Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity

Abstract

The active form of Escherichia coli DNA polymerase V responsible for damage-induced mutagenesis is a multiprotein complex (UmuD'(2)C-RecA-ATP), called pol V Mut. Optimal activity of pol V Mut in vitro is observed on an SSB-coated single-stranded circular DNA template in the presence of the β/γ complex and a transactivated RecA nucleoprotein filament, RecA*. Remarkably, under these conditions, wild-type pol V Mut efficiently incorporates ribonucleotides into DNA. A Y11A substitution in the 'steric gate' of UmuC further reduces pol V sugar selectivity and converts pol V Mut into a primer-dependent RNA polymerase that is capable of synthesizing long RNAs with a processivity comparable to that of DNA synthesis. Despite such properties, Y11A only promotes low levels of spontaneous mutagenesis in vivo. While the Y11F substitution has a minimal effect on sugar selectivity, it results in an increase in spontaneous mutagenesis. In comparison, an F10L substitution increases sugar selectivity and the overall fidelity of pol V Mut. Molecular modeling analysis reveals that the branched side-chain of L10 impinges on the benzene ring of Y11 so as to constrict its movement and as a consequence, firmly closes the steric gate, which in wild-type enzyme fails to guard against ribonucleoside triphosphates incorporation with sufficient stringency.

Figure 1.

Gel images of primer extension reactions catalyzed by wild-type pol V in the presence of dNTPs (A–D) or rNTPs (E–H). Reactions in the presence of each individual nucleotide were carried out for 5 min. The identity of nucleotide incorporated is shown below the respective lanes. The extended sequence of templates with five consecutive Ts (A and E), As (B and F), Cs (C and G) or Gs (D and H), are indicated to the right of each gel panel. Determination of the specificity of nucleotide incorporation is complicated due to the efficient incorporation of ATP present in the reactions at 1 mM concentration. Lanes with reactions lacking polymerase are indicated as ‘pr’ (short for primer) and reactions with no additional nucleotide are indicated by dash (–).

Figure 2.

Role of UmuC F10 and Y11 residues on sugar discrimination by pol V. Primer extension reactions catalyzed by wild-type pol V (A and B), F10L (C and D), Y11A (E and F) and Y11F (G and H) were compared in the presence of 100 µM dNTPs (A, C, E and G) or rNTPs (B, D, F and H). Reactions were performed for 20 s, 1, 2, 4, 8 or 16 min (lanes 1–6, respectively) under optimal conditions for pol V Mut. The numbers shown next to the nucleotide identity represent primer elongation as a percent of total primer termini.

Figure 3.

Specificity of nucleotide incorporation by mutant pol Vs. Reactions catalyzed by F10L (A–D), Y11A (E–H) or Y11F (I–L) in the presence 100 µM of each individual dNTP (A, B, E, F, I, J) or rNTP (C, D, G, H, K, L) were carried out for 5 min. All reactions contained 1 mM ATP. The identity of the nucleotide incorporated is shown below each lane and the extended sequence of templates with five consecutive Ts (A, C, E, G, I, K) or As (B, D, F, H, J, L) is indicated to the right of each gel panel. All lanes with reactions lacking polymerase are indicated as ‘pr’ and reactions with no additional nucleotide are indicated by dash (–).

Figure 4.

Discrimination against rNMP insertion by pol V mutants. Insertion of dA versus rA and T versus U by wild-type pol V (A), F10L (B), Y11A (C) or Y11F (D) were analyzed using a template containing five consecutive As. Insertion of dA/rA and dC/rC by wild-type pol V (E), F10L (F), Y11A (G) or Y11F (H) were analyzed using a template containing five consecutive Ts followed by two Gs. Reactions were performed in the presence of 100 µM dNTPs and/or 1 mM rNTPs for 10 min; half of each reaction mixture was subjected to alkaline hydrolysis (OH⁻) (lanes 2, 4, 6, 8 and 10 in each panel) under conditions that completely hydrolyze DNA chains at the positions of rNTP insertion. The identity of the nucleotide present in the reaction is shown below each track (I NTP, II NTP). The extended sequence of the templates with five consecutive As (A–D) or Ts (E–H) is indicated to the right of the gel panels. Reactions in lanes 1 and 2 contained only ATP; lanes 3, 4 ATP and dATP; lanes 5, 6 ATP with UTP (A–D) or with CTP (E–H); lanes 7, 8 ATP and dATP with UTP and dTTP (A–D) or with CTP and dCTP (E–H); lanes 9, 10 ATP and dATP with dTTP (A–D) or with dCTP (E–H). Bands corresponding to incorporation of UTP (in A and D) or dTTP (in C) are indicated by stars.

