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)

Very Fast Product Release and Catalytic Turnover of DNA Photolyase

“…The found substrate binding rate constant k 1 is close to literature values 1. 12, 13 However, our product release rate, k 2 , is more than four orders of magnitude faster than what we had expected based on the low catalytic turnover numbers (0.1 to 1 s −1 ) reported in the literature9–11 for saturating illumination (see above).…”

“…The overall rate k repair of these photochemical repair steps is ∼10 9 s −1 6–8. Despite the fast repair reaction, the catalytic turnover number of photolyase under saturating continuous light has been reported to be only in the order of 0.1 to 1 s −1 ;9–11 this suggests that exchange of repaired DNA (product) for damaged DNA (substrate) is slow and rate limiting. While substrate binding was found to be rather fast ( k 1 ∼10 6 M −1 s −1 12, 13), the rate of product release ( k 2 ) has not been established as yet, and this step might well be rate limiting.…”

Energy transduction during catalysis by Escherichia coli DNA photolyase

“…Native enzyme (3.1 /*M) was reduced with dithionite (12.5 mM) and then titrated with TT*TT. Binding was monitored by measuring the quenching of FADH2 fluorescence emission at 509 nm (excite 390 nm), similar to that described in previous studies with enzyme containing only FADH2 Jordan et al, 1989), except that the data was corrected for a small (5%) increase in pterin fluorescence, which accompanies the binding of substrate to native enzyme (Lipman & Jorns, 1992). The latter was estimated by monitoring fluorescence emission at 470 nm, a wavelength where emission from FADH2 is negligible (Lipman & Jorns, 1992).…”

DNA repair by photolyases

“…This binding occurs around the cyclobutane dimer in a light-independent manner. Upon binding, PL flips the dimer out into the active site cavity to make contact with the flavin (Mees et al, 2004;Park et al, 1995) which leads to stable enzyme-substrate complex (Jordan, Alderfer, Chanderkar, & Jorns, 1989;Jorns, Sancar, & Sancar, 1985). Exposure of this complex to light initiates catalysis; the FADH and MTHF photoantenna absorb a photon and MTHF transfers the excitation energy to fully reduce FADHby Förster dipole-dipole resonance energy transfer to increase the efficiency of the overall DNA repair.…”