Steric gate variants of UmuC confer UV hypersensitivity on Escherichia coli

Abstract

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.

FIG. 1.

The steric gate residue of UmuC is predicted to approach the incoming nucleotide. (Top) Homology model of UmuC (4) showing the template (green), primer and incoming nucleotide (blue), and residues F10 and steric gate Y11 (red). The backbone of UmuC is shown in yellow. The illustration was prepared using VMD (28). (Bottom) Amino acid sequences of representative Y family polymerases showing conserved residues aligned with F10 and Y11 of UmuC (boxed) (7). Ec, Escherichia coli; Sa, Sulfolobus acidocaldarius; Ss, Sulfolobus solfataricus; Sc, Saccharomyces cerevisiae; Hs, Homo sapiens.

FIG. 2.

The steric gate Y11 and F10 variants in UmuC cause UV hypersensitivity. Assays were performed with the pGY9738 plasmid and the following derivatives in GW8017: pGY9738 (umuD′C; ⧫); pGB2 (empty vector; ×); pGY9738-C104 (cat-) (umuD′C C104; −); pGY9738-F10V (umuD′C F10V; ▪); pGY9738-Y11V (umuD′C Y11V; •); pGY9738-Y11A (umuD′C Y11A; ▴).

FIG. 3.

(A) A strain expressing the steric gate variant of UmuC exhibits a growth phenotype similar to wild type. Assays were performed with plasmid pGY9738 and derivatives in GW8017: pGY9738 (umuD′C; ▪); pGB2 (empty vector; ⧫); pGY9738-Y11A (umuD′C Y11A; ▴). (B) Immunoblot showing steady-state levels of UmuC expressed from variant umuDC plasmids (in GW8017) and from the chromosome in GW2771. Relative UmuC protein levels are indicated below the blot.

FIG. 4.

The extreme UV sensitivity is suppressed by mutations in the β clamp interaction motifs. Assays were performed with plasmid pGY9738 and derivatives in GW8017: pGY9738 (umuD′C; ⧫); pGB2 (empty vector; ×); pGY9738-Y11A β1&2 (umuD′C Y11A β1&2; −); pGY9738-Y11A β2 (umuD′C Y11A β2; ▪); pGY9738-Y11A-β1 (umuD′C Y11A β1; •); pGY9738-Y11A (umuD′C Y11A; ▴).

FIG. 5.

Steric gate variants of UmuC are dominant negative. Assays were performed with plasmid pGY9738 and derivatives in AB1157 (umuDC⁺). (A) pGY9738 (umuD′C; ⧫); pGB2 (empty vector; ▪); pGY9738-F10V (umuD′C F10V; ▴); pGY9738-Y11V (umuD′C Y11V; ×); pGY9738-Y11A (umuD′C Y11A; •). (B) pGY9738 (umuD′C; ⧫); pGB2 (empty vector; ▪); pGY9738-Y11A-β1&2 (umuD′C Y11A β1&2; ▴); pGY9738-Y11A-β1 (umuD′C Y11A β1; −); pGY9738-Y11A-β2 (umuD′C Y11A β2; ×); pGY9738-Y11A (umuD′C Y11A; •).

FIG. 6.

UV-induced and uninduced arg⁺ revertants of AB1157 harboring plasmids expressing the umuD′C variants indicated. Black bars, +UV (10 J/m²); white bars, −UV.

FIG. 7.

Steric gate variants confer sensitivity to the DNA-damaging agents nitrofurazone (A), MMS (B), and UV (C). (A and B) Assays were performed with plasmid pGY9738 and derivatives in AB1157 (umuDC⁺): pGY9738 (umuD′C; ⧫); pGB2 (empty vector; ▴); pGY9738-Y11A (umuD′C Y11A; ▪). (C) The DinB steric gate variant (F13V) confers sensitivity to UV light on AB1157 (umuDC⁺) and derivatives: pYG768 (dinB; ▪); pWSK30 (empty vector, ⧫); pYG768-F13V (dinB F13V, ▴).

FIG. 8.

(A) Disruption of dnaQ, the proofreading subunit, suppresses the UV hypersensitivity of the UmuC steric gate variant. Assays were performed with plasmid pGY9738-Y11A in GW2771 and derivatives: GW2771 spq-2 (▪); GW2771 (⧫); GW2771 spq-2 dnaQ903 (▴). (B) UV-induced arg⁺ revertants of GW2771 spq-2 dnaQ903 harboring the plasmids indicated (irradiated at 10 J/m²).