Figure 5.

The inhibitory effect of cytosine arabinoside incorporation on DNA synthesis by wild-type pol V (A) and the Y11A variant (B). Insertions of dCTP (lanes 2, 4, 6) versus ara-C (lanes 3, 5, 7) with, or without, dGTP alone (4, 5), or in combination with dATP and dTTP (6, 7) were analyzed using a substrate containing five consecutive Gs in the template. Reactions were carried out in the presence of 100 µM nucleotides for 5 min; all reactions contained 1 mM ATP. The identity of the nucleotide incorporated is shown below each track. The extended sequence of the template is indicated to the right of the gel. The reaction shown in lane 0 contained no polymerase and the reactions in lane 1 contained no other nucleotides except the 1 mM ATP. The well and primer locations are indicated by arrows at the top and bottom of the gel, respectively.

Figure 6.

Qualitative analysis of spontaneous mutagenesis. Spontaneous pol V-dependent mutagenesis was measured in the E. coli strain, RW584 [recA730 lexA(Def) ΔumuDC] harboring low-copy-number vector control (pGB2) or a plasmid expressing wild-type pol V (pRW134), umuC F10L (pJM964), UmuC Y11A (pJM963) or UmuC Y11F (pJM952) by assaying reversion of the hisG4 (ochre) allele (leading to Histidine prototophy).

Figure 7.

Spectrum of spontaneous rpoB mutations arising in a mutL recA730 lexA(Def) ΔdinB ΔumuDC strain expressing pol V variants. The types of base-pair substitutions observed in the rpoB gene in E. coli strain RW710 [recA730 lexA(Def) ΔdinB ΔumuDC mutL], that result in rifampicin resistance are color coded as shown in the figure. The arrows indicate mutagenic hot spots discussed in the article.

Figure 8.

Structural models of UmuC and variants. Models of wild-type UmuC or steric gate mutants inserting dATP or ATP opposite a template T. These models are based on the structure of human DNA pol η (PDB 3MR3), and amino acid substitutions were made following the alignment by HHpred (toolkit.tubebingen.mpg.de/hhpred). Residues surrounding the incoming nucleotide are modeled and labeled according to the UmuC sequence. (A) Wild-type UmuC showing that Y11 coordinates a conserved water molecule (red sphere) that interacts with the incoming nucleotide, dATP (shown in yellow). (B) Wild-type pol V can accommodate ATP (shown in blue) by rotation of the benzene ring of Y11 (alternate positions shown in pink and grey). (C) Substitution of Y11 with Ala leaves a void in the active site of UmuC that can readily accommodate ATP. (D) Modeling of Phe10 (shown in grey), and Leu10 (shown in blue), reveals that the side chain of Leu presses on the benzene ring of Y11, so as to close the distance between the C2 position of the incoming deoxyribonucleotide (dATP, shown in yellow), and concomitantly precludes ribonucleotide incorporation.

Figure 9.

Model of UmuC Y11F. UmuC Y11 normally coordinates a conserved water molecule that interacts with the incoming nucleotide. However, in the UmuC Y11F mutant, the absence of the hydroxyl group destabilizes the water molecule and in its absence allows for the misincorporation of dGTP (shown in blue) and the formation of a G:T mispair (dotted lines). For comparison, the correct base (dATP) with the water molecule is shown in light yellow.