Abstract
Nucleotide excision repair is distinguished from other DNA repair pathways by its ability to process a wide range of structurally unrelated DNA lesions. In bacteria, damage recognition is achieved by the UvrA.UvrB ensemble. Here, we report the structure of the complex between the interaction domains of UvrA and UvrB. These domains are necessary and sufficient for full-length UvrA and UvrB to associate and thereby form the DNA damage-sensing complex of bacterial nucleotide excision repair. The crystal structure and accompanying biochemical analyses suggest a model for the complete damage-sensing complex.
Highlights
Nucleotide excision repair is distinguished from other DNA repair pathways by its ability to process a diverse set of lesions
UvrC and the damage-containing oligonucleotide are removed by UvrD, whereas UvrB remains bound to the gapped DNA and recruits DNA polymerase I for repair synthesis
NUMBER 19 handle a diverse set of lesions while maintaining specificity? How do UvrA and UvrB cooperate during damage recognition, and what is the precise role of ATP? Ongoing studies in the field, including those described below, aim to address these issues
Summary
Expression and Purification of G. stearothermophilus UvrA and UvrB Interaction Domain Complex—The DNA sequences encoding the interaction domains (Fig. 1) were amplified from the plasmids containing the genes for full-length UvrA and UvrB [5], cloned into pET-28a (ϩ) (Novagen; see Table 3), and confirmed by sequencing. The resulting proteins were further purified by size exclusion chromatography (Superdex 75; GE Healthcare; 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM -ME). The complex was purified from excess UvrB 149 –250 by size exclusion chromatography (Superdex 75; GE Healthcare; 25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM -ME; Fig. 2). For x-ray diffraction, the crystals were transferred to a 10-l drop of crystallization buffer containing 15% (Ϯ) 1, 2-propanediol for cryoprotection and flash frozen in liquid nitrogen. The processed data (HKL2000 [7]) revealed that the crystal belonged to the tetragonal space group P41212 with cell parameters a ϭ b ϭ 84.49 Å, c ϭ 60.87 Å, ␣ ϭ  ϭ ␥ ϭ 90.0°, and contained one complex in the asymmetric unit. The coordinates and structure factors have been deposited to the Protein Data Bank with the accession code 3FPN
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