Thumb-domain dynamics modulate the functional repertoire of DNA-Polymerase IV (DinB)

Abstract

In order to cope with the risk of stress-induced mutagenesis, cells in all kingdoms of life employ Y-family DNA polymerases to resolve resulting DNA lesions and thus maintaining the integrity of the genome. In Escherichia coli, the DNA polymerase IV, or DinB, plays this crucial role in coping with these type of mutations via the so-called translesion DNA synthesis. Despite the availability of several high-resolution crystal structures, important aspects of the functional repertoire of DinB remain elusive. In this study, we use advanced solution NMR spectroscopy methods in combination with biophysical characterization to elucidate the crucial role of the Thumb domain within DinB's functional cycle. We find that the inherent dynamics of this domain guide the recognition of double-stranded (ds) DNA buried within the interior of the DinB domain arrangement and trigger allosteric signals through the DinB protein. Subsequently, we characterized the RNA polymerase interaction with DinB, revealing an extended outside surface of DinB and thus not mutually excluding the DNA interaction. Altogether the obtained results lead to a refined model of the functional repertoire of DinB within the translesion DNA synthesis pathway.

Graphical Abstract

Figure 1.

(A) Scheme of the used constructs indicating the domain-structure of the DinB. (B) Crystal structure of the E. coli DNA polymerase IV (PDB-ID: 4Q45) with the Fingers (orange), Palm (yellow), Thumb (orange-red) and PAD (dark-red) domains as well as magnesium ions (white) indicated. (C) 2D [¹⁵N, ¹H]-NMR spectra of the [U-²H,¹⁵N]-DinB, [U-²H,¹⁵N]-DinBΔPAD, [U-¹⁵N]-DinB-Thumb and [U-¹⁵N]-DinB-PAD measured in NMR-buffer at 298K. (D) Secondary structure elements of the DinB-domains in solution (color) as derived from the secondary backbone ¹³C chemical shifts. The secondary structure elements of DinB within the X-ray structure (PDB-ID:4Q45) are indicated in grey (β-strands) and dark-grey (α-helices).

Figure 2.

(A) Dynamics on the ps–ns timescale plotted on the DinB structure (PDB-ID: 4Q45). The amide moieties are shown as spheres and the hetNOE values are indicated by the green to blue gradient. (B) The hetNOE (top) as well as the longitudinal relaxation rate R₁ are plotted against the DinB residue number. (C) The R_2(R1ρ) (top) as well as the rotational correlation time τ_c, obtained from the analysis of the R₁ and R_2(R1ρ) relaxation rates, are plotted against the DinB residue number. The broken line indicates the average value of 21.5 ns. (D) Dynamics on the microsecond timescale plotted on the DinB structure (PDB-ID: 4Q45). The amide moieties are shown as spheres and the R_2(R1ρ) values are indicated by the yellow to red gradient. Relaxation data was measured at 18.8 T.

Figure 3.

(A) Distribution and assignment of isoleucine, alanine, methionine, leucine and valine methyl groups using an [U-²H, Ile-δ₁–¹³CH₃, Leu-δ₂–¹³CH₃, Val-γ₂–¹³CH₃, Ala-β-¹³CH₃, Met-ϵ-¹³CH₃] stereospecific labelled DinB measured in NMR buffer at 298 K. * and ^# denote leucine residues in the overlapping central region. (B) Representative NOE strips from a 3D ¹³C_methyl-¹³C_methyl-¹H_methyl SOFAST NOESY focusing on the interdomain stabilization between Palm (yellow) and Thumb (orange). (C) NOE network, indicated by the black lines, stabilizing the Thumb on the Palm surface plotted on the DinB structure (PDB-ID: 4Q45). The respective orientation relative to panel A is indicated. (D) Flareplot visualization of the complete methyl-methyl NOE network detected for the MALVI^proS-DinB illustrating the connectivity between the individual domains. (E, F) ΔR_2eff values for the methyl-groups obtained from the difference of R_2,eff at the lowest and highest CPMG frequency υ_CPMG (E). Structural view of the amplitude of the CPMG relaxation dispersion profiles ΔR_2eff at 18.8 T (F). Exchange broadened residues are indicated.

Figure 4.

(A) Titration of increasing amounts of dsDNA to either [U-²H,¹⁵N]-DinB, [U-²H,¹⁵N]-DinBΔPAD or [U-¹⁵N]-DinB-PAD as indicated by the schematics on the top. Overlay of 2D [¹⁵N,¹H]-NMR spectra of different DinB constructs in the absence (blue or red, respectively) and in the presence of increasing amounts of DNA as indicated by the green gradient acquired in NMR-buffer at 298 K. (B) The ratio of the individual peak intensities in the presence of by the green gradient indicated equivalents of dsDNA to the respective apoDinB-forms plotted against the DinB residue number. (C, D) Intensity changes upon dsDNA interaction were plotted on the DinB as well as the DinBΔPAD (PDB: 4Q45) structures by the indicated color gradient. The amide moieties of the individual construct are shown as spheres. (E) Near-surface electrostatic potential (ϕ_ENS) of the DinB:dsDNA complex by X-ray crystallography (PDB: 4Q45) using the Analytical Poisson-Boltzmann Solver (APBS) (61), with positively and negatively-charged surfaces represented in blue and red, respectively.

Figure 5.

(A, B) Bio-layer interferometry (BLI) data analysis of DinB (A) and DinBΔPAD (B) binding to dsDNA. Analyte concentrations are indicated. Non-linear least quare fits to the experimental data are indicated by the black lines. (C) Table of the obtained dissociation constant K_D, the kinetic parameters k_on and k_off, as well as the χ² and R² parameters indicating the quality of the non-linear least squares fits. (D) Generalized order parameter, S², for holoDinB (0.1:1 dsDNA:protein ratio) plotted on the DinB structure (PDB-ID: 4Q45). The amide moieties are shown as spheres and the S² values are indicated by the yellow to blue gradient. (E) The S² (top) as well as conformational exchange contributions, R_ex, are plotted against the DinB residue number. (F) Residues exhibiting conformational exchange on the micro- to millisecond timescale are plotted on the DinB structure (PDB-ID: 4Q45). The amide moieties are shown as spheres and the R_ex values are indicated by the yellow to red gradient.

Figure 6.

(A) Titration of increasing amounts of RNAP to either [U-²H,¹⁵N]-DinB, [U-²H,¹⁵N]-DinBΔPAD or [U-¹⁵N]-DinB-PAD as indicated by the schematics on the top. Overlay of 2D [¹⁵N,¹H]-NMR spectra of different DinB constructs in the absence (blue or red, respectively) and in the presence of increasing amounts of RNAP as indicated by the orange to yellow gradient acquired in NMR-buffer at 298 K. (B) The ratio of the individual peak intensities in the presence of the indicated equivalents of RNAP to the respective apo DinB-forms highlighted by the orange to yellow gradient plotted against the DinB residue number. (C–E) Intensity changes upon RNAP interaction were plotted on the DinB as well as the DinBΔPAD and DinB-PAD (PDB: 4Q45) structures, respectively, by the indicated color gradient. The amide moieties of the individual construct are shown as spheres.

Figure 7.

(A) Titration of increasing amounts of RNAP to MALVI^proS-DinB. Overlay of 2D [¹³C,¹H]-NMR spectra of in the absence (blue) and in the presence of increasing amounts of RNAP as indicated by the orange to yellow gradient acquired in NMR-buffer at 298 K. (B) Intensity changes upon RNAP interaction were plotted on the DinB (PDB: 4Q45) structure, respectively, by the indicated color gradient. The methyl groups are shown as spheres. (C) Rotated view of panel B, indicating a large interaction surface on DinB encompassing the Fingers, Palm and Thumb domains. The dimensions of the surface highlighted in grey are indicated. (D) The ratio of the individual peak intensities of the indicated methyl groups in the presence of 0.1 equivalents of RNAP. The fully annotated plot is provided in Supplementary Figure 5. (E–G) BLI data analysis of DinB (E), DinBΔPAD (F) and DinB-PAD (G) binding to RNAP. Analyte concentrations are indicated. Non-linear least square fits to the experimental data are indicated by the black lines. (H) Table of the obtained dissociation constant K_D, the kinetic parameters k_on and k_off, as well as the χ² and R² parameters indicating the quality of the non-linear least squares fits. n.b. indicates non-binding.

Figure 8.

(A) DinB is a highly dynamic protein in its apo-state and the PAD domain is flexibly attached to the core of the protein. Upon encountering DNA, the core part of DinB consisting of the Fingers, Palm and Thumb domain facility the initial binding. In a second step the PAD-domain locks the DNA within the DNA-binding cavity stabilizing the complex. (B) DinB can bind RNAP directly, suggesting the possibility of hooking up during the transcription cycle and in this state directly acting on encountered DNA-lesions as the RNAP-interaction does not impair DinB’s DNA binding capabilities.