umuD Gene Products Research Articles

Identification of the Dimer Exchange Interface of the Bacterial DNA Damage Response Protein UmuD.

The Escherichia coli SOS response, an induced DNA damage response pathway, confers survival on bacterial cells by providing accurate repair mechanisms as well as the potentially mutagenic pathway translesion synthesis (TLS). The umuD gene products are upregulated after DNA damage and play roles in both nonmutagenic and mutagenic aspects of the SOS response. Full-length UmuD is expressed as a homodimer of 139-amino-acid subunits, which eventually cleaves its N-terminal 24 amino acids to form UmuD'. The cleavage product UmuD' and UmuC form the Y-family polymerase DNA Pol V (UmuD'2C) capable of performing TLS. UmuD and UmuD' exist as homodimers, but their subunits can readily exchange to form UmuDD' heterodimers preferentially. Heterodimer formation is an essential step in the degradation pathway of UmuD'. The recognition sequence for ClpXP protease is located within the first 24 amino acids of full-length UmuD, and the partner of full-length UmuD, whether UmuD or UmuD', is degraded by ClpXP. To better understand the mechanism by which UmuD subunits exchange, we measured the kinetics of exchange of a number of fluorescently labeled single-cysteine UmuD variants as detected by Förster resonance energy transfer. Labeling sites near the dimer interface correlate with increased rates of exchange, indicating that weakening the dimer interface facilitates exchange, whereas labeling sites on the exterior decrease the rate of exchange. In most but not all cases, homodimer and heterodimer exchange exhibit similar rates, indicating that somewhat different molecular surfaces mediate homodimer exchange and heterodimer formation.

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.

Directed Evolution of DinB in Escherichia coli by Hydroxylamine Mutagenesis and UV Selection

DNA polymerase III (pol III) replicates undamaged DNA in E. coli under normal conditions. However, pol III is unable to replicate past DNA damage and when this happens, the SOS response is induced. The SOS response induces the expression of at least 57 genes that help repair or tolerate the damage. Some of the genes expressed during the SOS response include those encoding the Y‐family polymerases, which are capable of translesion synthesis (TLS). Translesion synthesis is the process by which DNA polymerases replicate damaged DNA without correcting the damage.The dinB gene product (pol IV) is capable of replicating DNA containing minor groove deoxyguanine adducts, such as N2‐furfuryl‐dG and N2‐benzo[a]pyryl‐dG adducts, whereas pol V, a product of the umuC and umuD gene products, is capable of replicating DNA containing lesions caused by ultraviolet (UV) light. Our goal was to induce TLS across UV lesions with variants of pol IV. We used DinB and chimeras of DinB containing fragments of UmuC, with additional random mutations via treatment with hydroxylamine, to identify variants of DinB that have obtained the ability to replicate past UV lesions. This work is supported by the American Cancer Society.

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.

Interactions Between the E. Coli Sos Response Protein Umud and DNA Polymerase III Alpha Subunit Have Implications for Regulating Replication in Response to DNA Damage

Faithful and efficient DNA replication is critical for the survival of all organisms; however, DNA is constantly subject to damage from endogenous and exogenous sources. Attempts to replicate damaged DNA can result in harmful mutations. The bacterial SOS response system regulates DNA repair and other pathways responsible for the cellular response to DNA damage. Among genes upregulated during the SOS response is the umuD gene, encoding the manager protein UmuD that subsequently undergoes self-cleavage to remove its N-terminal 24 amino acids to form UmuD'. The umuD gene products are involved in managing the cellular response to DNA damage through their interactions with damage-bypass DNA polymerases DNA pol IV and pol V. UmuD and UmuD' also interact with the alpha DNA polymerase and beta processivity clamp subunits of the replicative DNA polymerase III. We probed the interactions between UmuD or UmuD' and the alpha polymerase subunit and determined that the umuD gene products interact with alpha at two sites: the N-terminal PHP domain, which is responsible for interactions with the epsilon proofreading subunit, and the C-terminal domain, which harbors interaction sites for the beta clamp, the tau subunit of the clamp loader, and single-stranded DNA (ssDNA). The C-terminal domain of alpha preferentially binds to full-length UmuD. With FRET experiments, we subsequently showed that UmuD but not UmuD' inhibits the interaction between alpha and beta, suggesting that early in the SOS response to DNA damage, UmuD could displace alpha from the beta processivity clamp. Furthermore, optical tweezers experiments revealed that UmuD specifically inhibits the interaction between alpha and ssDNA. Together, these observations suggest that UmuD plays a key role in regulating the replication fork after DNA damage.

Dimer exchange and cleavage specificity of the DNA damage response protein UmuD

The cellular response to DNA damage in Escherichia coli is controlled in part by the activity of the umuD gene products. The full-length dimeric UmuD2 is the initial product that is expressed shortly after the induction of the SOS response and inhibits bacterial mutagenesis, allowing for error-free repair to occur. Over time, the slow auto-cleavage of UmuD2 to UmuD′2 promotes mutagenesis to ensure cell survival. The intracellular levels of UmuD2 and UmuD′2 are further regulated by degradation in vivo, returning the cell to a non-mutagenic state. To further understand the dynamic regulatory roles of the umuD gene products, we monitored the kinetics of exchange and cleavage of the UmuD2 and UmuD′2 homodimers as well as of the UmuDD′ heterodimer under equilibrium conditions. We found that the heterodimer is the preferred but not exclusive protein form, and that both the heterodimer and homodimers exhibit slow exchange kinetics which is further inhibited in the presence of interacting partner DinB. In addition, the heterodimer efficiently cleaves to form UmuD′2. Together, this work reveals an intricate UmuD lifecycle that involves dimer exchange and cleavage in the regulation of the DNA damage response.

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.

Selective disruption of the DNA polymerase III - 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.

Conformational Dynamics of the E. Coli SOS Mutagenesis Protein UmuD

The SOS response in Escherichia coli involves the induction of at least 57 genes in response to DNA damage, including the umuD gene. UmuD2 is a homodimer of 139-amino acid subunits that interacts with RecA/ssDNA nucleoprotein filaments, resulting in cleavage of its N-terminal 24-amino acids to yield UmuD’2. The full-length form UmuD plays a role in a primitive DNA damage checkpoint and prevents mutagenesis, whereas the cleaved form facilitates mutagenesis by Y family DNA polymerase UmuC. The goal of our research is to determine the conformation and dynamics of UmuD in order to understand its regulatory role in response to DNA damage. UmuD2 and UmuD’2 are both exceptionally tight dimers, with picoMolar dissociation constants. However, we find that a monomeric variant of UmuD maintains essentially all biological roles of UmuD2. This surprising finding suggests that UmuD can cleave in either a cis or trans conformation. By forming alternate dimeric forms of UmuD, we determined that cleavage in trans is more efficient than cleavage in cis. UmuD2 and UmuD’2 readily exchange monomers to form the heterodimer UmuDD’, which is the most stable dimeric form. We are investigating the kinetics and mechanism of exchange of UmuD dimers and the UmuDD’ heterodimer by using FRET assays and native PAGE. The different dimeric forms of the umuD gene products exchange on the minute time scale, and each dimer can continue to exchange once formed. Designed site-directed mutations are expected to allow us to determine the mechanism of UmuD dimer exchange.

Heterodimer formation in the DNA damage response protein UmuD

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′. UmuD and UmuD′ have been shown to perform distinct functions in both preventing and facilitating mutagenesis. The umuD gene products are highly dynamic 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. It has been proposed that the UmuD′D heterodimer is the predominant conformer of the umuD gene products. We are designing mutations at the dimer interface of UmuD that will allow us to determine the kinetics of dimer exchange as well as of heterodimer formation. 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. 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.

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.

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.

Evidence for multiple active forms of the DNA damage response protein UmuD

All organisms experience DNA damage and have evolved multiple pathways to maintain genomic stability. In Escherichia coli, the SOS response to DNA damage involves the induction of 57 genes including the umuD gene products. The UmuD homodimer of 139‐amino acid subunits binds a RecA/ssDNA nucleoprotein filament which stimulates the latent ability of UmuD to cleave its N‐terminal 24‐amino acids, producing UmuD′2. UmuD2 and UmuD′2 play differential roles in the DNA damage response. Several models have been proposed for the conformations of UmuD2 including two versions with the N‐terminal arms in trans and two versions with the arms in cis (Beuning, et al. 2006 J. Biol. Chem 281:9633). These conformations expose different binding surfaces that may interact with other proteins. The goal of our research is to determine the conformation and dynamics of the umuD gene products in order to understand how their differential protein‐protein interactions regulate cellular responses to DNA damage. Our preliminary results suggest that a single amino acid substitution, N41G, weakens the KD for dimerization by over five orders of magnitude. This N41G mutation yields a UmuD monomer that is cleavable in the presence and absence of RecA/ssDNA. These results suggest that UmuD2 exists in multiple active conformations and that the cis conformation may be physiologically relevant. Supported by NSF Career Award MCB‐0845033.

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.

The Roles of UmuD in Regulating Mutagenesis

All organisms are subject to DNA damage from both endogenous and environmental sources. DNA damage that is not fully repaired can lead to mutations. Mutagenesis is now understood to be an active process, in part facilitated by lower-fidelity DNA polymerases that replicate DNA in an error-prone manner. Y-family DNA polymerases, found throughout all domains of life, are characterized by their lower fidelity on undamaged DNA and their specialized ability to copy damaged DNA. Two E. coli Y-family DNA polymerases are responsible for copying damaged DNA as well as for mutagenesis. These DNA polymerases interact with different forms of UmuD, a dynamic protein that regulates mutagenesis. The UmuD gene products, regulated by the SOS response, exist in two principal forms: UmuD2, which prevents mutagenesis, and UmuD2′, which facilitates UV-induced mutagenesis. This paper focuses on the multiple conformations of the UmuD gene products and how their protein interactions regulate mutagenesis.

Regulation of DNA damage responses by the polymerase manager proteins UmuD and UmuD′

Organisms experience DNA damage from numerous sources. When bacterial cells experience DNA damage and other stresses, the SOS response is induced, leading to upregulation of at least 50 genes in E. coli. Among the upregulated genes are specialized Y family DNA polymerases, which replicate damaged DNA and play a role in tolerance to DNA damage. The activity of these potentially mutagenic proteins is tightly regulated. The manager proteins UmuD2 and the cleaved form UmuD'2 provide one regulatory mechanism. The umuD gene products interact with both Y family polymerases, the replicative DNA polymerase, and the beta processivity clamp. The goal of this study is to determine how the conformation and dynamics of the umuD gene products regulate the cellular response to DNA damage. We are characterizing the effects of UmuD and UmuD' on DNA polymerase III activity and probing their conformations using fluorescence resonance energy transfer (FRET) and hydrogen‐deuterium exchange mass spectrometry (HXMS). In HXMS experiments, backbone amide hydrogens that are solvent‐accessible exchange with deuterium over time, whereas inaccessible hydrogens do not exchange. Our HXMS results reveal that the N‐terminal arm of UmuD, which is not present in the cleaved form UmuD′, exhibits local partial unfolding. Additionally, there are substantial regions of stable conformation in both proteins.This work is funded by a Dreyfus Foundation New Faculty Award.

Regulation Of The DNA Damage Response By The DNA Polymerase Manager Protein UmuD

Organisms experience DNA damage from environmental as well as endogenous sources. When bacterial cells experience DNA damage DNA and other stresses, the SOS response is induced, leading to upregulation of at least 50 genes in E. coli. Many of the genes whose expression is induced as part of the SOS response are responsible for DNA repair and cell cycle regulation. Another group of genes, specialized Y family DNA polymerases with the ability to replicate damaged DNA, play a role in tolerance to DNA damage at a potentially mutagenic cost. Multiple layers of regulation control the activity of these potentially mutagenic proteins. The function of Y family DNA polymerases is regulated by UmuD, a manager protein, and its cleaved form, UmuD'. The umuD gene products directly interact with both Y family polymerases as well as the beta processivity clamp. The goal of this study is to determine the conformation and dynamics of the umuD gene products in order to understand how they regulate the cellular response to DNA damage. We are using fluorescence resonance energy transfer (FRET) and hydrogen-deuterium exchange mass spectrometry (HXMS) to probe the conformations of UmuD and UmuD'. In HXMS experiments, backbone amide hydrogens that are solvent-accessible become labeled with deuterium over time, whereas those are are not accessible do not become labeled. Our HXMS results reveal that the N-terminal arm of UmuD, which is not present in the cleaved form UmuD', exhibits local partial unfolding. Residues that contact the N-terminal arm show large differences between UmuD and UmuD'. Additionally, there are substantial regions of stable conformation in both proteins. Complete characterization of UmuD and UmuD' dynamics is currently in progress.

