were assayed for SOS mutator activity as described in Materials and Methods. SOS mutator activity is expressed as the number of rifampin-resistant colonies per 107 survivors.

UmuD1012 is active in SOS mutagenesis in vivo in a recA allele-specific fashion. In a parallel experiment, we compared the abilities of the UmuD and UmuD1012 proteins to participate in SOS mutagenesis as a function of the recA alleles discussed above. This was done by plating aliquots of the same UV-irradiated cultures used for the analysis of the levels of the umuD (or umuD1012) gene products onto solid medium containing rifampin. Quantitation of the rifampin-resistant CFU per survivor for each strain allowed a direct comparison of the umuD and umuD1012 alleles with respect to their activity in SOS mutagenesis. The presence of the wild-type umuDC operon efficiently complemented the umuDC allele of the host strains, as indicated by the increased frequency of rifampin resistance with all recA alleles tested except for recA430, which is defective in promoting cleavage of UmuD (Fig. 4B to E). Furthermore, the frequency of umuDC-dependent rifampin resistance was itself enhanced relative to the recA⁺ strain by recA alleles exhibiting enhanced coprotease activity (i.e., recA441 and recA730 [Fig. 4, compare panels B, D, and E]), as expected (reviewed in references 28 and 66).

UmuD1012 can undergo RecA-ssDNA-facilitated cleavage in vitro. To follow up on our observation that wild-type RecA protein could not facilitate efficient cleavage of UmuD1012 in vivo, we investigated whether highly purified UmuD1012 would be cleaved when incubated with purified RecA protein and ssDNA in vitro. In our first experiment, we investigated the efficiency of self-cleavage of UmuD1012 relative to that of wild-type UmuD as a function of time using a fixed concentration of RecA protein (4.6 µM as a monomer) and ssDNA (1.7 µM). Under these conditions, the ssDNA was slightly more than twofold in excess of that necessary for complete nucleation of RecA (~6 RecA molecules can bind to a single 20-mer ssDNA) (28). In striking contrast to our expectations based on in vivo observations, we did observe rather efficient cleavage of UmuD1012 to yield UmuD'1012 in vitro (Fig. 5A). Densitometric analysis of the Coomassie blue-stained gel relative to appropriate standard curves indicated that although UmuD1012 was activated for cleavage by RecA-ssDNA in vitro, it nevertheless underwent self-cleavage less efficiently than did wild-type UmuD under the identical conditions (Fig. 5B).

View larger version (33K): [in this window] [in a new window]   FIG. 5.   Relative in vitro cleavage efficiencies of UmuD and UmuD1012. RecA-ssDNA-facilitated cleavage of UmuD (lanes 1 to 6) or UmuD1012 (lanes 7 to 12) as a function of either time (A and B) or the RecA protein concentration (C and D) were measured in vitro as described in Materials and Methods. In both panels A and C, lanes 1 and 7 correspond to the 0-min and 0-µg RecA protein controls, respectively. Note that UmuD1012 runs slightly ahead of UmuD+ in SDS-polyacrylamide gel electrophoresis. The molar ratio of UmuD2 (as a dimer) to RecA (as a monomer) in panel A was 1:1.4. The molar ratios of UmuD2 (as a dimer) to RecA (as a monomer) in panel C are indicated (UmuD2/RecA). Densitometric analysis (B and D) of the results shown in panels A and C was performed using the Molecular Analyst software package (Bio-Rad).

UmuD1012 can interact directly with the RecA-ssDNA nucleoprotein filament in vitro. Cleavage of purified UmuD1012 in vitro was very sensitive to the concentration of RecA protein (Fig. 5C and D), suggesting that UmuD1012 might have a reduced affinity for the RecA-ssDNA nucleoprotein filament. To test whether UmuD1012 was affected in interaction with RecA, we employed a cross-linking assay developed by Frank et al. (14). Under the conditions used in this analysis, both UmuD and UmuD1012 were essentially comparable with respect to their abilities to be cross-linked to RecA-ssDNA nucleoprotein filaments in vitro (Fig. 6). Our finding that UmuD1012 was able to interact with the RecA-ssDNA nucleoprotein filament in a manner grossly similar to that of wild-type UmuD is consistent with our finding that purified UmuD1012 can be cleaved by RecA and ssDNA in vitro.

View larger version (57K): [in this window] [in a new window]   FIG. 6.   The ability of UmuD or UmuD1012 to interact with RecA-ssDNA was measured in vitro essentially as described previously (14). Reaction mixtures (10 µl) contained 100 ng of M13mp18 ssDNA and 8 µg of RecA protein (enough to coat ~50% of the ssDNA) as indicated, as well as 0.3 (lanes 3 and 8), 0.6 (lanes 4 and 9), or 1.2 (lanes 5 and 10) µg of UmuD or UmuD1012. After glutaraldehyde cross-linking (0.01% glutaraldehyde for 10 min at room temperature), aliquots of each reaction mixture were electrophoresed in 0.9% TGE (TGE is 25 mM Tris-HCl, 190 mM glycine, and 1 mM EDTA)-agarose gels (20), transferred to polyvinylidene difluoride, and probed with affinity-purified anti-UmuD-UmuD' antibodies as described elsewhere (45, 46). The positions of free and cross-linked UmuD and UmuD1012 are indicated.

UmuD'1012 is defective in inhibition of RecA-ssDNA-facilitated homologous recombination. Although UmuD'1012 was active in SOS mutagenesis (Fig. 3), it was only ~60% as active as wild-type UmuD', suggesting that Leu101 and Arg102 might contribute in some relatively modest way towards TLS. Another biological property of UmuD' and UmuC is their ability to inhibit RecA-mediated homologous recombination (53, 59, 63). In vivo, this inhibition requires that their expression be elevated to higher than physiological levels (3). Sommer and coworkers have proposed that this antirecombination activity of UmuD' together with UmuC is physiologically relevant and important for regulating the activity of RecA protein such that homologous recombination can be attenuated to permit TLS once UmuD' and UmuC have accumulated to sufficient levels (59).

View larger version (16K): [in this window] [in a new window]   FIG. 7.   The effect of plasmids carrying either wild-type or mutant umuDC or umuD'C operons on RecA-dependent homologous recombination in vivo. The efficiency of P1vir-mediated transduction of the crcA280::Tn10 locus was measured as a function of the indicated plasmid-carried umuDC operon (40). The extent of inhibition of homologous recombination is expressed relative to that observed for the umuDC control strain GW8024 bearing pBR322kan. The efficiency of transducing GW8024(pBR322kan) to tetracycline resistance by P1vir grown on the crcA280::Tn10 strain CAG12077 was 4.67 × 106 CFU per P1vir PFU. This value was normalized to 1.0, and the values obtained for GW8024 bearing the indicated plasmids were expressed relative to it as fold effects. For example, homologous recombination was inhibited ~4.3-fold by pSE117 (umuD+C+) relative to inhibition by the pBR322kan control. Plasmid genotypes are as follows: pBR322kan, umuDC; pSE117, umuD+C+; pSE117-1012, umuD1012C+; pGW3751, umuD'+C+; and pGW3751-1012, umuD'1012C+.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Model for RecA-ssDNA-facilitated cleavage of UmuD. In this report we describe our genetic and biochemical characterizations of the umuD1012 and umuD'1012 alleles, each encoding glycines in place of Leu101 and Arg102. Our findings that UmuD1012 undergoes RecA-ssDNA-facilitated cleavage less efficiently than wild-type UmuD, both in vivo and in vitro, and that umuD'1012C⁺ is severely impaired in inhibition of RecA-ssDNA-mediated homologous recombination in vivo indicate that Leu101 and Arg102 are important for interaction of the umuD gene products with RecA-ssDNA nucleoprotein filaments. In contrast, Leu101 and Arg102 are of only modest importance for TLS in vivo; umuD'1012C⁺ was ~60% as proficient as wild-type umuD'C in SOS mutagenesis.

View larger version (24K): [in this window] [in a new window]   FIG. 8.   A model to describe a possible role for Leu101 and Arg102 in RecA-ssDNA-facilitated cleavage of UmuD2. (A) Ribbon diagram of UmuD' (residues 40 to 136) indicating the relative positions of the Ser60-Lys97 catalytic dyad and Leu101-Arg102 based on the crystal structure reported by Peat et al. (10, 50). (B and C) Cartoon representation of the Ser60-Lys97 catalytic dyad and Leu101-Arg102 region of UmuD (gray) alone (B) and in complex with RecA-ssDNA (blue) (C). In the absence of RecA-ssDNA (B), Lys97 is relatively far away from Ser60 (Ferentz et al., unpublished data). In the model (C), RecA-ssDNA pushes on Leu101-Arg102, leading to the repositioning of Lys97 relative to Ser60. This repositioning of Lys97, together with numerous other contacts between UmuD and RecA-ssDNA (19, 31, 32), leads to the activation of Ser60 and subsequent cleavage of UmuD to yield UmuD'. For simplicity, the cartoons depict the catalytic dyad and Leu101-Arg102 of a single UmuD protomer; its intradimer partner and the amino-terminal arms containing the cleavage sites are not shown. See the text for further details.

The UmuD-like mutagenesis proteins and the LexA-like transcriptional repressors utilize nonidentical sets of contacts with RecA-ssDNA to facilitate their cleavage. Our finding that Leu101 and Arg102 are critical for cleavage of UmuD yet are not conserved among the structurally related transcriptional repressors that also undergo RecA-ssDNA-facilitated cleavage (1, 17) raises the question of why the UmuD-like and LexA-like subfamilies employ nonidentical sets of contacts to enable self-cleavage. Three possible explanations for this difference are as follows: (i) the UmuD-like proteins have a different and smaller amino-terminal domain than do the transcriptional repressors (52) and therefore require a different set of contacts with the RecA-ssDNA nucleoprotein filament to facilitate their cleavage; (ii) UmuD-like proteins undergo physical interactions with partner proteins (i.e., UmuC and its homologs) (4, 22, 70), as well as with other proteins involved in recombination (5, 32) and replication (62), that collectively demand that the UmuD-like proteins undergo interaction with RecA-ssDNA nucleoprotein filaments via a different set of contacts; and (iii) the use of a nonidentical set of contacts to differentially activate cleavage constitutes a device whereby the cell is able to achieve radically distinct cleavage kinetics for LexA relative to UmuD.

Relationship between the roles of the umuD'C gene products in SOS mutagenesis and in inhibition of RecA-mediated homologous recombination. In addition to its role in TLS, elevated levels of UmuD' together with UmuC act to antagonize RecA-mediated homologous recombination in vivo (59). It has been suggested that this inhibition of homologous recombination might result from an effect on the formation of RecA-ssDNA filaments and may constitute a general mechanism by which RecA-mediated homologous recombination is attenuated to allow TLS (59, 63). Our finding that umuD'1012 is able to efficiently promote SOS mutagenesis, despite its inability to efficiently inhibit RecA-mediated homologous recombination in vivo, indicates that this inhibition of recombination is not a prerequisite for SOS mutagenesis under our experimental conditions. This interpretation is consistent with the finding that a recA mutation with an N113K change was refractory to the inhibitory effect of elevated levels of UmuD'₂C but was nonetheless proficient in SOS mutagenesis (60). Although other recA alleles shown to be similarly refractory to the inhibitory effect of elevated levels of UmuD'₂C were less active in SOS mutagenesis (60), it is possible that their defect in SOS mutagenesis was due to a deficiency of the mutant RecA proteins in TLS rather than to their reduced sensitivity to UmuD'₂C.