RecA-mediated Cleavage Research Articles

Integration of molecular modelling and in vitro studies to inhibit LexA proteolysis.

As antibiotic resistance has become more prevalent, the social and economic impacts are increasingly pressing. Indeed, bacteria have developed the SOS response which facilitates the evolution of resistance under genotoxic stress. The transcriptional repressor, LexA, plays a key role in this response. Mutation of LexA to a non-cleavable form that prevents the induction of the SOS response sensitizes bacteria to antibiotics. Achieving the same inhibition of proteolysis with small molecules also increases antibiotic susceptibility and reduces drug resistance acquisition. The availability of multiple LexA crystal structures, and the unique Ser-119 and Lys-156 catalytic dyad in the protein enables the rational design of inhibitors. We pursued a binary approach to inhibit proteolysis; we first investigated β-turn mimetics, and in the second approach we tested covalent warheads targeting the Ser-119 residue. We found that the cleavage site region (CSR) of the LexA protein is a classical Type II β-turn, and that published 1,2,3-triazole compounds mimic the β-turn. Generic covalent molecule libraries and a β-turn mimetic library were docked to the LexA C-terminal domain using molecular modelling methods in FlexX and CovDock respectively. The 133 highest-scoring molecules were screened for their ability to inhibit LexA cleavage under alkaline conditions. The top molecules were then tested using a RecA-mediated cleavage assay. The β-turn library screen did not produce any hit compounds that inhibited RecA-mediated cleavage. The covalent screen discovered an electrophilic serine warhead that can inhibit LexA proteolysis, reacting with Ser-119 via a nitrile moiety. This research presents a starting point for hit-to-lead optimisation, which could lead to inhibition of the SOS response and prevent the acquisition of antibiotic resistance.

Zinc blocks SOS-induced antibiotic resistance via inhibition of RecA in Escherichia coli.

Zinc inhibits the virulence of diarrheagenic E. coli by inducing the envelope stress response and inhibiting the SOS response. The SOS response is triggered by damage to bacterial DNA. In Shiga-toxigenic E. coli, the SOS response strongly induces the production of Shiga toxins (Stx) and of the bacteriophages that encode the Stx genes. In E. coli, induction of the SOS response is accompanied by a higher mutation rate, called the mutator response, caused by a shift to error-prone DNA polymerases when DNA damage is too severe to be repaired by canonical DNA polymerases. Since zinc inhibited the other aspects of the SOS response, we hypothesized that zinc would also inhibit the mutator response, also known as hypermutation. We explored various different experimental paradigms to induce hypermutation triggered by the SOS response, and found that hypermutation was induced not just by classical inducers such as mitomycin C and the quinolone antibiotics, but also by antiviral drugs such as zidovudine and anti-cancer drugs such as 5-fluorouracil, 6-mercaptopurine, and azacytidine. Zinc salts inhibited the SOS response and the hypermutator phenomenon in E. coli as well as in Klebsiella pneumoniae, and was more effective in inhibiting the SOS response than other metals. We then attempted to determine the mechanism by which zinc, applied externally in the medium, inhibits hypermutation. Our results show that zinc interferes with the actions of RecA, and protects LexA from RecA-mediated cleavage, an early step in initiation of the SOS response. The SOS response may play a role in the development of antibiotic resistance and the effect of zinc suggests ways to prevent it.

RecA-Mediated Cleavage of λ cI Repressor Accepts Repressor Dimers: Probable Role of Prolyl Cis-Trans Isomerization and Catalytic Involvement of H163, K177, and K232 of RecA

The λ cI repressor is found to be cleaved in the presence of activated RecA in its DNA- bound dimeric form at a rate similar to that in the absence of operator DNA in contrast to previous studies inferring repressor monomer as a preferred substrate. Though activated RecA does not possess any measurable isomerase activity against a standard peptide substrate, prolyl isomerase inhibitors cyclosporin A and rapamycin do inhibit RecA-mediated cleavage. Histidine and lysine to a smaller extent, are shown to cleave cI repressor in a non- enzymatic fashion whereas arginine and glutamate do not. When activated RecA filament is covalently modified by using an excess of diethyl pyrocarbonate or maleic anhydride, RecA- mediated cleavage of cI repressor is inhibited. Combining our chemical modification data with model building and earlier mutagenesis data, it is argued that H163, K177, and K232 in RecA are crucial residues involved in cI repressor cleavage by combining with the catalytic Ser149 and K192 in the repressor. It is suggested by model building that subunits n, n+4, and n+5 in the RecA filament contribute one loop each for holding the C-terminal domain of the repressor during cleavage within the RecA helical groove, explaining why its ADP-form is inactive and its ATP-form is active regarding repressor cleavage.

Structure of a Hyper-cleavable Monomeric Fragment of Phage λ Repressor Containing the Cleavage Site Region

The key event in the switch from lysogenic to lytic growth of phage lambda is the self-cleavage of lambda repressor, which is induced by the formation of a RecA-ssDNA-ATP filament at a site of DNA damage. Lambda repressor cleaves itself at the peptide bond between Ala111 and Gly112, but only when bound as a monomer to the RecA-ssDNA-ATP filament. Here we have designed a hyper-cleavable fragment of lambda repressor containing the hinge and C-terminal domain (residues 101–229), in which the monomer–monomer interface is disrupted by two point mutations and a deletion of seven residues at the C terminus. This fragment crystallizes as a monomer and its structure has been determined to 1.8 Å resolution. The hinge region, which bears the cleavage site, is folded over the active site of the C-terminal oligomerization domain (CTD) but with the cleavage site flipped out and exposed to solvent. Thus, the structure represents a non-cleavable conformation of the repressor, but one that is poised for cleavage after modest rearrangements that are presumably stabilized by binding to RecA. The structure provides a unique snapshot of lambda repressor in a conformation that sheds light on how its self-cleavage is tempered in the absence of RecA, as well as a framework for interpreting previous genetic and biochemical data concerning the RecA-mediated cleavage reaction.

LexA Analog (dra0074) Is a Regulatory Protein That Is Irrelevant to recA Induction

The protein DRA0074 is suggested to be another LexA in Deinococcus radiodurans, having similar motifs and RecA-mediated cleavage activity to D. radiodurans LexA (dra0344). However, its function has not been studied. We disrupted the gene dra0074 and measured its effect on RecA induction using fusion translation, immunoblot, and proteomic analysis. Results showed that the product of gene dra0074 is not involved in RecA induction, but is a regulator of other metabolisms in D. radiodurans.

PH-dependent autocleavage of lambda repressor occurs in the operator-bound form: characterization of lambda repressor autocleavage.

The first-order rate constants for the RecA-independent, spontaneous, pH-dependent autocleavage of the lambda cI repressor was measured in the present study at pH 10.6 at 27, 37 and 42 degrees C respectively. Autocleavage of the repressor occurs also at pH 9 and 8, although at progressively slower rates. We demonstrate that the spontaneous autocleavage occurs also in the operator-bound state, at a rate either higher than or equal to the rate in solution, depending on the pH value. Owing to the near equality of the rate constant in both operator-free and operator-bound repressors, it can be inferred that the cleavage site has a similar structure and dynamics with respect to the catalytic site in both forms at neutral pH. Covalent modification using PMSF, brought about by a large molar excess of the reagent, inhibits autocleavage of the lambda repressor. The difficulty in obtaining this covalent modification is rationalized using our recent lambda repressor models. Bimolecular type II trans -cleavage was observed previously for mutant LexA repressors lacking a crucial catalytic serine or lysine residue [Kim and Little (1993) Cell (Cambridge, Mass.) 73, 1165-1173], but it could still be cleaved by an 85-202 'enzyme' fragment possessing an improved or hypercleavable character lacking its own cleavage site. Such a type II trans -cleavage was not observed with the covalently modified intact lambda repressor used as substrate and the purified wild-type lambda repressor 112-236 fragment used as the 'enzyme'. All these results show that for the wild-type lambda repressor, the catalytic site is close to the cleavage site in both operator-free and -bound states. In the lytic pathway, the repressor is mainly cleaved via RecA-mediated cleavage, which occurs much faster than the spontaneous autocleavage; the possible biological significance of this slow, spontaneous, but constant, autocleavage is related to the lysogenic state, when RecA-mediated cleavage is absent.