Translesion DNA Synthesis by Human DNA Polymerase η on Templates Containing a Pyrimidopurinone Deoxyguanosine Adduct, 3-(2′-Deoxy-β-d-erythro-pentofuranosyl)pyrimido-[1,2-a]purin-10(3H)-one

M1dG (3-(2′-deoxy-β-d-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one) lesions are mutagenic in bacterial and mammalian cells, leading to base substitutions (mostly M1dG to dT and M1dG to dA) and frameshift mutations. M1dG is produced endogenously through the reaction of peroxidation products, base propenal or malondialdehyde, with deoxyguanosine residues in DNA. The mutagenicity of M1dG in Escherichia coli is dependent on the SOS response, specifically the umuC and umuD gene products, suggesting that mutagenic lesion bypass occurs by the action of translesion DNA polymerases, like DNA polymerase V. Bypass of DNA lesions by translesion DNA polymerases is conserved in bacteria, yeast, and mammalian cells. The ability of recombinant human DNA polymerase η to synthesize DNA across from M1dG was studied. M1dG partially blocked DNA synthesis by polymerase η. Using steady-state kinetics, we found that insertion of dCTP was the least favored insertion product opposite the M1dG lesion (800-fold less efficient than opposite dG). Extension from M1dG·dC was equally as efficient as from control primer-templates (dG·dC). dATP insertion opposite M1dG was the most favored insertion product (8-fold less efficient than opposite dG), but extension from M1dG·dA was 20-fold less efficient than dG·dC. The sequences of full-length human DNA polymerase η bypass products of M1dG were determined by LC-ESI/MS/MS. Bypass products contained incorporation of dA (52%) or dC (16%) opposite M1dG or −1 frameshifts at the lesion site (31%). Human DNA polymerase η bypass may lead to M1dG to dT and frameshift but likely not M1dG to dA mutations during DNA replication.

Regulation of Escherichia coli SOS mutagenesis by dimeric intrinsically disordered umuD gene products.

Products of the umuD gene in Escherichia coli play key roles in coordinating the switch from accurate DNA repair to mutagenic translesion DNA synthesis (TLS) during the SOS response to DNA damage. Homodimeric UmuD(2) is up-regulated 10-fold immediately after damage, after which slow autocleavage removes the N-terminal 24 amino acids of each UmuD. The remaining fragment, UmuD'(2), is required for mutagenic TLS. The small proteins UmuD(2) and UmuD'(2) make a large number of specific protein-protein contacts, including three of the five known E. coli DNA polymerases, parts of the replication machinery, and RecA recombinase. We show that, despite forming stable homodimers, UmuD(2) and UmuD'(2) have circular dichroism (CD) spectra with almost no alpha-helix or beta-sheet signal at physiological concentrations in vitro. High protein concentrations, osmolytic crowding agents, and specific interactions with a partner protein can produce CD spectra that resemble the expected beta-sheet signature. A lack of secondary structure in vitro is characteristic of intrinsically disordered proteins (IDPs), many of which act as regulators. A stable homodimer that lacks significant secondary structure is unusual but not unprecedented. Furthermore, previous single-cysteine cross-linking studies of UmuD(2) and UmuD'(2) show that they have a nonrandom structure at physiologically relevant concentrations in vitro. Our results offer insights into structural characteristics of relatively poorly understood IDPs and provide a model for how the umuD gene products can regulate diverse aspects of the bacterial SOS response.