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

Abstract

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.

Fig. 1.

E. coli UmuC is a Y-family DNA polymerase that shares little homology with other members of the Y family. (A) Homology model of UmuC (6). UmuC loop 1 (residues 31 to 38) is shown in red, and loop 2 (residues 50 to 54) is shown in blue. UmuC residues N32 (black), N33 (green), and D34 (purple) are predicted to determine the gap size that dictates which lesions can fit into the active site (13, 14, 62). The backbone of UmuC is shown in yellow. DNA is rendered as sticks and colored by atom identity. The illustration was prepared using the VMD package (28). (B) Amino acid sequences of representative Y-family polymerases showing conserved residues aligned with loop 1 (residues 31 to 38, red box) and loop 2 (residues 50 to 54, blue box). Secondary structure based on the crystal structure of Dpo4 is shown above the alignment (11, 39). Ec, Escherichia coli; St, Salmonella enterica serovar Typhimurium; Hs, Homo sapiens; Ss, Sulfolobus solfataricus; Sc, Saccharomyces cerevisiae. The first five sequences are UmuC and its homologs, which share almost 100% homology in loop 1.

Fig. 2.

N-terminal loop 1 variants 32, 33, and 34 cause hypersensitivity to UV radiation in a strain that is not normally sensitive to UV. (A) Assays were performed with the pGY9738 plasmid and the following derivatives in GW8017: pGY9738 (umuD′C wild type [WT]; ■), pGB2 (empty vector [EV]; ◆), pGY9738-N32A (umuD′C N32A; ×), pGY9738-N33A (umuD′C N33A; ♢), and pGY9738-D34A (umuD′C D34A; ○). (B) Conservative mutations of N32, N33, and D34 conferred hypersensitivity to UV radiation. Assays were performed with the pGY9738 plasmid and the following derivatives in GW8017: pGY9738 (umuD′C, wild type; ■), pGB2 (empty vector; ◆), pGY9738-D34N (umuD′C D34N; ▴), pGY9738-D34E (umuD′C D34E; ×), pGY9738-N32Q (umuD′C N32Q; ●), pGY9738-N33Q (umuD′C N33Q; ▵), pGY9738-N33D (umuD′C N33D; +), and pGY9738-N32D (umuD′C N32D; □). (C) Immunoblot showing steady-state levels of UmuC variants expressed from plasmids encoding the umuD′C genes in GW8017. The wild-type plasmid was pGY9738, and the empty vector was pGB2. Xreact, cross-reaction.

Fig. 3.

N-terminal loop 1 variants 32, 33, and 34 cause hypersensitivity to UV radiation in a wild-type strain. (A) Assays were performed with the pGY9738 plasmid and the following derivatives in AB1157: pGY9738 (umuD′C wild type; ■), pGB2 (empty vector; ◆), pGY9738-N32A (umuD′C N32A; ×), pGY9738-N33A (umuD′C N33A; ♢), and pGY9738-D34A (umuD′C D34A; ○). (B) Conservative mutations of N32, N33, and D34 conferred hypersensitivity to UV radiation. Assays were performed with the pGY9738 plasmid and the following derivatives in AB1157: pGY9738 (umuD′C wild type; ■), pGB2 (empty vector; ◆), pGY9738-D34N (umuD′C D34N; ▴), pGY9738-D34E (umuD′C D34E; ×), pGY9738-N32Q (umuD′C N32Q; ●), pGY9738-N33Q (umuD′C N33Q; ▵), pGY9738-N32D (umuD′C N32D; □), and pGY9738-N33D (umuD′C N33D; +). (C) UV (5 J/m²)-induced mutation frequency of selected variants in plasmid pGY9738 (umuD′C) in strain AB1157. The wild-type plasmid was pGY9738, and the empty vector was pGB2. Frequencies were as follows: for the empty vector, induced mutant, 2.78 × 10⁻⁷; spontaneous mutants, 2.99 × 10⁻⁷; mutation frequency, −2.16 × 10⁻⁸ (set equal to 0); for the wild type, induced mutants, 9.53 × 10⁻⁶; spontaneous mutants, 3.40 × 10⁻⁶; mutation frequency, 6.13 × 10⁻⁶; for N32A, induced mutants, 1.73 × 10⁻⁶; spontaneous mutants, 7.89 × 10⁻⁷; mutation frequency, 9.50 × 10⁻⁷; for N33A, induced mutants, 8.20 × 10⁻⁷; spontaneous mutants, 5.83 × 10⁻⁷; mutation frequency, 2.37 × 10⁻⁷; and for D34A, induced mutants, 6.67 × 10⁻⁷; spontaneous mutants, 5.01 × 10⁻⁷; mutation frequency, 1.66 × 10⁻⁷.

Fig. 4.

