title: Reaction of Escherichia coli and yeast photolyases with homogeneous short-chain oligonucleotide substrates.
abstract: Similar rates have been observed for dimer repair with Escherichia coli photolyase and the heterogeneous mixtures generated by UV irradiation of oligothymidylates [UV-oligo(dT)n, n greater than or equal to 4] or DNA. Comparable stability was observed for ES complexes formed with UV-oligo(dT)n, (n greater than or equal to 9) or dimer-containing DNA. In this paper, binding studies with E. coli photolyase and a series of homogeneous oligonucleotide substrates (TpT, TpTp, pTpT, TpTpT, TpTpT, TpTpTpT, TpTpTpT, TpTpTpT, TpTpTpT) show that about 80% of the binding energy observed with DNA as substrate (delta G approximately 10 kcal/mol) can be attributed to the interaction of the enzyme with a dimer-containing region that spans only four nucleotides in length. This major binding determinant (TpTpTpT) coincides with the major conformational impact region of the dimer and reflects contributions from the dimer itself (TpT, delta G = 4.6 kcal/mol), adjacent phosphates (5'p, 0.8 kcal/mol; 3'p, 1.1 kcal/mol), and adjacent thymine residues (5'T, 0.8 kcal/mol; 3'T, 1.3 kcal/mol). Similar turnover rates (average kcat = 6.7 min-1) are observed with short-chain oligonucleotide substrates and UV-oligo(dT)18, despite a 25,000-fold variation in binding constants (Kd). In contrast, the ratio Km/Kd decreases as binding affinity decreases and appears to plateau at a value near 1. Turnover with oligonucleotide substrates occurs at a rate similar to that estimated for the photochemical step (5.1 min-1), suggesting that this step is rate determining. Under these conditions, Km will approach Kd when the rate of ES complex dissociation exceeds kcat.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Biological Chemistry, Hahnemann University School of Medicine, Philadelphia, Pennsylvania 19102.
authors.forename: S P
authors.initials: SP
authors.lastname: Jordan
authors.forename: J L
authors.initials: JL
authors.lastname: Alderfer
authors.forename: L P
authors.initials: LP
authors.lastname: Chanderkar
authors.forename: M S
authors.initials: MS
authors.lastname: Jorns
mesh_terms: D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D009841:Oligonucleotides; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase