Structure of UvrA nucleotide excision repair protein in complex with modified DNA

Abstract

One of the primary pathways for removal of DNA damage is nucleotide excision repair (NER). In bacteria, the UvrA protein is the component of NER that locates the lesion. A notable feature of NER is its ability to act on many DNA modifications that vary in chemical structure. So far, the mechanism underlying this broad specificity has been unclear. Here, we report the first crystal structure of a UvrA protein in complex with a chemically modified oligonucleotide. The structure shows that the UvrA dimer does not contact the site of lesion directly, but rather binds the DNA regions on both sides of the modification. The DNA region harboring the modification is deformed, with the double helix bent and unwound. UvrA uses damage-induced deformations of the DNA and a less rigid structure of the modified double helix for indirect readout of the lesion.

Figure 1

Binding and processing of the palindromic DNA substrate. (a) DNA binding by Tm-UvrA. Binding curves were determined in filter binding assays. Values are mean ± s.e.m. of four independent measurements. Modified DNA was the palindromic 32-mer with fluorescein modification in position 14 used for crystallization (see Online Methods for sequence). The unmodified oligonucleotide had the same sequence and length but did not contain the fluorescein moiety. WT, wild type. (b) K_d values determined in the filter binding assays. (c,d) Incision assay. The oligonucleotides were radiolabeled on 5′ end (c) or 3′ end (d). Oligonucleotides added to each reaction are specified for 10-32, the labeled strand is indicated. UvrA, UvrB and/or UvrC were added to the reaction as indicated and the reaction products were resolved on the TBE-urea denaturing PAGE. DNA was visualized by phosphorimaging. Positions of DNA size markers are at right of each gel (nt, nucleotides). (e) Sequences of oligonucleotides used in incision assays. Sequences added to the 32-mer palindromic duplex are underlined. Fluorescein-modified thymines are boxed. Arrows indicate observed cleavage sites.

Figure 2

Structure of complex. (a) Domain organization of UvrA protein. The domain names are the same as in reference 19. Numbers indicate Tm-UvrA residues at the domain boundaries. (b) Two views of the structure of the Tm-UvrA–DNA complex. Protein is in ribbon representation, and domains of one subunit of the dimer are colored as in a. DNA, blue. Structural zinc ions, orange and gray spheres.

Figure 3

DNA binding. (a) Surface representation of Tm-UvrA with surface potential in red (negative) and blue (positive) (±20 kT e⁻¹). DNA is yellow. (b) Details of key protein-DNA interactions mediated by signature domain II. DNA, purple; protein residues, cyan.

Figure 4

Deformation of the DNA. (a) Two views of the DNA from structure of the Tm-UvrA–DNA complex (purple and blue) superimposed on the model of ideal B-form DNA (white) of identical length and sequence. Superimposition was carried out using the positions of the phosphate groups of residues 20–29 from the strand in blue. Fluorescein-modified bases, orange sticks. Numbers of selected residues in both models (blue, purple and orange for DNA from the Tm-UvrA–DNA complex and black for the ideal DNA model) indicate the unwinding of the strand shown in purple. Trailing of the DNA, purple arrow. Unstacking of central bases in DNA from the Tm-UvrA–DNA complex, double-headed arrows. (b) Stereoview showing midpoint of DNA from Tm-UvrA–DNA complex superimposed on NMR solution structure of a psoralen monoadduct (DNA, green; modification, cyan; PDB 203D). DNA from the Tm-UvrA–DNA structure is blue.

Figure 5

Comparison of Bst-UvrA–ADP and Tm-UvrA–DNA structures. (a) Structure of Tm-UvrA–DNA complex (Tm-UvrA–DNA colored by domain as in Fig. 2, Bst-UvrA–ADP in orange) was superimposed on the Bst-UvrA structure using the positions of Cα atoms of the two ATP-binding domains. Only the most invariant core of the structures, ATP-binding domains I and II, signature domains I and UvrB-binding domains, are shown. Only one subunit of the dimer is shown for clarity. (b) Comparison of positions of DNA-binding domains (purple, Tm-UvrA–DNA; orange, Bst-UvrA–ADP). ATP-binding domains are light gray for Tm-UvrA and dark gray for Bst-UvrA. (c) Comparison of position of signature II domains (cyan, Tm-UvrA–DNA; orange, Bst-UvrA–ADP). In a–c the structures are in the same orientation. (d) Position of signature II domains from Bst-UvrA–ADP structure (orange) and Tm-UvrA–DNA structure (cyan) relative to DNA from the latter model. The zinc finger, its loop and the helical core of the domains’ structure are in ribbon representation for both subunits of the dimers. Zinc ions are gray spheres with darker shade for Bst-UvrA. The DNA is blue. (e) Close-up of ATP-binding sites from Tm-UvrA (domains colored as in Fig. 2) and Bst-UvrA (orange). Backbone trace of helices containing Walker A motif (left) and signature motif (right) is in ribbon representation. Catalytic lysine from Walker A motif, conserved serine from signature motif and ADP from Bst-UvrA structure are sticks. (f) Position of conserved arginine located near the of DNA interface and proximal active site. Protein structure is colored as in e.