The role of conserved amino acids in substrate binding and discrimination by photolyase.
DNA photolyases catalyze the light-dependent repair of pyrimidine dimers in DNA. We have utilized chemical modification and site-directed mutagenesis to probe the interactions involved in substrate recognition by the yeast photolyase Phr1. Lys517 was protected from reductive methylation in the presence of substrate, but not in its absence, and the specific and nonspecific association constants for substrate binding by Phr1 (Lys517-->Ala) were decreased 10-fold. These results establish a role for Lys517 in substrate binding. Mutations at Arg507, Lys463, and Trp387 reduced both the overall affinity for substrate and substrate discrimination. Sites of altered interactions in ES complexes were identified by methylation and ethylation interference techniques. Interaction with the base immediately 3' to the dimer was altered in the Phr1(Lys517-->Ala). DNA complex, whereas interactions with the phosphate and base immediately 5' to the dimer were reduced when Phr1(Arg507-->Ala) bound substrate. Multiple interactions 5' and 3' to the dimer were perturbed in complexes containing Phr1(Trp387-->Ala) or Phr1(Lys463-->Ala). In addition the quantum yield for dimer photolysis by Phr1(Trp387-->Ala) was reduced 3-fold. The locations of these mutations establish that a portion of the DNA binding domain is comprised of residues in the highly conserved carboxyl-terminal half of the enzyme.
Baer
M E
ME
Department of Biochemistry and Biophysics, School of Medicine, University of North Carolina, Chapel Hill 27599-7260.
Sancar
G B
GB
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence; D003488:Cyanogen Bromide; D004247:DNA / Q000378:metabolism; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D013379:Substrate Specificity
D000596:Amino Acids; D010710:Phosphates; D004247:DNA; D004255:Deoxyribodipyrimidine Photo-Lyase; D003488:Cyanogen Bromide