UmuD Protein Research Articles

Deciphering proteolysis pathways for the error-prone DNA polymerase in cyanobacteria.

Protein quality control pathways require AAA+ proteases, such as Clp and Lon. Lon protease maintains UmuD, an important component of the error-prone DNA repair polymerase (Pol V), at very low levels in E. coli. Most members of the phylum Cyanobacteria lack Lon (including the model cyanobacterium, Synechocystis sp. PCC6803), so maintenance of UmuD at low levels must employ different proteases. We demonstrate that the first 19 residues from the N-terminus of UmuD (Sug1-19 ) fused to a reporter protein are adequate to trigger complete proteolysis and that mutation of a single leucine residue (L6) to aspartic acid inhibits proteolysis. This process appears to follow the N-end rule and is mediated by ClpA/P protease and the ClpS adaptor. Additionally, mutations of arginine residues in the Sug1-19 tag suggest that the ClpX/P pathway also plays a role in proteolysis. We propose that there is a dual degron at the N-terminus of the UmuD protein in Synechocystis sp. PCC6803, which is distinct from the degron required for degradation of UmuD in E. coli. The use of two proteolysis pathways to tune levels of UmuD might reflect how a photosynthetic organism responds to multiple environmental stressors.

Site-Specific Reversible Protein and Peptide Modification: Transglutaminase-Catalyzed Glutamine Conjugation and Bioorthogonal Light-Mediated Removal.

Dynamic photoswitches in proteins that impart spatial and temporal control are important to manipulate and study biotic and abiotic processes. Nonetheless, approaches to install these switches into proteins site-specifically are limited. Herein we describe a novel site-specific method to generate photoremovable protein conjugates. Amine-containing chromophores (e.g., venerable o-nitrobenzyl and less-explored o-nitrophenylethyl groups) were incorporated via transamidation into a glutamine side chain of α-gliadin, LCMV, and TAT peptides, as well as β-casein and UmuD proteins by transglutaminase (TGase, EC 2.3.2.13). Subsequently, photolysis regenerated the native peptides and proteins. When this modification leads to the reduction or abolishment of certain activities, the process is referred to as caging, as in the case for E. coli polymerase manager protein UmuD. Importantly, this method is simple, robust, and easily adaptable, e.g., all components are commercially available.

Impact of UVA pre-radiation on UVC disinfection performance: Inactivation, repair and mechanism study

Ultraviolet (UV) light emission diode (LED), which is mercury free and theoretically more energy efficient, has now become an alternative to conventional UV lamps in water disinfection industry. In this research, the disinfection performance of a novel sequential process, UVA365nm LED followed by UVC265nm LED (UVA-UVC), was evaluated. The results revealed that the responses of different bacterial strains to UVA-UVC varied. Coupled with appropriate dosages of UVC, a 20 min UVA pre-radiation provided higher inactivations (log inactivation) of E.coli ATCC 11229, 15597 and 700891 by 1.2, 1.4 and 1.2 times, respectively than the sum of inactivations by UVA alone and UVC alone. On the contrary, the inactivation of E.coli ATCC 25922, the most UVC sensitive strain, decreased from 3 log to 1.8 log after UVA pre-radiation. A 30 min UVA pre-radiation did not affect the photo repair capacity of the four strains (n = 23, p > 0.1), but their dark repair ability was significantly inhibited (n = 14, p < 0.05). Mechanism study was conducted for two representative strains, E.coli ATCC 15597 and 25922 to understand the observed effect. The hypothesis that UVA pre-radiation promoted the yield of reactive oxygen species (ROS) was rejected. ELISA results indicated that 18% more cyclobutane pyrimidine dimers (CPD) were formed in E.coli ATCC 15597 with UVA pre-radiation (n = 3, p < 0.01), however, the CPD levels of E.coli ATCC 25922 was the same with or without UVA pre-radiation (n = 3, p > 0.01). Considering the results of both dark repair and CPD formation, it was concluded that the increased UV sensitivity of E.coli 15597 was originated from the increased CPD. For E.coli ATCC 25922, the enhanced UV resistance was attributed to the strain's adoption of a survival strategy, translesion DNA synthesis (TLS), when triggered by UVA pre-radiation. The study on UmuD protein, which is a key protein during TLS, confirmed this hypothesis.

Multiple forms of the E. coli SOS response protein UmuD

Escherichia coli SOS mutagenesis involves the induction of at least 57 genes in response to DNA damage, including the umuD gene products. UmuD2 is a homodimer of 139‐amino acid subunits that interacts with RecA:ssDNA resulting in cleavage of its N‐terminal 24‐amino acids, yielding UmuD’2. The goal of our research is to determine the conformation and dynamics of the UmuD proteins in order to understand its regulatory role in response to DNA damage. We have discovered an N‐terminally truncated version of the UmuD protein that is similar to full‐length UmuD2 in cleavage activity, but differs in its conformation. In vivo characterization reveals that this newly identified form is active in facilitating UV mutagenesis and displays a cold sensitive phenotype. This suggests that the length of the arms of the UmuD proteins is critical in regulating function. Indeed, we have shown by EPR that UmuD2 and UmuD’2 arm dynamics are different. Additionally, full‐length UmuD2 and cleaved UmuD’2 interact differentially with partner proteins resulting in dramatically different cellular outcomes. Our preliminary work points to yet another level of regulation in which the umuD gene products coordinate the DNA damage response. Research supported by NSF Career MCB‐0845033.

Multple Active Forms of UmuD Protein that Regulates Bacterial Mutagenesis

The SOS mutagenesis pathway in Escherichia coli involves the induction of at least 57 genes in response to DNA damage, including the umuD gene products. The homodimeric umuD gene products play key roles in regulating the cellular response to DNA damage. UmuD2 is composed of 139-amino acid subunits and is upregulated as part of the SOS response. Subsequently, damage-induced RecA/ssDNA nucleoprotein filaments mediate the slow autocleavage of the N-terminal 24-amino acid arms of UmuD2 yielding UmuD′2. Several models have been proposed wherein the N-terminal arms of UmuD2 are in the cis or trans conformations, with elbows up or down. These conformational variants expose multiple binding surfaces that may interact with other proteins. UmuD2 and UmuD′2 make a number of distinct protein-protein contacts with DNA polymerases and other proteins that both prevent and facilitate potentially mutagenic translesion synthesis. Wild-type UmuD2 and UmuD′2 form exceptionally tight dimers in solution; however, we show that the single amino-acid change N41D generates stable, active UmuD and UmuD′ monomers that functionally mimic the dimeric wild-type proteins. The UmuD N41D monomer is proficient for cleavage and interacts physically with DNA polymerase IV (DinB). Furthermore, the N41D variants facilitate UV-induced mutagenesis and promote overall cell viability. Taken together, these observations show that a monomeric form of UmuD retains substantial function in vivo and in vitro.

DNA damage response protein UmuD displays conformational dynamics

DNA damage is universal and all organisms have systems in place to respond to such damage. The Escherichia coli umuD gene products are essential to SOS‐induced mutagenesis in response to DNA damage. UmuD is a dynamic 139‐amino acid homodimeric protein that binds to a RecA/ssDNA nucleoprotein filament, which stimulates the latent ability of UmuD to cleave its N‐terminal 24‐amino acids to form UmuD’. The umuD gene products have been shown to perform distinct functions in both preventing and facilitating mutagenesis. Several models have been proposed for the conformation of the UmuD protein including two versions with the arms in trans conformation and two models with the arms in cis conformation. Each conformation exposes different binding surfaces through which UmuD could interact with other proteins. Through the use of models and structural data, it was concluded that structural rearrangement of UmuD occurs upon binding to the RecA filament and upon cleavage. The goal of our research is to determine the conformation and dynamics of the umuD gene products in order to understand how their protein‐protein interactions regulate cellular responses to DNA damage. Electron paramagnetic resonance spectroscopy (EPR) has been employed to probe the conformations of UmuD and UmuD’. Our EPR experiments suggest that the UmuD N‐terminal arms exhibit a large degree of motion. Research supported by NSF Career MCB‐0845033.

Characterization of Novel Alleles of the Escherichia coli umuDC Genes Identifies Additional Interaction Sites of UmuC with the Beta Clamp

Translesion synthesis is a DNA damage tolerance mechanism by which damaged DNA in a cell can be replicated by specialized DNA polymerases without being repaired. The Escherichia coli umuDC gene products, UmuC and the cleaved form of UmuD, UmuD', comprise a specialized, potentially mutagenic translesion DNA polymerase, polymerase V (UmuD'(2)C). The full-length UmuD protein, together with UmuC, plays a role in a primitive DNA damage checkpoint by decreasing the rate of DNA synthesis. It has been proposed that the checkpoint is manifested as a cold-sensitive phenotype that is observed when the umuDC gene products are overexpressed. Elevated levels of the beta processivity clamp along with elevated levels of the umuDC gene products, UmuD'C, exacerbate the cold-sensitive phenotype. We used this observation as the basis for genetic selection to identify two alleles of umuD' and seven alleles of umuC that do not exacerbate the cold-sensitive phenotype when they are present in cells with elevated levels of the beta clamp. The variants were characterized to determine their abilities to confer the umuD'C-specific phenotype UV-induced mutagenesis. The umuD variants were assayed to determine their proficiencies in UmuD cleavage, and one variant (G129S) rendered UmuD noncleaveable. We found at least two UmuC residues, T243 and L389, that may further define the beta binding region on UmuC. We also identified UmuC S31, which is predicted to bind to the template nucleotide, as a residue that is important for UV-induced mutagenesis.

Dynamics of the polymerase manager protein UmuD: DNA damage tolerance in E. coli

In E. coli, the function of specialized Y family DNA polymerases is regulated by UmuD, a small manager protein, and its cleaved form, UmuD′. The different forms of the UmuD proteins play dramatically different roles in the cellular response to DNA damage: UmuD facilitates a primitive DNA damage checkpoint while UmuD′ enables DNA damage‐dependent mutagenesis. The β processivity clamp, which tethers DNA polymerases to the DNA, confers high processivity on normal DNA polymerases. UmuD is known to bind the β clamp and this may modulate the activity of β. The objective of this study is to understand the conformational dynamics of UmuD to determine its effect on DNA damage tolerance. We constructed a library of single cysteine derivatives of UmuD to facilitate labeling for fluorescence resonance energy transfer (FRET) and electron paramagnetic resonance (EPR) studies. All single cysteine derivatives of UmuD were shown to be active in UV‐induced mutagenesis, and will be labeled. Hydrogen‐deuterium exchange mass spectrometry experiments show that UmuD is flexible, while UmuD′ is slightly less so.This work is supported by a Dreyfus Foundation New Faculty Award and the Northeastern University Office of the Provost.

SSB Protein Limits RecOR Binding onto Single-stranded DNA

The RecO and RecR proteins form a complex that promotes the nucleation of RecA protein filaments onto SSB protein-coated single-stranded DNA (ssDNA). However, even when RecO and RecR proteins are provided at optimal concentrations, the loading of RecA protein is surprisingly slow, typically proceeding with a lag of 10 min or more. The rate-limiting step in RecOR-promoted RecA nucleation is the binding of RecOR protein to ssDNA, which is inhibited by SSB protein despite the documented interaction between RecO and SSB. Full activity of RecOR is seen only when RecOR is preincubated with ssDNA prior to the addition of SSB. The slow binding of RecOR to SSB-coated ssDNA involves the C terminus of SSB. When an SSB variant that lacks the C-terminal 8 amino acids is used, the capacity of RecOR to facilitate RecA loading onto the ssDNA is largely abolished. The results are used in an expanded model for RecOR action.

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.

A Non‐cleavable UmuD variant that acts as a UmuD’ mimic

Abstract UmuD2 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 β processivity clamp (“β”) of the replicative DNA polymerase, pol III. A possible β-binding motif has been predicted in the same region of UmuD shown to be important for its interaction with β. We performed alanine-scanning mutagenesis of this motif (14TFPLF18) 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 β-binding motif variant bind to β with similar Kd values as determined by changes in tryptophan fluorescence. However, these data also imply that the single tryptophan in β 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.

Genetic and biochemical characterization of a novel umuD mutation: insights into a mechanism for UmuD self-cleavage.

Most translesion DNA synthesis (TLS) in Escherichia coli is dependent upon the products of the umuDC genes, which encode a DNA polymerase, DNA polymerase V, with the unique ability to replicate over a variety of DNA lesions, including cyclobutane dimers and abasic sites. The UmuD protein is activated for its role in TLS by a RecA-single-stranded DNA (ssDNA)-facilitated self-cleavage event that serves to remove its amino-terminal 24 residues to yield UmuD'. We have used site-directed mutagenesis to construct derivatives of UmuD and UmuD' with glycines in place of leucine-101 and arginine-102. These residues are extremely well conserved among the UmuD-like proteins involved in mutagenesis but are poorly conserved among the structurally related LexA-like transcriptional repressor proteins. Based on both the crystal and solution structures of the UmuD' homodimer, these residues are part of a solvent-exposed loop. Our genetic and biochemical characterizations of these mutant UmuD and UmuD' proteins indicate that while leucine-101 and arginine-102 are critical for the RecA-ssDNA-facilitated self-cleavage of UmuD, they serve only a minimal role in enabling TLS. These results, and others, suggest that the interaction of RecA-ssDNA with leucine-101 and arginine-102, together with numerous other contacts between UmuD(2) and the RecA-ssDNA nucleoprotein filaments, serves to realign lysine-97 relative to serine-60, thereby activating UmuD(2) for self-cleavage.

Subunit-specific degradation of the UmuD/D' heterodimer by the ClpXP protease: the role of trans recognition in UmuD' stability.

The Escherichia coli UmuD' protein is a subunit of the recently described error-prone DNA polymerase, pol V. UmuD' is initially synthesized as an unstable and mutagenically inactive pro-protein, UmuD. Upon processing, UmuD' assumes a relatively stable conformation and becomes mutagenically active. While UmuD and UmuD' by themselves exist in vivo as homodimers, when together they preferentially interact to form heterodimers. Quite strikingly, it is in this context that UmuD' becomes susceptible to ClpXP-mediated proteolysis. Here we report a novel targeting mechanism designed for degrading the mutagenically active UmuD' subunit of the UmuD/D' heterodimer complex, while leaving the UmuD protein intact. Surprisingly, a signal that is essential and sufficient for targeting UmuD' for degradation was found to reside on UmuD not UmuD'. UmuD was also shown to be capable of channeling an excess of UmuD' to ClpXP for degradation, thereby providing a mechanism whereby cells can limit error-prone DNA replication.

The Escherichia coli SOS mutagenesis proteins UmuD and UmuD' interact physically with the replicative DNA polymerase.

The Escherichia coli umuDC operon is induced in response to replication-blocking DNA lesions as part of the SOS response. UmuD protein then undergoes an RecA-facilitated self-cleavage reaction that removes its N-terminal 24 residues to yield UmuD'. UmuD', UmuC, RecA, and some form of the E. coli replicative DNA polymerase, DNA polymerase III holoenzyme, function in translesion synthesis, the potentially mutagenic process of replication over otherwise blocking lesions. Furthermore, it has been proposed that, before cleavage, UmuD together with UmuC acts as a DNA damage checkpoint system that regulates the rate of DNA synthesis in response to DNA damage, thereby allowing time for accurate repair to take place. Here we provide direct evidence that both uncleaved UmuD and UmuD' interact physically with the catalytic, proofreading, and processivity subunits of the E. coli replicative polymerase. Consistent with our model proposing that uncleaved UmuD and UmuD' promote different events, UmuD and UmuD' interact differently with DNA polymerase III: whereas uncleaved UmuD interacts more strongly with beta than it does with alpha, UmuD' interacts more strongly with alpha than it does with beta. We propose that the protein-protein interactions we have characterized are part of a higher-order regulatory system of replication fork management that controls when the umuDC gene products can gain access to the replication fork.

A model for a umuDC-dependent prokaryotic DNA damage checkpoint.

The products of the Escherichia coli umuDC operon are required for translesion synthesis, the mechanistic basis of most mutagenesis caused by UV radiation and many chemicals. The UmuD protein shares homology with LexA, the repressor of SOS-regulated loci, and similarly undergoes a facilitated autodigestion on interaction with the RecA/single-stranded DNA nucleoprotein filaments formed after a cell experiences DNA damage. This cleavage, in which Ser-60 of UmuD acts as the nucleophile, produces UmuD', the form active in translesion synthesis. Expression of the noncleavable UmuD(S60A) protein and UmuC was found to increase survival after UV irradiation, despite the inability of the UmuD(S60A) protein to participate in translesion synthesis; this survival increase is uvr(+) dependent. Additional observations that expression of the UmuD(S60A) protein and UmuC delayed the resumption of DNA replication and cell growth after UV irradiation lead us to propose that the uncleaved UmuD protein and UmuC delay the resumption of DNA replication, thereby allowing nucleotide excision repair additional time to repair the damage accurately before replication is attempted. After a UV dose of 20 J/m(2), uncleaved UmuD is the predominant form for approximately 20 min, after which UmuD' becomes the predominant form, suggesting that the umuDC gene products play two distinct and temporally separated roles in DNA damage tolerance, the first in cell-cycle control and the second in translesion synthesis over unrepaired or irreparable lesions. The relationship of these observations to the eukaryotic DNA damage checkpoint is discussed.

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.

Effect of Tn10/Tn5 transposons on the survival and mutation frequency of halogen light-irradiated AB1157 Escherichia coli K-12

We show here that the Tn10/Tn5 transposon when inserted into the chromosome of strain AB1157 makes the bacteria more sensitive to and less mutable by halogen light irradiation. These effects are most probably caused by depletion of UmuD and UmuC proteins since: (i) transformation of the transposon-bearing bacteria with plasmids harbouring umuD'C (or umuDC) leads to recovery of the original survival and mutation frequencies; (ii) insertion of Tn10/Tn5 into chromosomal DNA has no effect on the level of mutation induced by ethyl methane-sulphonate treatment, a mutagen whose activity is umuDC-independent; (iii) the decline in survival is in about the same range for Tn10-bearing bacteria as for bacteria with deleted umuDC. However, whereas transformation of bacteria deleted in umuDC with plasmids carrying umuD'C/umuDC leads to full recovery of halogen light-induced mutability, recovery of survival is poor. This suggests that the mechanisms leading to umuDC-dependent mutagenesis and umuDC-dependent protection of cell survival are different. None of these effects occurs in bacteria bearing the Tn9 transposon in their DNA.

Lon-mediated proteolysis of the Escherichia coli UmuD mutagenesis protein: in vitro degradation and identification of residues required for proteolysis.

Most SOS mutagenesis in Escherichia coli is dependent on the UmuD and UmuC proteins. Perhaps as a consequence, the activity of these proteins is exquisitely regulated. The intracellular level of UmuD and UmuC is normally quite low but increases dramatically in lon- strains, suggesting that both proteins are substrates of the Lon protease. We report here that the highly purified UmuD protein is specifically degraded in vitro by Lon in an ATP-dependent manner. To identify the regions of UmuD necessary for Lon-mediated proteolysis, we performed 'alanine-stretch' mutagenesis on umuD and followed the stability of the mutant protein in vivo. Such an approach allowed us to localize the site(s) within UmuD responsible for Lon-mediated proteolysis. The primary signal is located between residues 15 and 18 (FPLF), with an auxiliary site between residues 26 and 29 (FPSP), of the amino terminus of UmuD. Transfer of the amino terminus of UmuD (residues 1-40) to an otherwise stable protein imparts Lon-mediated proteolysis, thereby indicating that the amino terminus of UmuD is sufficient for Lon recognition and the ensuing degradation of the protein.

Regulation of UmuD cleavage: role of the amino-terminal tail

An essential step in SOS mutagenesis is the RecA-mediated posttranslational processing of UmuD-like proteins to the shorter, but mutagenically active, UmuD′-like proteins. Interestingly, the UmuD-like proteins undergo posttranslational processing at different rates. For example, although the Escherichia coli UmuD (UmuD Ec) and the Salmonella typhimurium UmuD (UmuD St) proteins are 73% identical, UmuD Stis processed in vivo at a significantly faster rate than the UmuD Ec protein. Here, we report experiments aimed at investigating the molecular basis of these phenotypic differences. The faster rate of UmuDSt cleavage probably does not result solely from a better interaction with RecA, since we observed that, in vitro, UmuDStundergoes RecA-independent autocatalytic processing about four-times faster than UmuDEc. By constructing chimeric UmuD proteins, we determined that the amino-terminal tail of the UmuD proteins proximal to the Cys24-Gly25 cleavage site is mainly responsible for the difference in UmuDStand UmuDEc cleavage rates. Site-directed mutagenesis of the UmuDEc protein suggests that most of the enhanced cleavage observed with the UmuDSt protein can be attributed to the presence of a Pro23 residue, juxtaposed to the cleavage site in UmuDSt. Furthermore, this proline residue appears to result in a UmuD protein that is a much better substrate for intermolecular cleavage. These findings clearly implicate the N-terminal tail of the UmuD-like proteins as playing an important and unexpected regulatory function in the maturation of the mutagenically active UmuD′-like mutagenesis proteins.

Structural insights into the regulation of SOS mutagenesis.

The Escherichia coli Umu proteins are best characterized by their role in damage inducible mutagenesis. Recently, we discovered that the intracellular levels of the UmuD and UmuC proteins are kept to a minimum by the Lon serine protease. Studies with the Salmonella typhimurium UmuD protein (which is 73% homologous with its E. coli counterpart) revealed that it too is degraded by Lon, suggesting that both UmuD proteins share conserved structural motifs. In contrast, E. coli UmuD' is removed from the cell by the ClpXP serine protease, but only when it is in a heterodimer complex with UmuD. We have generated deletion mutants of UmuD' and have coexpressed the mutant proteins with UmuD1 (a non-cleavable UmuD protein). By assaying the sensitivity of the mutant UmuD'-UmuD1 complex to ClpXP, we have been able to map regions of UmuD' that appear essential for efficient UmuD'-UmuD heterodimer formation. Previous experiments have suggested that the in vivo posttranslational processing of UmuD to UmuD' is inefficient. We have, however, discovered that limited cleavage occurs in an undamaged cell, but that these small amounts of UmuD' are rapidly degraded by ClpXP, thus giving rise to the appearance of inefficient cleavage. The ClpXP protease therefore plays dual roles in regulating SOS mutagenesis: it keeps the basal levels of UmuD' to a minimum in undamaged cells but it also acts in damaged cells to reduce the elevated levels of mutagenically active UmuD' protein, thereby returning the cell to a resting non-mutable state.