Cleavage Of UmuD Research Articles

Altering the N-terminal arms of the polymerase manager protein UmuD modulates protein interactions.

Escherichia coli cells that are exposed to DNA damaging agents invoke the SOS response that involves expression of the umuD gene products, along with more than 50 other genes. Full-length UmuD is expressed as a 139-amino-acid protein, which eventually cleaves its N-terminal 24 amino acids to form UmuD′. The N-terminal arms of UmuD are dynamic and contain recognition sites for multiple partner proteins. Cleavage of UmuD to UmuD′ dramatically affects the function of the protein and activates UmuC for translesion synthesis (TLS) by forming DNA Polymerase V. To probe the roles of the N-terminal arms in the cellular functions of the umuD gene products, we constructed additional N-terminal truncated versions of UmuD: UmuD 8 (UmuD Δ1–7) and UmuD 18 (UmuD Δ1–17). We found that the loss of just the N-terminal seven (7) amino acids of UmuD results in changes in conformation of the N-terminal arms, as determined by electron paramagnetic resonance spectroscopy with site-directed spin labeling. UmuD 8 is cleaved as efficiently as full-length UmuD in vitro and in vivo, but expression of a plasmid-borne non-cleavable variant of UmuD 8 causes hypersensitivity to UV irradiation, which we determined is the result of a copy-number effect. UmuD 18 does not cleave to form UmuDʹ, but confers resistance to UV radiation. Moreover, removal of the N-terminal seven residues of UmuD maintained its interactions with the alpha polymerase subunit of DNA polymerase III as well as its ability to disrupt interactions between alpha and the beta processivity clamp, whereas deletion of the N-terminal 17 residues resulted in decreases in binding to alpha and in the ability to disrupt the alpha-beta interaction. We find that UmuD 8 mimics full-length UmuD in many respects, whereas UmuD 18 lacks a number of functions characteristic of UmuD.

Regulation of Mutagenic DNA Polymerase V Activation in Space and Time.

Spatial regulation is often encountered as a component of multi-tiered regulatory systems in eukaryotes, where processes are readily segregated by organelle boundaries. Well-characterized examples of spatial regulation are less common in bacteria. Low-fidelity DNA polymerase V (UmuD′2C) is produced in Escherichia coli as part of the bacterial SOS response to DNA damage. Due to the mutagenic potential of this enzyme, pol V activity is controlled by means of an elaborate regulatory system at transcriptional and posttranslational levels. Using single-molecule fluorescence microscopy to visualize UmuC inside living cells in space and time, we now show that pol V is also subject to a novel form of spatial regulation. After an initial delay (~ 45 min) post UV irradiation, UmuC is synthesized, but is not immediately activated. Instead, it is sequestered at the inner cell membrane. The release of UmuC into the cytosol requires the RecA* nucleoprotein filament-mediated cleavage of UmuD→UmuD′. Classic SOS damage response mutants either block [umuD(K97A)] or constitutively stimulate [recA(E38K)] UmuC release from the membrane. Foci of mutagenically active pol V Mut (UmuD′2C-RecA-ATP) formed in the cytosol after UV irradiation do not co-localize with pol III replisomes, suggesting a capacity to promote translesion DNA synthesis at lesions skipped over by DNA polymerase III. In effect, at least three molecular mechanisms limit the amount of time that pol V has to access DNA: (1) transcriptional and posttranslational regulation that initially keep the intracellular levels of pol V to a minimum; (2) spatial regulation via transient sequestration of UmuC at the membrane, which further delays pol V activation; and (3) the hydrolytic activity of a recently discovered pol V Mut ATPase function that limits active polymerase time on the chromosomal template.

Point mutations in Escherichia coli DNA pol V that confer resistance to non-cognate DNA damage also alter protein–protein interactions

Y-family DNA polymerases are important for conferring cellular resistance to DNA damaging agents in part due to their specialized ability to copy damaged DNA. The Escherichia coli Y-family DNA polymerases are encoded by the umuDC and dinB genes. UmuC and the cleaved form of UmuD, UmuD′, form UmuD′2C (pol V), which is able to bypass UV photoproducts such as cyclobutane pyrimidine dimers and 6-4 thymine–thymine dimers, whereas DinB is specialized to copy N2-dG adducts, such as N2-furfuryl-dG. To better understand this inherent specificity, we used hydroxylamine to generate a random library of UmuC variants from which we then selected those with the ability to confer survival to nitrofurazone (NFZ), which is believed to cause N2-furfuryl-dG lesions. We tested the ability of three of the selected UmuC variants, A9V, H282P, and T412I, to bypass N2-furfuryl-dG in vitro, and discovered that pol V containing UmuC A9V has overall modestly better primer extension activity than WT pol V, whereas the UmuC T412I and UmuC H282P mutations result in much lower primer extension efficiency. Upon further characterization, we found that the ability of the UmuC variant A9V to render cells UV-mutable is dependent on the proper length of the arm of UmuD′. Cells harboring UmuC variants T412I and H282P show enhanced cleavage of UmuD to form UmuD′, which, together with our other observations, suggests that this may be due to a disruption of a direct interaction between UmuC and UmuD. Thus, we find that protein interactions as well as protein conformation appear to be crucial for resistance to specific types of DNA damage.

Characterization of the N‐terminal Arms of the Polymerase Manager Protein UmuD

Escherichia coli cells that are exposed to exogenous or endogenous DNA damaging agents invoke the SOS response that involves expression of the umuD gene products. Full-length UmuD is expressed as a 139-amino-acid protein, which eventually cleaves its N-terminal 24 amino acids to form UmuD′. Both UmuD and UmuD′ exist alone as homodimers, but can also exchange to form UmuDD′ heterodimers. The N-termini of UmuD exist as conformationally-dynamic arms, and contain a number of recognition sites for partner proteins. Cleavage of UmuD to UmuD′ dramatically affects the function of the protein, and activates UmuC for translesion synthesis (TLS). We have constructed additional N-terminal truncations of UmuD, UmuD 8 (UmuD Δ1-7) and UmuD 18 (UmuD Δ1-17), and are probing the conformation of the N-terminal arms, their effect on cleavage as well as their effect on protein-protein interactions. We found that the loss of just the N-terminal seven amino acids of UmuD results in significant changes in conformation of the N-terminal arms and in relative preference for binding sites on the partner protein DNA polymerase III α subunit. UmuD 8 is cleaved as efficiently as full-length UmuD in vitro and in vivo, but expression of plasmid-borne UmuD 8 makes cells hypersensitive to UV irradiation. UmuD 18 does not cleave to form UmuDʹ, but confers resistance to UV irradiation. We used site-directed spin labeling for analysis by electron paramagnetic resonance (EPR) and covalent cross-linking to characterize the local motion of the N-terminal arms of the different UmuD variants. This investigation will lead to understanding the mechanism by which UmuD 8 causes UV hypersensitivity. This work is supported by the National Science Foundation and American Cancer Society.

Conformational Dynamics of the Escherichia coli DNA Polymerase Manager Proteins UmuD and UmuD′

The expression of Escherichia coli umuD gene products is upregulated as part of the SOS response to DNA damage. UmuD is initially produced as a 139-amino-acid protein, which subsequently cleaves off its N-terminal 24 amino acids in a reaction dependent on RecA/single-stranded DNA, giving UmuD′. The two forms of the umuD gene products play different roles in the cell. UmuD is implicated in a primitive DNA damage checkpoint and prevents DNA polymerase IV-dependent −1 frameshift mutagenesis, while the cleaved form facilitates UmuC-dependent mutagenesis via formation of DNA polymerase V (UmuD′2C). Thus, the cleavage of UmuD is a crucial switch that regulates replication and mutagenesis via numerous protein–protein interactions. A UmuD variant, UmuD3A, which is noncleavable but is a partial biological mimic of the cleaved form UmuD′, has been identified. We used hydrogen–deuterium exchange mass spectrometry (HXMS) to probe the conformations of UmuD, UmuD′, and UmuD3A. In HXMS experiments, backbone amide hydrogens that are solvent accessible or not involved in hydrogen bonding become labeled with deuterium over time. Our HXMS results reveal that the N-terminal arm of UmuD, which is truncated in the cleaved form UmuD′, is dynamic. Residues that are likely to contact the N-terminal arm show more deuterium exchange in UmuD′ and UmuD3A than in UmuD. These observations suggest that noncleavable UmuD3A mimics the cleaved form UmuD′ because, in both cases, the arms are relatively unbound from the globular domain. Gas-phase hydrogen exchange experiments, which specifically probe the exchange of side-chain hydrogens and are carried out on shorter timescales than solution experiments, show that UmuD′ incorporates more deuterium than either UmuD or UmuD3A. This work indicates that these three forms of the UmuD gene products are highly flexible, which is of critical importance for their many protein interactions.

A Non-cleavable UmuD Variant That Acts as a UmuD′ Mimic

UmuD(2) cleaves and removes its N-terminal 24 amino acids to form UmuD'(2), which activates UmuC for its role in UV-induced mutagenesis in Escherichia coli. Cells with a non-cleavable UmuD exhibit essentially no UV-induced mutagenesis and are hypersensitive to killing by UV light. UmuD binds to the beta processivity clamp ("beta") of the replicative DNA polymerase, pol III. A possible beta-binding motif has been predicted in the same region of UmuD shown to be important for its interaction with beta. We performed alanine-scanning mutagenesis of this motif ((14)TFPLF(18)) in UmuD and found that it has a moderate influence on UV-induced mutagenesis but is required for the cold-sensitive phenotype caused by elevated levels of wild-type UmuD and UmuC. Surprisingly, the wild-type and the beta-binding motif variant bind to beta with similar K(d) values as determined by changes in tryptophan fluorescence. However, these data also imply that the single tryptophan in beta is in strikingly different environments in the presence of the wild-type versus the variant UmuD proteins, suggesting a distinct change in some aspect of the interaction with little change in its strength. Despite the fact that this novel UmuD variant is non-cleavable, we find that cells harboring it display phenotypes more consistent with the cleaved form UmuD', such as resistance to killing by UV light and failure to exhibit the cold-sensitive phenotype. Cross-linking and chemical modification experiments indicate that the N-terminal arms of the UmuD variant are less likely to be bound to the globular domain than those of the wild-type, which may be the mechanism by which this UmuD variant acts as a UmuD' mimic.

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.

Mutational analysis of the RecA protein L1 region identifies this area as a probable part of the co-protease substrate binding site.

Previous mutational analysis of the L1 region of the RecA protein suggested that Gly-157 and Glu-158 are 'hot-spots' for the occurrence of constitutive LexA co-protease mutants (coprt[c]). In the present study, we clearly establish that position 157 is a hot-spot for the occurrence of such mutants, as 12 of 14 and 10 of 14 substitutions result in this phenotype for UmuD and LexA cleavage respectively. The frequency of such mutations at position 158 is somewhat lower, 8 of 13 and 5 of 13 for UmuD and LexA respectively. Comparison of the UmuD vs. LexA co-protease activity for all single mutants with substitutions at positions 154, 155, 156, 157 and 158 (47 in total) reveals that, although there is good agreement among most mutants regarding their ability to cleave both LexA and UmuD, there are two in particular (Glu-154-->Asp and Glu-154-->Gln) that show a clear preference for cleavage of UmuD. We also show that three second-site mutations that completely suppress coprt(c) activity toward LexA have little or no effect on the coprt(c) activity of the primary mutant toward UmuD. In addition, we observe a high frequency of second-site suppressor mutations, suggesting a functional interaction among side-chains in this region. Together, these results support the idea that the L1 region of RecA makes up part of the co-protease substrate-binding site.

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).

Escherichia coli umuDC mutants: DNA sequence alterations and UmuD cleavage.

The products of the chromosomally encoded umuDC genes are directly required for mutagenesis in Escherichia coli. Strains with either umuD or umuC mutations are rendered phenotypically non-mutable. To ascertain the molecular basis of this non-mutability, we determined the DNA sequence alterations of seven chromosomal umuDC mutants. Six mutants (umuD1, umuD44, umuD77, umuC36, umuC25, and umuC104) were found to be single base-pair substitutions that resulted in missense mutations. The Tn5 transposon insertion mutation (umuC122) resulted in a missense mutation followed immediately by a termination codon, producing a truncated UmuC protein lacking 102 carboxyl-terminal amino acids. All of the mutations were found to reside in regions of the UmuD and UmuC proteins that share high homology with analogous proteins. Chemiluminescent immunoassays revealed that the umuD1, umuD44, and umuD77 mutations all resulted in a non-cleavable UmuD protein. Because UmuD cleavage is a prerequisite for mutagenesis, the lack of UmuD processing appears to be the molecular basis for the non-mutable phenotype in these strains. These studies re-emphasize the critical nature of the RecA-mediated cleavage of UmuD for inducible mutagenesis and provide insights into the functional domains of the UmuC protein.

Coexpression of UmuD' with UmuC suppresses the UV mutagenesis deficiency of groE mutants.

The GroE proteins of Escherichia coli are heat shock proteins which have also been shown to be molecular chaperone proteins. Our previous work has shown that the GroE proteins of E. coli are required for UV mutagenesis. This process requires the umuDC genes which are regulated by the SOS regulon. As part of the UV mutagenesis pathway, the product of the umuD gene, UmuD, is posttranslationally cleaved to yield the active form, UmuD'. In order to investigate what role the groE gene products play in UV mutagenesis, we measured UV mutagenesis in groE+ and groE strains which were expressing either the umuDC or umuD'C genes. We found that expression of umuD' instead of umuD will suppress the nonmutability conferred by the groE mutations. However, cleavage of UmuD to UmuD' is unaffected by mutations at the groE locus. Instead we found that the presence of UmuD' increased the stability of UmuC in groE strains. In addition, we obtained evidence which indicates that GroEL interacts directly with UmuC.

Alleviation of EcoK DNA restriction in Escherichia coli and involvement of umuDC activity.

The activity of the EcoK DNA restriction system of Escherichia coli reduces both the plating efficiency of unmodified phage lambda and the transforming ability of unmodified pBR322 plasmid DNA. However, restriction can be alleviated in wild-type cells, by UV irradiation and expression of the SOS response, so that 10(3)- to 10(4)-fold increases in phage growth and fourfold increases in plasmid transformation occurred with unmodified DNA. Restriction alleviation was found to be a transient effect because induced cells, which initially failed to restrict unmodified plasmid DNA, later restricted unmodified phage lambda. Although the SOS response was needed for restriction alleviation, constitutive SOS induction, elicited genetically with a recA730 mutation, did not alleviate restriction and UV irradiation was still needed. A hitherto unsuspected involvement of the umuDC operon in this alleviation of restriction is characterized and, by differential complementation, was separated from the better known role of umuDC in mutagenic DNA repair. The need for cleavage of UmuD for restriction alleviation was shown with plasmids encoding cleavable, cleaved, and non-cleavable forms of UmuD. However, UV irradiation was still needed even when cleaved UmuD was provided. The possibility that restriction alleviation occurs by a general inhibition of the EcoK restriction/modification complex was tested and discounted because modification of lambda was not reduced by UV irradiation. An alternative idea, that restriction activity was competitively reduced by an increase in EcoK modification, was also discounted by the lack of any increase in the modification of lambda Ral-, a naturally undermodified phage. Other possible mechanisms for restriction alleviation are discussed.

Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage.

Most of the inducible mutagenesis observed in Escherichia coli after treatment with many DNA damaging agents is dependent upon the products of the umuD,C operon. RecA-mediated proteolytic processing of UmuD yields a carboxyl-terminal fragment (UmuD') that is active for mutagenesis. Processing of UmuD is therefore a critical step in the fixation of mutations. In this paper we have analyzed the requirements for UmuD processing in vivo. Standard immuno-detection assays, coupled with a sensitive chemiluminescence detection assay, have been utilized to probe levels of chromosomally encoded Umu proteins from whole-cell E. coli extracts. We found that the derepression of additional SOS gene products, other than RecA, was not required for UmuD processing. Moreover, efficient cleavage of UmuD was observed only in the presence of elevated levels of activated RecA, suggesting that efficient processing would occur only under conditions of severe DNA damage. Detection of chromosomally encoded Umu proteins has allowed us, for the first time, to measure directly the cellular steady-state levels of these proteins under various SOS inducing conditions. UmuD was present at approximately 180 copies per uninduced cell and was measured at approximately 2400 copies per cell in strains that lacked a functional repressor. Induced levels of UmuC were approximately 12-fold lower than UmuD with approximately 200 molecules per cell. These levels of cellular UmuC protein suggest that it functions through specific protein-DNA or protein-protein interactions, possibly as a lesion recognition protein or by interacting with DNA polymerase III.

Mutagenic DNA repair in Escherichia coli, XX. Overproduction of UmuD′ protein results in suppression of the umuC36 mutation in excision defective bacteria

Overproduction of Umu+ or UmuD′ protein by means of a gene carried on a multicopy plasmid suppressed the umuC36 phenotype and permitted induction of mutations by ultraviolet light. The umuC122::Tn5 phenotype was not suppressed. Suppression of the umuC36 phenotype was only seen when excision repair was blocked by acriflavine or by an uvrA or uvrB mutation. Cleavage of UmuD to UmuD′ in SOS-induced cells was not dependent upon the presence of UmuC protein. The results are interpreted in terms of a revised model in which UmuC protein is envisaged as guiding UmuD′ to ReA protein which has recognized and become bound to an appropriate DNA lesion. It is suggested that the umuC36 mutation gives rise to a protein with reduced affinity for UmuD′ and that the effect of this can be compensated by an excess of UmuD′.

Requirements for bypass of UV-induced lesions in single-stranded DNA of bacteriophage phi X174 in Salmonella typhimurium.

According to the current model for mutagenic bypass of UV-induced lesions, efficient bypass requires three proteins: activated RecA (RecA*) and either activated UmuD (UmuD') and UmuC or their plasmid-encoded analogues, MucA' and MucB. RecA* aids synthesis of UmuD' and UmuC (and MucA'/MucB) at two levels: by inactivation of the LexA transcriptional repressor of these genes and by cleavage of UmuD (and MucA) to produce the active fragments, UmuD' (MucA'). A third role for RecA is revealed when these two roles are otherwise satisfied in a suitably engineered strain. An often-suggested possible role for RecA in bypass is inhibition of editing by the epsilon subunit of DNA polymerase III. Here, by demonstrating that elimination of epsilon by deletion of its gene, dnaQ, does not relieve the requirement for the third function of RecA, we show that RecA must perform some function other than, or in addition to, inhibition of epsilon. We also show that elimination of epsilon does not relieve the requirement for either Muc protein. Moreover, we observed reactivation of irradiated phi X174 in unirradiated cells expressing MucA' and MucB. This finding makes it unlikely that the additional role of recA involves derepression of an unidentified gene or cleavage of an unidentified protein and makes it more likely that RecA participates directly in bypass.

UmuC mutagenesis protein of Escherichia coli: purification and interaction with UmuD and UmuD'.

The introduction of a replication-inhibiting lesion into the DNA of Escherichia coli produces a marked elevation in mutation rate. The mutation pathway is a component of the induced, multigene SOS response. SOS mutagenesis is a tightly regulated process dependent on two RecA-mediated proteolytic events: cleavage of the LexA repressor to induce the UmuC and UmuD mutagenesis proteins, and cleavage of UmuD to UmuD' to activate the mutation pathway. To investigate the protein-protein interactions responsible for SOS mutagenesis, we have studied the interaction of UmuC, UmuD, and UmuD'. To probe intracellular interaction, we have used immunoprecipitation techniques with antibodies against UmuC or UmuD and UmuD'. We have found that antibody to UmuC precipitates UmuD' from cell extracts, and antibody to UmuD and UmuD' precipitates UmuC. Thus we conclude that UmuC probably associates tightly with UmuD' in cells. For biochemical studies, we have purified the UmuC and UmuD' proteins to use with the previously purified UmuD. UmuC associates strongly with an affinity column of UmuD and UmuD', eluting only under strongly dissociating conditions (2 M urea or 1.5 M KSCN). UmuC also associates efficiently with UmuD or UmuD' in solution, as judged by velocity sedimentation in a glycerol gradient. The likely stoichiometry is one UmuC with a dimeric UmuD or UmuD'. From these experiments and previous work, we infer that SOS mutagenesis depends on the action of the UmuC-UmuD' complex and probably RecA to rescue a stalled DNA polymerase III holoenzyme at the DNA lesion.

Genetic control of AAF-induced mutagenesis at alternating GC sequences: An additional role for RecA

In a previous study, the forward mutation spectrum induced by the chemical carcinogen N-acetoxy-N-2-acetylaminofluorene was determined (Koffel-Schwartz et al. 1984). It was found that 90% of the induced mutations are frameshift mutations located within specific sequences (mutation hot spots). Two classes of mutation hot spots were found: (i) -1 frameshift mutations occurring within runs of guanines (i.e. GGGG----GGG; (ii) -2 frameshift mutations occurring within the NarI recognition sequence (GGCGCC----GGCC). In the present work, we further investigate the genetic requirements of these frameshift events by using specific reversion assays. Like UV-induced mutagenesis, frameshift mutations occurring within runs of G's (also referred to as the "slippage pathway") require the activated form of the RecA protein (RecA*). On the other hand, frameshift mutations occurring at the NarI site (the "NarI mutation pathway") require a LexA-controlled function(s) that is not UmuDC. The LexA-controlled gene(s) that is (are) involved in this pathway remain to be identified. Moreover, this pathway does not require RecA* for the proteolytic processing of a protein other than LexA (like the cleavage of UmuD in UV-induced mutagenesis). An "additional" role of RecA can be defined as follows: (i) The non-activated form of the RecA protein acts as an inhibitor in the NarI mutation pathway. (ii) This inhibition is relieved upon activation of RecA by UV irradiation of the bacteria. (iii) A recA deletion mutant is totally proficient in the NarI mutation pathway provided the SOS system is derepressed [lexA (Def) allele]. Therefore, RecA does not actively participate in the fixation of the mutation. A molecular model for this "additional" role of RecA is proposed.

UmuD mutagenesis protein of Escherichia coli: overproduction, purification, and cleavage by RecA.

The mutation rate of Escherichia coli increases approximately 100-fold after treatment with replication-inhibiting agents such as UV light. This enhanced mutation rate requires the action of the UmuD and UmuC proteins, which are induced as part of the SOS response to DNA damage. To initiate a biochemical characterization of the role of these proteins, we have developed a plasmid system that gives efficient expression of the umuD and umuC genes. The umuD and umuC genes were placed under the control of a regulated phage lambda PL promoter and a synthetic ribosome-binding site, and the distance to the UmuD start was adjusted to maximize gene expression. Starting from this overproduction system, we have purified the UmuD protein and studied its interaction with RecA. The SOS response is turned on by the capacity of RecA protein to mediate cleavage of the LexA repressor for SOS-controlled operons. Others have shown that UmuD exhibits sequence homology to LexA around the cleavage site, suggesting a possible cleavage reaction for UmuD. We show that RecA mediates cleavage of UmuD, probably at this site. As with LexA, UmuD also undergoes a self-cleavage reaction. We infer that RecA-mediated cleavage of UmuD is another role for RecA in SOS mutagenesis, probably activating UmuD for its mutagenic function.

RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation.

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. It has been shown that the UmuD protein shares homology with LexA, the repressor of the SOS genes. In this paper we describe a series of genetic experiments that indicate that the purpose of RecA-mediated cleavage of UmuD at its bond between Cys-24 and Gly-25 is to activate UmuD for its role in mutagenesis and that the COOH-terminal fragment of UmuD is necessary and sufficient for the role of UmuD in UV mutagenesis. Other genetic experiments are presented that (i) support the hypothesis that the primary role of Ser-60 in UmuD function is to act as a nucleophile in the RecA-mediated cleavage reaction and (ii) raise the possibility that RecA has a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD.