Abstract
The UvrB protein is the central recognition protein in bacterial nucleotide excision repair. We have shown previously that the highly conserved beta-hairpin motif in Bacillus caldotenax UvrB is essential for DNA binding, damage recognition, and UvrC-mediated incision, as deletion of the upper part of the beta-hairpin (residues 97-112) results in the inability of UvrB to be loaded onto damaged DNA, defective incision, and the lack of strand-destabilizing activity. In this work, we have further examined the role of the beta-hairpin motif of UvrB by a mutational analysis of 13 amino acids within or in the vicinity of the beta-hairpin. These amino acids are predicted to be important for the interaction of UvrB with both damaged and non-damaged DNA strands as well as the formation of salt bridges between the beta-hairpin and domain 1b of UvrB. The resulting mutants were characterized by standard functional assays such as oligonucleotide incision, electrophoretic mobility shift, strand-destabilizing, and ATPase assays. Our data indicated a direct role of Tyr96, Glu99, and Arg123 in damage-specific DNA binding. In addition, Tyr93 plays an important but less essential role in DNA binding by UvrB. Finally, the formation of salt bridges between the beta-hairpin and domain 1b, involving amino acids Lys111 bound to Glu307 and Glu99 bound to Arg367 or Arg289, are important but not essential for the function of UvrB.
Highlights
Nucleotide excision repair (NER)1 is a universal and highly conserved DNA repair pathway found in bacteria and eukaryotic cells
In our padlock binding model for the UvrB1⁄7DNA preincision complex, we have proposed that the -hairpin is inserted between the two DNA strands, and the non-damaged strand is locked between the -hairpin and domain 1b (3)
Goosen and colleagues (5) have probed the role of hydrophobic residues in the -hairpin of E. coli UvrB through a series of double mutants to show that (a) Tyr92/Tyr93 function in damage recognition by preventing UvrB binding to non-damaged sites, (b) Tyr95/Tyr96 at the base of -hairpin have a direct role in damage recognition and are positioned in the vicinity of the lesion in the UvrB1⁄7DNA complex, and (c) Tyr101 and Phe108 in the tip of -hairpin are important for the strand-separating activity of UvrB
Summary
Nucleotide excision repair (NER) is a universal and highly conserved DNA repair pathway found in bacteria and eukaryotic cells. Goosen and colleagues (5) have probed the role of hydrophobic residues in the -hairpin of E. coli UvrB through a series of double mutants to show that (a) Tyr92/Tyr function in damage recognition by preventing UvrB binding to non-damaged sites, (b) Tyr95/Tyr at the base of -hairpin have a direct role in damage recognition and are positioned in the vicinity of the lesion in the UvrB1⁄7DNA complex, and (c) Tyr101 and Phe108 in the tip of -hairpin are important for the strand-separating activity of UvrB. We further examined the role of amino acid residues in UvrB, which we predicted to be important in the process of damage recognition, namely in the interaction of UvrB with both the damaged and non-damaged DNA strands, and in the formation of salt bridges between the -hairpin and domain 1b
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