PMID:15456749
| Citation |
Skorvaga, M, DellaVecchia, MJ, Croteau, DL, Theis, K, Truglio, JJ, Mandavilli, BS, Kisker, C and Van Houten, B (2004) Identification of residues within UvrB that are important for efficient DNA binding and damage processing. J. Biol. Chem. 279:51574-80 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1074/jbc.M409266200 |
| Keywords |
Adenosine Triphosphatases/chemistry; Adenosine Triphosphate/chemistry; Amino Acid Motifs; Bacillus/genetics; Bacillus/metabolism; Base Sequence; Binding Sites; Cholesterol/chemistry; DNA/chemistry; DNA Damage; DNA Helicases/chemistry; DNA Helicases/genetics; DNA Helicases/metabolism; DNA Repair; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Glutamic Acid/chemistry; Hydrolysis; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Oligonucleotides/chemistry; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Tyrosine/chemistry |
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