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 UmuD(2) 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 UmuD(2) to UmuD'(2) promotes mutagenesis to ensure cell survival. The intracellular levels of UmuD(2) 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 UmuD(2) 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.

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 ß 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.

Steric gate variants of UmuC confer UV hypersensitivity on Escherichia coli.

Y family DNA polymerases are specialized for replication of damaged DNA and represent a major contribution to cellular resistance to DNA lesions. Although the Y family polymerase active sites have fewer contacts with their DNA substrates than replicative DNA polymerases, Y family polymerases appear to exhibit specificity for certain lesions. Thus, mutation of the steric gate residue of Escherichia coli DinB resulted in the specific loss of lesion bypass activity. We constructed variants of E. coli UmuC with mutations of the steric gate residue Y11 and of residue F10 and determined that strains harboring these variants are hypersensitive to UV light. Moreover, these UmuC variants are dominant negative with respect to sensitivity to UV light. The UV hypersensitivity and the dominant negative phenotype are partially suppressed by additional mutations in the known motifs in UmuC responsible for binding to the beta processivity clamp, suggesting that the UmuC steric gate variant exerts its effects via access to the replication fork. Strains expressing the UmuC Y11A variant also exhibit decreased UV mutagenesis. Strikingly, disruption of the dnaQ gene encoding the replicative DNA polymerase proofreading subunit suppressed the dominant negative phenotype of a UmuC steric gate variant. This could be due to a recruitment function of the proofreading subunit or involvement of the proofreading subunit in a futile cycle of base insertion/excision with the UmuC steric gate variant.

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.

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 UmuD', 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.

Electron spin labeling reveals the highly dynamic N-terminal arms of the SOS mutagenesis protein UmuD.

Electron paramagnetic resonance (EPR) spectroscopy was used to probe the conformational dynamics of the N-terminal arms of the umuD gene products. We determined that the arms of UmuD(2) display a large degree of motion, are largely unbound from the globular C-terminal domain, and that the free energy of dissociation is +2.1 kJ mol(-1).

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: UmuD(2), which prevents mutagenesis, and UmuD(2)', which facilitates UV-induced mutagenesis. This paper focuses on the multiple conformations of the UmuD gene products and how their protein interactions regulate mutagenesis.