Mechanism of damage recognition by Escherichia coli DNA photolyase.
Escherichia coli DNA photolyase binds to DNA containing pyrimidine dimers with high affinity and then breaks the cyclobutane ring joining the two pyrimidines of the dimer in a light- (300-500 nm) dependent reaction. In order to determine the structural features important for this level of specificity, we have constructed a 43 base pair (bp) long DNA substrate that contains a thymine dimer at a unique location and studied its interaction with photolyase. We find that the enzyme protects a 12-16-bp region around the dimer from DNase I digestion and only a 6-bp region from methidium propyl-EDTA-Fe (II) digestion. Chemical footprinting experiments reveal that photolyase contacts the phosphodiester bond immediately 5' and the 3 phosphodiester bonds immediately 3' to the dimer but not the phosphodiester bond between the two thymines that make up the dimer. Methylation protection and interference experiments indicate that the enzyme makes major groove contacts with the first base 5' and the second base 3' to the dimer. These data are consistent with photolyase binding in the major groove over a 4-6-bp region. However, major groove contacts cannot be of major significance in substrate recognition as the enzyme binds equally well to a thymine dimer in a 44-base long single strand DNA and protects a 10-nucleotide long region around the dimer from DNase I digestion. It is therefore concluded that the unique configuration of the phosphodiester backbone in the strand containing the pyrimidine dimer, as well as the cyclobutane ring of the dimer itself are the important structural determinants of the substrate for recognition by photolyase.
Husain
I
I
Department of Biochemistry, University of North Carolina School of Medicine, Chapel Hill 27514.
Sancar
G B
GB
Holbrook
S R
SR
Sancar
A
A
D001483:Base Sequence; D003196:Computer Graphics; D004249:DNA Damage; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008190:Lyases / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011740:Pyrimidine Dimers; D013379:Substrate Specificity
D011740:Pyrimidine Dimers; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase