The dimeric SOS mutagenesis protein UmuD is active as a monomer

The homodimeric umuD gene products play key roles in regulating the cellular response to DNA damage in Escherichia coli. UmuD(2) is composed of 139-amino acid subunits and is up-regulated as part of the SOS response. Subsequently, damage-induced RecA·ssDNA nucleoprotein filaments mediate the slow self-cleavage of the N-terminal 24-amino acid arms yielding UmuD'(2). UmuD(2) and UmuD'(2) make a number of distinct protein-protein contacts that both prevent and facilitate mutagenic translesion synthesis. Wild-type UmuD(2) 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) and the β clamp. 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.     

Visualization of two binding sites for the Escherichia coli UmuD'(2)C complex (DNA pol V) on RecA-ssDNA filaments

The heterotrimeric UmuD'(2)C complex of Escherichia coli has recently been shown to possess intrinsic DNA polymerase activity (DNA pol V) that facilitates error-prone translesion DNA synthesis (SOS mutagenesis). When overexpressed in vivo, UmuD'(2)C also inhibits homologous recombination. In both activities, UmuD'(2)C interacts with RecA nucleoprotein filaments. To examine the biochemical and structural basis of these reactions, we have analyzed the ability of the UmuD'(2)C complex to bind to RecA-ssDNA filaments in vitro. As estimated by a gel retardation assay, binding saturates at a stoichiometry of approximately one complex per two RecA monomers. Visualized by cryo-electron microscopy under these conditions, UmuD'(2)C is seen to bind uniformly along the filaments, such that the complexes are completely submerged in the deep helical groove. This mode of binding would impede access to DNA in a RecA filament, thus explaining the ability of UmuD'(2)C to inhibit homologous recombination. At sub-saturating binding, the distribution of UmuD'(2)C complexes along RecA-ssDNA filaments was characterized by immuno-gold labelling with anti-UmuC antibodies. These data revealed preferential binding at filament ends (most likely, at one end). End-specific binding is consistent with genetic models whereby such binding positions the UmuD'(2)C complex (pol V) appropriately for its role in SOS mutagenesis.     

Selective disruption of the DNA polymerase III α-β complex by the umuD gene products

DNA polymerase III (DNA pol III) efficiently replicates the Escherichia coli genome, but it cannot bypass DNA damage. Instead, translesion synthesis (TLS) DNA polymerases are employed to replicate past damaged DNA; however, the exchange of replicative for TLS polymerases is not understood. The umuD gene products, which are up-regulated during the SOS response, were previously shown to bind to the α, β and ε subunits of DNA pol III. Full-length UmuD inhibits DNA replication and prevents mutagenic TLS, while the cleaved form UmuD' facilitates mutagenesis. We show that α possesses two UmuD binding sites: at the N-terminus (residues 1-280) and the C-terminus (residues 956-975). The C-terminal site favors UmuD over UmuD'. We also find that UmuD, but not UmuD', disrupts the α-β complex. We propose that the interaction between α and UmuD contributes to the transition between replicative and TLS polymerases by removing α from the β clamp.     

Identification of specific amino acid residues in the E. coli beta processivity clamp involved in interactions with DNA polymerase III, UmuD and UmuD'

Variants of a pentapeptide sequence (QL[S/F]LF), referred to as the eubacterial clamp-binding motif, appear to be required for certain proteins to bind specifically to the Escherichia coli beta sliding clamp, apparently by making contact with a hydrophobic pocket located at the base of the C-terminal tail of each beta protomer. Although both UmuC (DNA pol V) and the alpha catalytic subunit of DNA polymerase III (pol III) each bear a reasonable match to this motif, which appears to be required for their respective interactions with the clamp, neither UmuD not UmuD' do. As part of an ongoing effort to understand how interactions involving the different E. coli umuDC gene products and components of DNA polymerase III help to coordinate DNA replication with a DNA damage checkpoint control and translesion DNA synthesis (TLS) following DNA damage, we characterized the surfaces on beta important for its interactions with the two forms of the umuD gene product. We also characterized the surface of beta important for its interaction with the alpha catalytic subunit of pol III. Our results indicate that although UmuD, UmuD' and alpha share some common contacts with beta, each also makes unique contacts with the clamp. These findings suggest that differential interactions of UmuD and UmuD' with beta impose a DNA damage-responsive conditionality on how beta interacts with the translesion DNA polymerase UmuC. This is formally analogous to how post-translational modification of the eukaryotic PCNA clamp influences mutagenesis. We discuss the implications of our findings in terms of how E. coli might coordinate the actions of the umuDC gene products with those of pol III, as well as for how organisms in general might manage the actions of their multiple DNA polymerases.     

Error-prone repair and translesion synthesis III: the activation of UmuD (or less is more)

Following DNA damage to Escherichia coli bacteria, RecA protein is activated by binding to single stranded DNA and cleaves its own gene repressor (LexA protein). Two papers from Graham Walker's laboratory showed that several bacterial genes in addition to RecA are repressed by the LexA repressor and are inducible following DNA damage [C.J. Keyon, G.C. Walker, DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli, in: Proceedings of the National Academy of Sciences of the United States of America 77, 1980, pp. 2819--2823] and predicted that one of them (UmuD) might itself be subject to activation by a further cleavage reaction involving activated RecA protein [K.L. Perry, S.J. Elledge, B.B. Mitchell, L. Marsh, G.C. Walker, umuD,C and mucA,B operans whose products are required for UV light- and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology, in: Proceedings of the National Academy of Sciences of the United States of America 82, 1985, pp. 4331--4335]. The processed form of UmuD, termed UmuD', later proved to be a subunit of DNA polymerase V, a key enzyme involved in translesion synthesis.     

DNA polymerase V and RecA protein, a minimal mutasome

A hallmark of the Escherichia coli SOS response is the large increase in mutations caused by translesion synthesis (TLS). TLS requires DNA polymerase V (UmuD'2C) and RecA. Here, we show that pol V and RecA interact by two distinct mechanisms. First, pol V binds to RecA in the absence of DNA and ATP and second, through its UmuD' subunit, requiring DNA and ATP without ATP hydrolysis. TLS occurs in the absence of a RecA nucleoprotein filament but is inhibited in its presence. Therefore, a RecA nucleoprotein filament is unlikely to be required for SOS mutagenesis. Pol V activity is severely diminished in the absence of RecA or in the presence of RecA1730, a mutant defective for pol V mutagenesis in vivo. Pol V activity is strongly enhanced with RecA mutants constitutive for mutagenesis in vivo, suggesting that RecA is an obligate accessory factor that activates pol V for SOS mutagenesis.     

umuDC-mediated cold sensitivity is a manifestation of functions of the UmuD(2)C complex involved in a DNA damage checkpoint control

The umuDC genes are part of the Escherichia coli SOS response, and their expression is induced as a consequence of DNA damage. After induction, they help to promote cell survival via two temporally separate pathways. First, UmuD and UmuC together participate in a cell cycle checkpoint control; second, UmuD'(2)C enables translesion DNA replication over any remaining unrepaired or irreparable lesions in the DNA. Furthermore, elevated expression of the umuDC gene products leads to a cold-sensitive growth phenotype that correlates with a rapid inhibition of DNA synthesis. Here, using two mutant umuC alleles, one that encodes a UmuC derivative that lacks a detectable DNA polymerase activity (umuC104; D101N) and another that encodes a derivative that is unable to confer cold sensitivity but is proficient for SOS mutagenesis (umuC125; A39V), we show that umuDC-mediated cold sensitivity can be genetically separated from the role of UmuD'(2)C in SOS mutagenesis. Our genetic and biochemical characterizations of UmuC derivatives bearing nested deletions of C-terminal sequences indicate that umuDC-mediated cold sensitivity is not due solely to the single-stranded DNA binding activity of UmuC. Taken together, our analyses suggest that umuDC-mediated cold sensitivity is conferred by an activity of the UmuD(2)C complex and not by the separate actions of the UmuD and UmuC proteins. Finally, we present evidence for structural differences between UmuD and UmuD' in solution, consistent with the notion that these differences are important for the temporal regulation of the two separate physiological roles of the umuDC gene products.     

Escherichia coli DNA polymerase V subunit exchange: a post-SOS mechanism to curtail error-prone DNA synthesis

DNA polymerase V consisting of a heterotrimer composed of one molecule of UmuC and two molecules of UmuD' (UmuD'2C) is responsible for SOS damage-induced mutagenesis in Escherichia coli. Here we show that although the UmuD'2C complex remains intact through multiple chromatographic steps, excess UmuD, the precursor to UmuD', displaces UmuD' from UmuD'2C by forming a UmuDD' heterodimer, while UmuC concomitantly aggregates as an insoluble precipitate. Although soluble UmuD'2C is readily detected when the two genes are co-transcribed and translated in vitro, soluble UmuD2C or UmuDD'C are not detected. The subunit exchange between UmuD'2C and UmuD offers a biological means to inactivate error-prone polymerase V following translesion synthesis, thus preventing mutations from occurring on undamaged DNA.     

Genetic interactions between the Escherichia coli umuDC gene products and the beta processivity clamp of the replicative DNA polymerase

The Escherichia coli umuDC gene products encode DNA polymerase V, which participates in both translesion DNA synthesis (TLS) and a DNA damage checkpoint control. These two temporally distinct roles of the umuDC gene products are regulated by RecA-single-stranded DNA-facilitated self-cleavage of UmuD (which participates in the checkpoint control) to yield UmuD' (which enables TLS). In addition, even modest overexpression of the umuDC gene products leads to a cold-sensitive growth phenotype, apparently due to the inappropriate expression of the DNA damage checkpoint control activity of UmuD(2)C. We have previously reported that overexpression of the epsilon proofreading subunit of DNA polymerase III suppresses umuDC-mediated cold sensitivity, suggesting that interaction of epsilon with UmuD(2)C is important for the DNA damage checkpoint control function of the umuDC gene products. Here, we report that overexpression of the beta processivity clamp of the E. coli replicative DNA polymerase (encoded by the dnaN gene) not only exacerbates the cold sensitivity conferred by elevated levels of the umuDC gene products but, in addition, confers a severe cold-sensitive phenotype upon a strain expressing moderately elevated levels of the umuD'C gene products. Such a strain is not otherwise normally cold sensitive. To identify mutant beta proteins possibly deficient for physical interactions with the umuDC gene products, we selected for novel dnaN alleles unable to confer a cold-sensitive growth phenotype upon a umuD'C-overexpressing strain. In all, we identified 75 dnaN alleles, 62 of which either reduced the expression of beta or prematurely truncated its synthesis, while the remaining alleles defined eight unique missense mutations of dnaN. Each of the dnaN missense mutations retained at least a partial ability to function in chromosomal DNA replication in vivo. In addition, these eight dnaN alleles were also unable to exacerbate the cold sensitivity conferred by modestly elevated levels of the umuDC gene products, suggesting that the interactions between UmuD' and beta are a subset of those between UmuD and beta. Taken together, these findings suggest that interaction of beta with UmuD(2)C is important for the DNA damage checkpoint function of the umuDC gene products. Four possible models for how interactions of UmuD(2)C with the epsilon and the beta subunits of DNA polymerase III might help to regulate DNA replication in response to DNA damage are discussed.     

Lesion bypass by the Escherichia coli DNA polymerase V requires assembly of a RecA nucleoprotein filament

Translesion replication is carried out in Escherichia coli by the SOS-inducible DNA polymerase V (UmuC), an error-prone polymerase, which is specialized for replicating through lesions in DNA, leading to the formation of mutations. Lesion bypass by pol V requires the SOS-regulated proteins UmuD' and RecA and the single-strand DNA-binding protein (SSB). Using an in vitro assay system for translesion replication based on a gapped plasmid carrying a site-specific synthetic abasic site, we show that the assembly of a RecA nucleoprotein filament is required for lesion bypass by pol V. This is based on the reaction requirements for stoichiometric amounts of RecA and for single-stranded gaps longer than 100 nucleotides and on direct visualization of RecA-DNA filaments by electron microscopy. SSB is likely to facilitate the assembly of the RecA nucleoprotein filament; however, it has at least one additional role in lesion bypass. ATPgammaS, which is known to strongly increase binding of RecA to DNA, caused a drastic inhibition of pol V activity. Lesion bypass does not require stoichiometric binding of UmuD' along RecA filaments. In summary, the RecA nucleoprotein filament, previously known to be required for SOS induction and homologous recombination, is also a critical intermediate in translesion replication.     

A model for the structure of the Escherichia coli SOS-regulated UmuD2 protein

The ubiquitous Y-family of DNA polymerases, exemplified by the Escherichia coli UmuC protein (the catalytic subunit of DNA Pol V), possess the remarkable ability to replicate imperfect DNA templates that cannot be replicated by other types of DNA polymerases. Since this ability comes at the cost of a reduced fidelity, it is important that organisms manage these unique polymerases to coordinate their actions with those of the replication machinery. In E. coli, it is becoming evident that a sophisticated series of protein-protein interactions involving the two forms of the umuD gene product, UmuD and UmuD' and components of the replicative DNA polymerase serve to manage the actions of the umuC-encoded DNA polymerase. The purpose of this study was to better understand how structural differences between UmuD2 and UmuD2' help to determine which biological role the umuDC gene products will play; the UmuD2C complex functions as a DNA damage checkpoint effector, while the UmuD2'C complex participates in translesion DNA synthesis, which serves as the mechanistic basis for most chemical and UV light mutagenesis. Based on the results of a combination of disulfide cross-linking experiments, measurements of solvent accessibility and electron paramagnetic spin resonance (EPR) studies, we have developed a refined model for the structure of the UmuD2 homodimer. In the model that we are proposing, the N-terminal arms of UmuD (residues 1-39) form an extended interface in the UmuD2 homodimer by folding down over the globular domains of their intradimer partners. As a result, significant portions of the surface of each globular domain are buried in the UmuD2 homodimer. Based on the structure of the UmuD2' homodimer, both in the crystal and in solution, these same surfaces are exposed. Implications of these structural differences between the UmuD2 and the UmuD2' homodimers with respect to their roles in managing the actions of the umuC-encoded DNA polymerase are discussed.     

A new model for SOS-induced mutagenesis: how RecA protein activates DNA polymerase V

In Escherichia coli, cell survival and genomic stability after UV radiation depends on repair mechanisms induced as part of the SOS response to DNA damage. The early phase of the SOS response is mostly dominated by accurate DNA repair, while the later phase is characterized with elevated mutation levels caused by error-prone DNA replication. SOS mutagenesis is largely the result of the action of DNA polymerase V (pol V), which has the ability to insert nucleotides opposite various DNA lesions in a process termed translesion DNA synthesis (TLS). Pol V is a low-fidelity polymerase that is composed of UmuD′₂C and is encoded by the umuDC operon. Pol V is strictly regulated in the cell so as to avoid genomic mutation overload. RecA nucleoprotein filaments (RecA*), formed by RecA binding to single-stranded DNA with ATP, are essential for pol V-catalyzed TLS both in vivo and in vitro. This review focuses on recent studies addressing the protein composition of active DNA polymerase V, and the role of RecA protein in activating this enzyme. Based on unforeseen properties of RecA*, we describe a new model for pol V-catalyzed SOS-induced mutagenesis.     