The preferred substrate for RecA-mediated cleavage of bacteriophage 434 repressor is the DNA-bound dimer.

Induction of a lysogen of a lambdoid bacteriophage usually involves RecA-stimulated autoproteolysis of the bacteriophage repressor protein. Previous work on the phage repressors showed that the monomeric form of the protein is the target of RecA. Our previous work indicated that in the case of bacteriophage 434, virtually none of the repressor is present as a monomer in vivo. Hence, if the repressor in a lysogen is present as a dimer, how can RecA-stimulated autoproteolysis play a role in bacteriophage induction? We examined this question by determining the rate of RecA-stimulated 434 repressor cleavage as a function of repressor concentration and added DNA. Our results show that binding of 434 repressor to a specific DNA binding site dramatically increases the velocity of repressor autocleavage compared to the velocity of cleavage of the monomer and concentration-induced dimer. DNA binding-deficient hemidimers formed between the intact repressor and its C-terminal domain fragment have a lower rate of cleavage than DNA-bound dimers. These results show that the DNA-bound 434 repressor dimer, which is the form of the repressor that is required for its transcriptional regulatory functions, is the preferred form for RecA-stimulated autocleavage. We also show that the rate of repressor autocleavage is influenced by the sequence of the bound DNA. Kinetic analysis of the autocleavage reaction indicated that the DNA sequence influences the velocity of 434 repressor autocleavage by affecting the affinity of the repressor-DNA complex for RecA, not the chemical cleavage step. Regardless of the mechanism, the finding that the presence and precise sequence of DNA modulate the autocleavage reaction shows that DNA allosterically affects the function of 434 repressor.

Stability puzzles in phage lambda.

In the absence of RecA-mediated cleavage of the repressor, the lambda prophage is exceptionally stable. We develop a stochastic model that predicts the stability of such epigenetic states from affinities of the molecular components. We find that the stability, in particular, depends on the maximum possible cI protein production, and on the number of translated cro proteins per transcribed mRNA. We apply the model to the behavior of recently published mutants of O(R) and find, in particular, that a mutant that overexpress cro behaves in a different way than what was predicted, thus suggesting that the current view of the O(R) switch is incomplete. The approach described here should be generally applicable to the stability of expressed states.

Analysis of Escherichia coli RecA interactions with LexA, lambda CI, and UmuD by site-directed mutagenesis of recA.

An early event in the induction of the SOS system of Escherichia coli is RecA-mediated cleavage of the LexA repressor. RecA acts indirectly as a coprotease to stimulate repressor self-cleavage, presumably by forming a complex with LexA. How complex formation leads to cleavage is not known. As an approach to this question, it would be desirable to identify the protein-protein interaction sites on each protein. It was previously proposed that LexA and other cleavable substrates, such as phage lambda CI repressor and E. coli UmuD, bind to a cleft located between two RecA monomers in the crystal structure. To test this model, and to map the interface between RecA and its substrates, we carried out alanine-scanning mutagenesis of RecA. Twenty double mutations were made, and cells carrying them were characterized for RecA-dependent repair functions and for coprotease activity towards LexA, lambda CI, and UmuD. One mutation in the cleft region had partial defects in cleavage of CI and (as expected from previous data) of UmuD. Two mutations in the cleft region conferred constitutive cleavage towards CI but not towards LexA or UmuD. By contrast, no mutations in the cleft region or elsewhere in RecA were found to specifically impair the cleavage of LexA. Our data are consistent with binding of CI and UmuD to the cleft between two RecA monomers but do not provide support for the model in which LexA binds in this cleft.

The bacteriophage P1 HumD protein is a functional homolog of the prokaryotic UmuD'-like proteins and facilitates SOS mutagenesis in Escherichia coli.

The Escherichia coli umuD and umuC genes comprise an operon and encode proteins that are involved in the mutagenic bypass of normally replication-inhibiting DNA lesions. UmuD is, however, unable to function in this process until it undergoes a RecA-mediated cleavage reaction to generate UmuD'. Many homologs of umuDC have now been identified. Most are located on bacterial chromosomes or on broad-host-range R plasmids. One such putative homolog, humD (homolog of umuD) is, however, found on the bacteriophage P1 genome. Interestingly, humD differs from other umuD homologs in that it encodes a protein similar in size to the posttranslationally generated UmuD' protein and not UmuD, nor is it in an operon with a cognate umuC partner. To determine if HumD is, in fact, a bona fide homolog of the prokaryotic UmuD'-like mutagenesis proteins, we have analyzed the ability of HumD to complement UmuD' functions in vivo as well as examined HumD's physical properties in vitro. When expressed from a high-copy-number plasmid, HumD restored cellular mutagenesis and increased UV survival to normally nonmutable recA430 lexA(Def) and UV-sensitive DeltaumuDC recA718 lexA(Def) strains, respectively. Complementing activity was reduced when HumD was expressed from a low-copy-number plasmid, but this observation is explained by immunoanalysis which indicates that HumD is normally poorly expressed in vivo. In vitro analysis revealed that like UmuD', HumD forms a stable dimer in solution and is able to interact with E. coli UmuC and RecA nucleoprotein filaments. We conclude, therefore, that bacteriophage P1 HumD is a functional homolog of the UmuD'-like proteins, and we speculate as to the reasons why P1 might require the activity of such a protein in vivo.

Mutations affecting the ability of the Escherichia coli UmuD' protein to participate in SOS mutagenesis.

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli, a process that results from a translesion synthesis mechanism. The UmuD protein is activated for its role in mutagenesis by a RecA-facilitated autodigestion that removes the N-terminal 24 amino acids. A previous genetic screen for nonmutable umuD mutants had resulted in the isolation of a set of missense mutants that produced UmuD proteins that were deficient in RecA-mediated cleavage (J. R. Battista, T. Ohta, T. Nohmi, W. Sun, and G. C. Walker, Proc. Natl. Acad. Sci. USA 87:7190-7194, 1990). To identify elements of the UmuD' protein necessary for its role in translesion synthesis, we began with umuD', a modified form of the umuD gene that directly encodes the UmuD' protein, and obtained missense umuD' mutants deficient in UV and methyl methanesulfonate mutagenesis. The D39G, L40R, and T51I mutations affect residues located at the UmuD'2 homodimer interface and interfere with homodimer formation in vivo. The D75A mutation affects a highly conserved residue located at one end of the central strand in a three-stranded beta-sheet and appears to interfere with UmuD'2 homodimer formation indirectly by affecting the structure of the UmuD' monomer. When expressed from a multicopy plasmid, the L40R umuD' mutant gene exhibited a dominant negative effect on a chromosomal umuD+ gene with respect to UV mutagenesis, suggesting that the mutation has an effect on UmuD' function that goes beyond its impairment of homodimer formation. The G129D mutation affects a highly conserved residue that lies at the end of the long C-terminal beta-strand and results in a mutant UmuD' protein that exhibits a strongly dominant negative effect on UV mutagenesis in a umuD+ strain. The A30V and E35K mutations alter residues in the N-terminal arms of the UmuD'2 homodimer, which are mobile in solution.

Novel Escherichia coli umuD' mutants: structure-function insights into SOS mutagenesis.

Although it has been 10 years since the discovery that the Escherichia coli UmuD protein undergoes a RecA-mediated cleavage reaction to generate mutagenically active UmuD', the function of UmuD' has yet to be determined. In an attempt to elucidate the role of UmuD' in SOS mutagenesis, we have utilized a colorimetric papillation assay to screen for mutants of a hydroxylamine-treated, low-copy-number umuD' plasmid that are unable to promote SOS-dependent spontaneous mutagenesis. Using such an approach, we have identified 14 independent umuD' mutants. Analysis of these mutants revealed that two resulted from promoter changes which reduced the expression of wild-type UmuD', three were nonsense mutations that resulted in a truncated UmuD' protein, and the remaining nine were missense alterations. In addition to the hydroxylamine-generated mutants, we have subcloned the mutations found in three chromosomal umuD1, umuD44, and umuD77 alleles into umuD'. All 17 umuD' mutants resulted in lower levels of SOS-dependent spontaneous mutagenesis but varied in the extent to which they promoted methyl methanesulfonate-induced mutagenesis. We have attempted to correlate these phenotypes with the potential effect of each mutation on the recently described structure of UmuD'.

Differential cleavage of LexA and UmuD mediated by recA pro67 mutants: implications for common LexA and UmuD binding sites on RecA

In Escherichia coli, RecA-mediated cleavage of LexA repressor is a key regulatory event required for expression of SOS genes involved in the repair of DNA damage. RecA also mediates the cleavage of UmuD protein to UmuD′, a form active in SOS mutagenesis. To determine whether LexA and UmuD have common binding determinants on RecA, we have compared the ability of several recA mutants to function in the cleavage of LexA versus UmuD in vivo. The data reveal that while some recA mutations at Pro67 have a similar effect on LexA and UmuD cleavage, others have striking differential effects. For example, a Pro67 → Trp mutation results in a high level of constitutive cleavage of both proteins. However, Pro67 → Asp and Glu mutations promote constitutive cleavage of LexA and reduce induction of UmuD cleavage to just 5 to 10% of wild-type activity. In contrast, Pro67 → Arg prevents LexA cleavage while allowing nearly 50% of wild-type induction of UmuD cleavage. These results are consistent with the idea that Pro67 is located at a site in the nucleoprotein filament where both LexA and UmuD contact RecA.

The Bacillus subtilis dinR Gene Codes for the Analogue of Escherichia coli LexA: PURIFICATION AND CHARACTERIZATION OF THE DinR PROTEIN

The Bacillus subtilis dinR gene encodes a 23-kDa protein that shares about 34% homology with the Escherichia coli LexA protein. We have purified the dinR gene product to near homogeneity, and we describe its activities. The purified DinR protein binds specifically to the promoter regions of three B. subtilis SOS genes: dinB, dinC, and recA. Electrophoretic mobility of DinR-promoter complexes in each case is identical to that of promoters bound by the B. subtilis SOS repressor (Lovett, et al., (1993) J. Bacteriol. 175, 6842-6849). Analysis of hydroxyl radical footprints of DinR bound to the dinC promoter indicates that DinR interacts with one side of the DNA providing access to the consensus operator site (5'-GAACN4GTTC-3') within two adjacent major grooves. Consistent with its proposed role as a transcriptional repressor, purified DinR displaces B. subtilis RNA polymerase from the recA promoter and represses transcription of the recA gene in vitro. We also show that purified DinR protein undergoes general base-catalyzed autodigestion as well as RecA-mediated cleavage at the peptide bond between Ala-91 and Gly-92. Corresponding to its cleavage by activated RecA following DNA damage, the level of DinR is significantly reduced in RecA+ B. subtilis cells following exposure to mitomycin C. Thus, the DinR protein is structurally and functionally analogous to the E. coli LexA protein, and accordingly, we propose renaming the protein B. subtilis LexA.

Analysis of the region between amino acids 30 and 42 of intact UmuD by a monocysteine approach.

On the basis of characterizations of a set of UmuD monocysteine derivatives, we had suggested that positions 24, 34, and 44 are closer to the intact UmuD homodimer interface than other positions tested (M. H. Lee, T. Ohta, and G. C. Walker, J. Bacteriol. 176:4825-4837, 1994). Because this region of UmuD also appeared to be important for interactions with RecA, we followed up on our previous study by constructing a second set of monocysteine UmuD derivatives with single cysteine substitutions at positions 30 to 42. We found that like the VC34 mutant, UmuD derivatives with monocysteine substitutions at positions 32 and 35 showed deficiencies in in vivo and in vitro RecA-mediated cleavage as well as in UV mutagenesis, suggesting that the position 32 to 35 region may be important for RecA-mediated cleavage of UmuD. Interestingly, UmuD with monocysteine substitutions at residues 33 and 40 showed a reduction in UV mutagenesis while retaining the ability to be cleaved by RecA in vivo, suggesting a deficiency in the subsequent role of the UmuD' derivatives in mutagenesis. All of the UmuD monocysteine derivatives in the position 30 to 42 series purified indistinguishably from the wild-type protein. The observations that purified proteins of the UmuD derivatives RC37 and IC38 could be disulfide cross-linked quantitatively upon addition of iodine and yet were poorly modified with iodoacetate led us to suggest that the pairs of residues at positions 37 and 38 are extremely close to the UmuD2 homodimer interface. These observations indicate that the structure of the UmuD2 homodimer in solution is very different from the crystal structure of the UmuD'2 homodimer reported by Peat et al. (T. S. Peat, E. G. Frank, J. P. McDonald, A. S. Levine, R. Woodgate, and W. A. Hendrickson, Nature [London] 380:727-730, 1996).

Inhibition of RecA-mediated cleavage in covalent dimers of UmuD.

Disulfide-cross-linked UmuD2 derivatives were cleaved poorly upon incubation with activated RecA. Reducing the disulfide bonds prior to incubating the derivatives with RecA dramatically increased their extent of cleavage. These observations suggest that the UmuD monomer is a better substrate for the RecA-mediated cleavage reaction than the dimer.

Mutant LexA proteins with specific defects in autodigestion.

In self-processing biochemical reactions, a protein or RNA molecule specifically modifies its own structure. Many such reactions are regulated in response to the needs of the cell by an interaction with another effector molecule. In the system we study here, specific cleavage of the Escherichia coli LexA repressor, LexA cleaves itself in vitro at a slow rate, but in vivo cleavage requires interaction with an activated form of RecA protein. RecA acts indirectly as a coprotease to stimulate LexA autodigestion. We describe here a new class of lexA mutants, lexA (Adg-; for autodigestion-defective) mutants, termed Adg- for brevity. Adg- mutants specifically interfered with the ability of LexA to autodigest but left intact its ability to undergo RecA-mediated cleavage. The data are consistent with a conformational model in which RecA favors a reactive conformation capable of undergoing cleavage. To our knowledge, this is the first example of a mutation in a regulated self-processing reaction that impairs the rate of self-processing without markedly affecting the stimulated reaction. Had wild-type lexA carried such a substitution, discovery of its self-processing would have been difficult; we suggest that, in other systems, a slow rate of self-processing has prevented recognition that a reaction is of this nature.

The genetic requirements for UmuDC-mediated cold sensitivity are distinct from those for SOS mutagenesis.

The umuDC operon of Escherichia coli, a member of the SOS regulon, is required for SOS mutagenesis. Following the posttranslational processing of UmuD to UmuD' by RecA-mediated cleavage, UmuD' acts in concert with UmuC, RecA, and DNA polymerase III to facilitate the process of translesion synthesis, which results in the introduction of mutations. Constitutive expression of the umuDC operon causes an inhibition of growth at 30 degrees C (cold sensitivity). The umuDC-dependent physiological phenomenon manifested as cold-sensitive growth is shown to differ from SOS mutagenesis in two respects. Intact UmuD, the form inactive in SOS mutagenesis, confers a significantly higher degree of cold sensitivity in combination with UmUC than does UmuD'. In addition, umuDC-mediated cold sensitivity, unlike SOS mutagenesis, does not require recA function. Since the RecA protein mediates the autodigestion of UnmD to UmuD', this finding supports the conclusion that intact UmuD is capable of conferring cold sensitivity in the presence of UmuC. The degree of inhibition of growth at 30 degrees C correlates with the levels of UmuD and UmuC, which are the only two SOS-regulated proteins required to observe cold sensitivity. Analysis of the cellular morphology of strains that exhibit cold sensitivity for growth led to the finding that constitutive expression of the umuDC operon causes a novel form of sulA- and sfiC-independent filamentation at 30 degrees C. This filamentation is observed in a strain constitutively expressing the single, chromosomal copy of umuDC and can be suppressed by overexpression of the ftsQAZ operon.

A monocysteine approach for probing the structure and interactions of the UmuD protein

UmuD participates in a variety of protein-protein interactions that appear to be essential for its role in UV mutagenesis. To learn about these interactions, we have initiated an approach based on the construction of a series of monocysteine derivatives of UmuD and have carried out experiments exploring the chemistry of the unique thiol group in each derivative. In vivo and in vitro characterizations indicate that these proteins have an essentially native structure. In proposing a model for the interactions of UmuD in the homodimer, we have made the following assumptions: (i) the conformations of the mutant proteins are similar to that of the wild type, and (ii) the differences in reactivity of the mutant proteins are predominantly due to the positional effects of the single cysteine substitutions. The model proposes the following. The region including the Cys-24-Gly-25 cleavage site, Val-34, and Leu-44 are closer to the interface than the other positions tested as suggested by the relative ease of dimer cross-linking of the monocysteine derivatives at these positions by oxidation with iodine (I2) and by reaction with bis-maleimidohexane. The mutant with a Ser-to-Cys change at position 60 (SC60) is similar in iodoacetate reactivity to the preceding derivatives but cross-links less efficiently by I2 oxidation. This suggests that Ser-60, the site of the putative nucleophile in the cleavage reaction, is located further from the dimer interface or in a cleft region. Both Ser-19, located in the N-terminal fragment of UmuD that is removed by RecA-mediated cleavage, and Ser-67 are probably not as close to the dimer interface, since they are cross-linked more easily with bis-maleimidohexane than with I2. The SC67 mutant phenotype also suggests that this position is less important in RecA-mediated cleavage but more important in a subsequent role for UmuD in mutagenesis. Ala-89, Gln-100, and Asp-126 are probably not particularly solvent accessible and may play important roles in protein architecture.

20] Cleavage of LexA repressor

Publisher Summary This chapter examines cleavage of LexA repressor. In normally growing cells, about 20 SOS genes are turned off by the LexA repressor. On inducing treatments, LexA undergoes specific proteolytic cleavage; cleavage inactivates LexA and leads to derepression of the SOS genes. This specific cleavage reaction is of biological interest as it controls the state of the SOS regulatory system. Lex A cleavage is catalyzed in vivo and at neutral pH in vitro , by interaction with another regulatory protein, RecA, which is activated in vivo by inducing treatments. The use of mutant proteins with an increased rate of cleavage allows the development of an intermolecular cleavage reaction in which one molecule of LexA acts as an enzyme to attack other molecules. LexA is 22.7 kDa in size and is organized into two structural and functional domains separated by a hinge region. LexA N-terminal region is the DNA binding domain; its C-terminal domain contains the contacts for dimerization, and all the elements necessary for specific cleavage. The cleavage site, Ala 84 -Gly 85 , lies in the hinge region. A tryptic fragment comprising residues 68-202 undergoes both autodigestion and RecA-mediated cleavage at normal rates.