Dissection of functional domains in Escherichia coli DNA photolyase by linker-insertion mutagenesis.
The phr gene, which encodes protein of 472 amino acid residues, is required for light-dependent photoreactivation and enhances light-independent excision repair of ultraviolet light (UV)-induced DNA damage. In this study, dodecamer HindIII linker insertions were introduced into the cloned phr gene and the functional effects of the resulting mutations on photoreactivation and light-independent dark repair in vivo were studied. Among 22 mutants obtained, 7 showed no photoreactivation as well as no enhancement of light-independent repair. Four of these were located in amino acid residues between Gln333 and Leu371 near the 3' end of the gene, two were located in a small region at Glu275 to Glu280 near the middle of the gene and the remaining one was between Pro49 and Arg50. Three mutants that had insertions located in the 42 bp segment from 399 to 441 bp of the phr coding sequence (corresponding to amino acid residues Ile134 to Lys149) lost the light-independent repair effect but retained photoreactivation. These results suggest that (i) Escherichia coli DNA photolyase contains several critical sites that are distributed over much of the enzyme molecule, and (ii) a functional domain required for the effect on light-independent repair is at least in part distinct from that necessary for light-dependent photoreactivation.
Yamamoto
K
K
Biological Institute, Faculty of Science, Tohoku University, Sendai, Japan.
D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004260:DNA Repair; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D015151:Immunoblotting; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation / Q000235:genetics; D011740:Pyrimidine Dimers / Q000378:metabolism; D016415:Sequence Alignment
D011740:Pyrimidine Dimers; D004255:Deoxyribodipyrimidine Photo-Lyase