Characterization of novel alleles of the Escherichia coli umuDC genes identifies additional interaction sites of UmuC with the beta clamp

Abstract

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.

FIG. 1.

Novel alleles of umuD′C suppress the cold-sensitive growth phenotype. The ratio of the number of CFU at the permissive temperature to the number of CFU at the nonpermissive temperature is plotted for (A) each allele in the presence of elevated levels of the β clamp (pJRC210) or (B) each single amino acid change in the absence of elevated levels of the β clamp. (A) The selected alleles suppress cold sensitivity due to elevated levels of umuD′C (pGY9738) and the β clamp (pJRC210). The wild-type plasmid is pGY9738. (B) All but two of the individual point mutations in umuDC suppress umuDC-mediated cold sensitivity in AB1157. The UmuC(T243M) and UmuC(L389F) variants confer a partial cold-sensitive phenotype compared to wild-type UmuC. The wild-type plasmid is pGY9739. In both panels A and B, the empty vector is pGB2. WT, wild type; cfu 42/30 C, 42°C/30°C ratio; cfu 37/30 C, 37°C/30°C ratio.

FIG. 2.

Steady-state levels of UmuC (A) and UmuD (B) variants as determined by immunoblotting. (A) UmuC was expressed from the pGY9739 (umuDC) plasmids in all cases except UmuC(C105Y), which was expressed from pGY9738 (umuD′C). (B) In vivo cleavage efficiency of UmuD variants expressed from the pGY9739 plasmids. Cells were harvested 3 h after UV irradiation. (C) Purified wild-type UmuD and UmuD(G129S) were assayed for proficiency for cleavage in vitro with a RecA-ssDNA nucleoprotein filament, the presence of which is indicated by a plus sign and the absence of which is indicated by a minus sign. We did not detect cleavage of UmuD(G129S). Because of the low yield of purified UmuD(G129S), the results were detected by immunoblotting. WT, wild type; D, UmuD; C, UmuC.

FIG. 3.

UV-induced mutation frequency of selected alleles in plasmid pGY9738 (umuD′C) in strain GW8017 (ΔumuDC). The wild-type plasmid was pGY9738, and the empty vector was pGB2. (A) Mutation frequency conferred by each single mutation in the umuD′C genes. (B to E) Mutation frequencies conferred by the double mutations found in the selection experiment together with the corresponding single mutations. WT, wild type; Mut. Freq., mutation frequency; D′, UmuD′.

FIG. 4.

Predicted locations of selected residues in homology models of UmuD (A) and UmuC (B, C, and D). (A) Homology model of UmuD (5) with Asp84 and Gly129 highlighted by space-filling rendering, with different colors indicating different kinds of atoms. The Ser60-Lys97 active site dyad is highlighted by green space-filling rendering. The N-terminal 24 amino acids that are removed to form UmuD′ are yellow, while the rest of the arm is orange. (B, C and D) Homology model for residues 1 to 353 of UmuC (3) with the DNA indicated by lines. (B) Eight of the selected residues are shown in red space-filling rendering and labeled explicitly. (C) Residues, shown in space-filling rendering, of UmuC implicated as residues that are important for binding to the β clamp. The model of UmuC with DNA is shown oriented along the DNA helical axis. The canonical β clamp binding motif (residues 357 to 361) is not included in the model; therefore, the C-terminal residue of the model, residue 353, is explicitly indicated in space-filling rendering. Residues 313 to 315 comprise the second β-interaction site. (D) Five of the selected residues, four of which lie along one β strand, are shown in space-filling rendering, with different colors indicating different kinds of atoms. The images were created with VMD (21).

FIG. 5.

UV survival of GW8017 strains harboring plasmids expressing UmuD variants (A) G129S and (B) D84N. (A) Noncleavable UmuD(G129S) suppresses UV sensitivity that is characteristic of noncleavable UmuD variants. UmuD(G129S) also suppresses the extreme UV sensitivity of UmuD(S60A). •, pGY9739-umuDC-D129 (pD129); ▪, pGY9738-umuD′C (pUmuD′C); −, pGB2 (pVector); □, pGY9739- umuDC-DS60A, G129S (pD129-S60A); ⧫, pGY9739-umuDC (pUmuDC); ▴, pGY9739-umuDC-DS60A (pS60A). (B) UmuD(D84N) is cleavable, and strains harboring plasmids expressing this variant are resistant to UV light. A strain expressing UmuD(S60A D84N) is sensitive to UV light, which is characteristic of a strain expressing UmuD(S60A). ▪, pGY9738-umuD′C (pUmuD′C); −, pGB2 (pVector); ⧫, pGY9739-umuDC (pUmuDC); •, pGY9739-umuDC-D84 (pD84); □, pGY9739-umuDC-D84, S60A (pD84-S60A); ▴, pGY9739-umuDC-DS60A (pS60A).

FIG. 6.

Peptide array blot showing selected UmuC residues and their corresponding mutations, probed with wild-type β protein. Only the UmuC residues predicted to be surface exposed are shown. The blots on the left contained the wild-type UmuC sequences shown, and the blots on the right contained the sequences with the underlined residue mutated, as indicated in Table 2. The “X” indicates a spot that is not meaningful, because the antibody control reacted at this position (data not shown).