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Structure and mechanism of the UvrA–UvrB DNA damage sensor

Nature Structural & Molecular Biology volume 19, pages 291–298 (2012)Cite this article

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Abstract

Nucleotide excision repair (NER) is used by all organisms to eliminate DNA lesions. We determined the structure of the Geobacillus stearothermophilus UvrA–UvrB complex, the damage-sensor in bacterial NER and a new structure of UvrA. We observe that the DNA binding surface of UvrA, previously found in an open shape that binds damaged DNA, also exists in a closed groove shape compatible with native DNA only. The sensor contains two UvrB molecules that flank the UvrA dimer along the predicted path for DNA, ~80 Å from the lesion. We show that the conserved signature domain II of UvrA mediates a nexus of contacts among UvrA, UvrB and DNA. Further, in our new structure of UvrA, this domain adopts an altered conformation while an adjacent nucleotide binding site is vacant. Our findings raise unanticipated questions about NER and also suggest a revised picture of its early stages.

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Figure 1: Deformed DNA conformations processed by NER.
Figure 2: Architecture of the UvrA–UvrB DNA damage sensor.
Figure 3: The DNA binding surface of UvrA is composed of two spatially distinct regions and undergoes considerable reconfiguration in the AB sensor.
Figure 4: Modeling of native and damaged DNA into the two dimer configurations of UvrA.
Figure 5: Determination of the disposition and number of UvrB molecules in the AB damage sensor.
Figure 6: Structure of UvrA in a unique domain conformation and its implications for dynamic contacts in the AB–DNA complex.
Figure 7: Three distinct conformations of UvrA imply a detailed model for the early events of NER.

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Acknowledgements

We thank G. Verdine and his research group for scientific discussions and for sharing experimental results. We are grateful to C. Harrison for help with isothermal titration calorimetry experiments, S. Akabayov for advice on SAXS and S. Berkovitch for assistance with illustrations. We thank N. Kleckner, G. Verdine, N. Francis, R. Gaudet, N. Goosen, G. Moolenaar, B. Hill, K. Mackenzie, R.-J. Sung, M. Lee and M. Spong for critical reading of the manuscript. We thank the staff members at NE-CAT and Bio-CAT, Advanced Photon Source, Argonne National Laboratory, for assistance. Bio-CAT (RR-08630) and NE-CAT (RR-15301) are supported by the US National Institutes of Health (NIH). The Advanced Photon Source is supported by the Department of Energy (Contract No. DE-AC02-06CH11357). This work was supported by the National Science Foundation (MCB 0918161) and the NIH (GM 084162).

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Authors and Affiliations

  1. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA

    Danaya Pakotiprapha, Martin Samuels, Koning Shen, Johnny H Hu & David Jeruzalmi

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Contributions

D.P. purified, crystallized and determined the structure of the AB complex, measured the SAXS data, and carried out DNA binding and UvrA-UvrB interaction studies. M.A.S. solved the structure of UvrA Δ118–419 and did filter binding assays. K.S. prepared UvrB mutants and analyzed their interaction with UvrA. J.H. assisted in protein purification. D.P. and D.J. analyzed the data and wrote the manuscript.

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Correspondence to David Jeruzalmi.

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Supplementary information

Supplementary Text and Figures

Supplementary Discussion, Supplementary Methods, Supplementary Figures 1–9 and Supplementary Tables 1–3. (PDF 14544 kb)

Supplementary Movie 1

Rearrangement of the UvrA dimer upon going from the 'closed groove' to 'open tray' conformation. Each UvrA protomer within the AB complex has undergone a 25° helical rotation (25° rotation with ~10-Å translation) relative to its position in the isolated UvrA structure (PDB 2R6F, 'open tray' conformation). The two UvrA protomers are shown in different shades of gray, with the signature domain II in cyan. An ideal B-form DNA model (green) was placed along the proposed DNA binding path, with a red sphere denoting the presumed location of the lesion. The views in Supplementary Movies 1 and 2 are related by a 90° rotation about the X-axis. (MOV 4138 kb)

Supplementary Movie 2

Rearrangement of the UvrA dimer upon going from the 'closed groove' to 'open tray' conformation. Each UvrA protomer within the AB complex has undergone a 25° helical rotation (25° rotation with ~10-Å translation) relative to its position in the isolated UvrA structure (PDB 2R6F, 'open tray' conformation). The two UvrA protomers are shown in different shades of gray, with the signature domain II in cyan. An ideal B-form DNA model (green) was placed along the proposed DNA binding path, with a red sphere denoting the presumed location of the lesion. The views in Supplementary Movies 1 and 2 are related by a 90° rotation about the X-axis. (MOV 3784 kb)

Supplementary Movie 3

The signature domain II of UvrA undergoes nucleotide-dependent conformational change. In comparison to the conformation observed in full-length UvrA structure (PDB 2R6F), the signature domain II of UvrA Δ118–419 has undergone a nucleotide-dependent conformational change. The change is best described by a ~16° rigid-body rotation away from the ATP-binding domain I. This change alters the distance between DNA binding residues on the signature domain II of the two UvrA protomers from ~95 Å to ~115 Å. The UvrA dimer is shown as Cα trace. The signature domain II is colored cyan, whereas the rest of the protein is shown in gray. Residues of the Region I DNA binding site are depicted as blue spheres. ADP molecules are shown as red sticks. The distance is measured between Cα atoms of Lys718 in the two protomers. (MOV 5361 kb)

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Pakotiprapha, D., Samuels, M., Shen, K. et al. Structure and mechanism of the UvrA–UvrB DNA damage sensor. Nat Struct Mol Biol 19, 291–298 (2012). https://doi.org/10.1038/nsmb.2240

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