UmuC N-terminal loop 1 variants S31, N32, N33, and D34 confer hypersensitivity to UV radiation in a ΔumuC ΔrecJ strain. (A) Mutation of N-terminal loop 1 residue N32 causes sensitivity to UV radiation. Assays were performed with pGY9738 plasmid and the following derivatives in PB102 (AB1157 ΔumuC ΔrecJ): pGY9738 (umuD′C wild type; ■), pGB2 (empty vector; ◆), pGY9738-N32A (umuD′C N32A; ×), pGY9738-N32Q (umuD′C N32Q; ●), and pGY9738-N32D (umuD′C N32D; □). (B) Mutation of N-terminal loop 1 residue N33 causes hypersensitivity to UV radiation. Assays were performed with pGY9738 plasmid and the following derivatives in PB102: pGY9738 (umuD′C wild type; ■), pGB2 (empty vector; ◆), pGY9738-N33Q (umuD′C N33Q; ▵), pGY9738-N33D (umuD′C N33D; +), and pGY9738-N33A (umuD′C N33A; ♢). (C) Mutation of N-terminal loop 1 residue D34 causes hypersensitivity to UV radiation. Assays were performed with pGY9738 plasmid and the following derivatives in PB102: pGY9738 (umuD′C wild type; ■), pGB2 (empty vector; ◆), pGY9738-D34E (umuD′C D34E; ×), pGY9738-D34A (umuD′C D34A; ○), and pGY9738-D34N (umuD′C D34N; ▴). (D) N-terminal loop 1 variant S31A causes hypersensitivity to UV radiation and confers upon strains a growth defect (G), a nongrowth defect (N), or an intermediate growth defect (I). Assays were performed with the pGY9738 plasmid and the following derivatives in PB102: pGY9738-S31A (N) (umuD′C S31A [N]; ▴), pGY9738 (umuD′C wild type; ■), pGY9738-S31A (I) (umuD′C S31A [I]; ×), pGB2 (empty vector; ◆), and pGY9738-S31A (G) (umuD′C S31A [G]; ▵).

Fig. 5.

Cells harboring UmuC variants N32A, N33A, and D34A are sensitive to 4-NQO (A), while only cells harboring UmuC variant N33A are sensitive to NFZ (B). (A and B) Assays were performed with the pGY9738 plasmid and the following derivatives in AB1157: pGB2 (empty vector; ◆), pGY9738 (umuD′C wild type; ■), pGY9738-N32A (umuD′C N32A; ●), pGY9738-N33A (umuD′C N33A; ♢), and pGY9738-D34A (umuD′C D34A; ○).

Fig. 6.

Deletion of the proofreading subunit of Pol III (dnaQ) or mutation of β clamp-binding sites of UmuC does not suppress sensitivity to UV radiation caused by mutation of N32, N33, and D34. (A to C) The respective plasmids were assayed in the following strains: GW2771 (×), GW2771 spq-2 (▴), and GW2771 spq-2 dnaQ903 (●). N-terminal loop 1 variants N32A (A), N33A (B), and D34A (C) confer sensitivity to UV radiation, despite the deletion of dnaQ. Assays were performed with derivatives of pGY9738 in the listed strains. (D) The hypersensitivity to UV radiation conferred by variants N32A, N33A, and D34A in GW8017 was not suppressed by the mutation of the β-binding motifs of UmuC. (E) The hypersensitivity to UV radiation conferred by UmuC variants N32A, N33A, and D34A in AB1157 was modestly suppressed by the mutation of the β-binding motifs of UmuC. (D and E) Assays were performed with plasmid pGY9738 and derivatives in GW8017 (D) and AB1157 (E): pGB2 (empty vector; ◆), pGY9738 (umuD′C wild type; ■), pGY9738-N32A (umuD′C N32A; ●), pGY9738-N32Aβ_{1 + 2} (umuD′C N32A β_{1 + 2}; □), pGY9738-N33A (umuD′C N33A; ♢), pGY9738-N33A β_{1 + 2} (umuD′C N33A β_{1 + 2}; ▵), pGY9738-D34A (umuD′C D34A; ○), and pGY9738-D34A β_{1 + 2} (umuD′C D34A β_{1 + 2}; ×).

Fig. 7.

UmuD′₂C inhibits homologous recombination facilitated by RecA. (A) Variants in plasmid pGY9738 (umuD′C wild type) were expressed in strain GW8017 (ΔumuDC). Cells harboring the variants N32A, N33A, and D34A show intermediate inhibition of RecA-mediated homologous recombination, whereas cells harboring other variants (Y11A, G52A, P54A; to the right of the vertical line) inhibit homologous recombination to a similar extent as wild-type umuD′C. Transduction efficiency is measured in CFU/PFU as a percentage of that for the empty vector (1.31 × 10⁻⁵ CFU/PFU, normalized to 100.0%): wild type, 2.09%; N32A, 58.1%; N33A, 48.4%; D34A, 38.1%; Y11A, 0.358%, G52A, 0.956%; P54A, 0.000%. (B) Homology model of UmuC (6). The backbone of UmuC is shown in yellow. UmuC loop 1 (residues 31 to 38) is shown in red, and loop 2 (residues 50 to 54) is shown in blue. UmuC residues N32, N33, and D34 (green) are near previously studied residues K342 and Y270 (purple) in the UmuC model. Previously studied residue Y11, as well as residues G52 and P54 (orange), is shown for comparison. Cells harboring variants Y270C and K342Q show a significant decrease in RecA-mediated homologous recombination. The image was prepared using VMD (28).

Fig. 8.

UmuC C-terminal loop 1 variants do not confer sensitivity to UV radiation and are proficient for mutagenesis. (A) UV (25 J/m²)-induced mutation frequency of selected variants in plasmid pGY9738 (umuD′C) in strain GW8017 (ΔumuDC). The wild-type plasmid was pGY9738, and the empty vector was pGB2. Frequencies were as follows: for the empty vector, induced mutants, 7.66 × 10⁻⁷; spontaneous mutants, 3.36 × 10⁻⁷; mutation frequency, 4.31 × 10⁻⁷; for the wild type, induced mutants, 1.85 × 10⁻⁵; spontaneous mutants, 6.48 × 10⁻⁶; mutation frequency, 1.21 × 10⁻⁵; for G35A, induced mutants, 1.73 × 10⁻⁵; spontaneous mutants, 7.58 × 10⁻⁶; mutation frequency, 9.68 × 10⁻⁶; for C36A, induced mutants, 3.25 × 10⁻⁵; spontaneous mutants, 2.30 × 10⁻⁶; mutation frequency, 3.03 × 10⁻⁵; for V37A, induced mutants, 2.24 × 10⁻⁵; spontaneous mutants; 5.41 × 10⁻⁶; mutation frequency, 1.70 × 10⁻⁵; and for I38A, induced mutants, 2.80 × 10⁻⁵; spontaneous mutants, 2.73 × 10⁻⁶; mutation frequency, 2.53 × 10⁻⁵. (B) C-terminal loop 1 variants do not confer sensitivity to UV radiation. Assays were performed with pGY9738 plasmid and the following derivatives in PB102: pGY9738-C36A (umuD′C C36A; ×), pGY9738 (umuD′C wild type; ■), pGY9738-I38A (umuD′C I38A; ▵), pGY9738-V37A (umuD′C V37A; ●), pGY9738-G35A (umuD′C G35A; □), and pGB2 (empty vector; ◆). (C) Immunoblot showing steady-state levels of UmuC expressed from variant umuDC plasmids (GW8017). The wild-type plasmid was pGY9738, and the empty vector was pGB2.

Fig. 9.

UmuC loop 2 variants do not confer sensitivity to UV radiation and are proficient for mutagenesis. (A) UV (25 J/m²)-induced mutation frequency of selected variants in plasmid pGY9738 (umuD′C) in strain GW8017 (ΔumuDC). The wild-type plasmid was pGY9738, and the empty vector was pGB2. Frequencies were as follows: for the empty vector, induced mutants, 7.66 × 10⁻⁷; spontaneous mutants, 3.36 × 10⁻⁷; mutation frequency, 4.31 × 10⁻⁷; for the wild type, induced mutants, 1.85 × 10⁻⁵; spontaneous mutants, 6.48 × 10⁻⁶; mutation frequency, 1.21 × 10⁻⁵; for K50A, induced mutants, 3.28 × 10⁻⁵; spontaneous mutants, 2.96 × 10⁻⁶; mutation frequency, 2.98 × 10⁻⁵; for M51A, induced mutants, 1.71 × 10⁻⁵; spontaneous mutants, 3.00 × 10⁻⁶; mutation frequency, 1.41 × 10⁻⁵; for G52A, induced mutants, 3.38 × 10⁻⁵; spontaneous mutants, 6.36 × 10⁻⁶; mutation frequency, 2.74 × 10⁻⁵; for D53A, induced mutants, 1.16 × 10⁻⁵; spontaneous mutants, 3.32 × 10⁻⁶; mutation frequency, 8.29 × 10⁻⁶; and for P54A, induced mutants, 3.77 × 10⁻⁵; spontaneous mutants, 7.22 × 10⁻⁶; mutation frequency, 3.05 × 10⁻⁵. (B) Loop 2 variants do not confer sensitivity to UV radiation. Assays were performed with pGY9738 plasmid and the following derivatives in PB102: pGY9738-K50A (umuD′C K50; ×), pGY9738 (umuD′C wild type; ■), pGY9738-G52A (umuD′C G52A; ○), pGY9738-M51A (umuD′C M51A; □), pGY9738-P54A (umuD′C P54A; ♢), pGY9738-D53A (umuD′C D53A; ●), and pGB2 (empty vector; ◆). (C) Immunoblot showing steady-state levels of UmuC expressed from variant umuDC plasmids (GW8017). The wild-type plasmid was pGY9738, and the empty vector was pGB2.