Characterization of Escherichia coli UmuC active-site loops identifies variants that confer UV hypersensitivity

DNA is constantly exposed to chemical and environmental mutagens, causing lesions that can stall replication. In order to deal with DNA damage and other stresses, Escherichia coli utilizes the SOS response, which regulates the expression of at least 57 genes, including umuDC. The gene products of umuDC, UmuC and the cleaved form of UmuD, UmuD', form the specialized E. coli Y-family DNA polymerase UmuD'2C, or polymerase V (Pol V). Y-family DNA polymerases are characterized by their specialized ability to copy damaged DNA in a process known as translesion synthesis (TLS) and by their low fidelity on undamaged DNA templates. Y-family polymerases exhibit various specificities for different types of DNA damage. Pol V carries out TLS to bypass abasic sites and thymine-thymine dimers resulting from UV radiation. Using alanine-scanning mutagenesis, we probed the roles of two active-site loops composed of residues 31 to 38 and 50 to 54 in Pol V activity by assaying the function of single-alanine variants in UV-induced mutagenesis and for their ability to confer resistance to UV radiation. We find that mutations of the N-terminal residues of loop 1, N32, N33, and D34, confer hypersensitivity to UV radiation and to 4-nitroquinoline-N-oxide and significantly reduce Pol V-dependent UV-induced mutagenesis. Furthermore, mutating residues 32, 33, or 34 diminishes Pol V-dependent inhibition of recombination, suggesting that these mutations may disrupt an interaction of UmuC with RecA, which could also contribute to the UV hypersensitivity of cells expressing these variants.     

Inhibition of Escherichia coli RecA coprotease activities by DinI.

In Escherichia coli, the SOS response is induced upon DNA damage and results in the enhanced expression of a set of genes involved in DNA repair and other functions. The initial step, self-cleavage of the LexA repressor, is promoted by the RecA protein which is activated upon binding to single-stranded DNA. In this work, induction of the SOS response by the addition of mitomycin C was found to be prevented by overexpression of the dinI gene. dinI is an SOS gene which maps at 24.6 min of the E.coli chromosome and encodes a small protein of 81 amino acids. Immunoblotting analysis with anti-LexA antibodies revealed that LexA did not undergo cleavage in dinI-overexpressed cells after UV irradiation. In addition, the RecA-dependent conversion of UmuD to UmuD' (the active form for mutagenesis) was also inhibited in dinI-overexpressed cells. Conversely, a dinI-deficient mutant showed a slightly faster and more extensive processing of UmuD and hence higher mutability than the wild-type. Finally, we demonstrated, by using an in vitro reaction with purified proteins, that DinI directly inhibits the ability of RecA to mediate self-cleavage of UmuD.     

Lyase activities intrinsic to Escherichia coli polymerases IV and V

Escherichia coli DNA polymerase IV and V (pol IV and pol V) are error-prone DNA polymerases that are induced as part of the SOS regulon in response to DNA damage. Both are members of the Y-family of DNA polymerases. Their principal biological roles appear to involve translesion synthesis (TLS) and the generation of mutational diversity to cope with stress. Although neither enzyme is known to be involved in base excision repair (BER), we have nevertheless observed apurinic/apyrimidinic 5'-deoxyribose phosphate (AP/5'-dRP) lyase activities intrinsic to each polymerase. Pols IV and V catalyze cleavage of the phosphodiester backbone at the 3'-side of an apurinic/apyrimidinic (AP) site as well as the removal of a 5'-deoxyribose phosphate (dRP) at a preincised AP site. The specific activities of the two error-prone polymerase-associated lyases are approximately 80-fold less than the associated lyase activity of human DNA polymerase beta, which is a key enzyme used in short patch BER. Pol IV forms a covalent Schiff's base intermediate with substrate DNA that is trapped by sodium borohydride, as proscribed by a beta-elimination mechanism. In contrast, a NaBH(4) trapped intermediate is not observed for pol V, even though the lyase specific activity of pol V is slightly higher than that of pol IV. Incubation of pol V (UmuD'(2)C) with a molar excess of UmuD drives an exchange of subunits to form UmuD'D+insoluble UmuC causing inactivation of polymerase and lyase activities. The concomitant loss of both activities is strong evidence that pol V contains a bona fide lyase activity.     

umuDC-dnaQ Interaction and its implications for cell cycle regulation and SOS mutagenesis in Escherichia coli

The Escherichia coli SOS-regulated umuDC gene products participate in a DNA damage checkpoint control and in translesion DNA synthesis. Specific interactions involving the UmuD and UmuD' proteins, both encoded by the umuD gene, and components of the replicative DNA polymerase, Pol III, appear to be important for regulating these two biological activities of the umuDC gene products. Here we show that overproduction of the epsilon proofreading subunit of Pol III suppresses the cold sensitivity normally associated with overexpression of the umuDC gene products. Our results suggest that this suppression is attributable to specific interactions between UmuD or UmuD' and the C-terminal domain of epsilon.     

Interactions of Escherichia coli UmuD with activated RecA analyzed by cross-linking UmuD monocysteine derivatives.

SOS mutagenesis in Escherichia coli requires the participation of a specialized system involving the activated form of UmuD (UmuD'), UmuC, RecA, and DNA polymerase III proteins. We have used a set of monocysteine derivatives of UmuD (M. H. Lee, T. Ohta, and G. C. Walker, J. Bacteriol. 176:4825-4837, 1994) and the cysteine-specific photoactive cross-linker p-azidoiodoacetanilide (AIA) to study not only the interactions of intact UmuD in the homodimer but also the interactions of UmuD with activated RecA. The reactivities of the individual UmuD monocysteine derivatives with AIA were similar to their reactivities with iodoacetate. The relative efficiencies of cross-linking of the AIA-modified monocysteine UmuD derivatives in the homodimer form are also consistent with our previous conclusions concerning the relative closeness of various UmuD residues to the dimer interface. With respect to the UmuD-RecA interface, the AIA-modified VC34 and SC81 monocysteine derivatives cross-linked most efficiently with RecA, indicating that positions 34 and 81 of UmuD are closer to the RecA interface than the other positions we tested. The AIA-modified SC57, SC67, and SC112 monocysteine derivatives cross-linked moderately efficiently with RecA. Neither C24, the wild-type UmuD that has a cysteine located at the Cys-24-Gly-25 cleavage site, nor SC60, the UmuD monocysteine derivative with a cysteine substitution at the position of the putative active-site residue, was able to cross-link with RecA, suggesting that RecA need not directly interact with residues involved in the cleavage reaction. SC19, located in the N-terminal fragment of UmuD that is cleaved, and LC44 also did not cross-link efficiently with RecA.     

Simple and efficient purification of Escherichia coli DNA polymerase V: cofactor requirements for optimal activity and processivity in vitro

Most damage induced mutagenesis in Escherichia coli is dependent upon the UmuD'(2)C protein complex, which comprises DNA polymerase V (pol V). Biochemical characterization of pol V has been hindered by the fact that the enzyme is notoriously difficult to purify, largely because overproduced UmuC is insoluble. Here, we report a simple and efficient protocol for the rapid purification of milligram quantities of pol V from just 4 L of bacterial culture. Rather than over producing the UmuC protein, it was expressed at low basal levels, while UmuD'(2)C was expressed in trans from a high copy-number plasmid with an inducible promoter. We have also developed strategies to purify the β-clamp and γ-clamp loader free from contaminating polymerases. Using these highly purified proteins, we determined the cofactor requirements for optimal activity of pol V in vitro and found that pol V shows robust activity on an SSB-coated circular DNA template in the presence of the β/γ-complex and a RecA nucleoprotein filament (RecA*) formed in trans. This strong activity was attributed to the unexpectedly high processivity of pol V Mut (UmuD'(2)C · RecA · ATP), which was efficiently recruited to a primer terminus by SSB.     

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.