Photolyase substrate binding

The binding of photolyase to its substrate has been extensively investigated with DNase and chemical footprinting methods (Baer and Sancar, 1989 Husain et al, 1987 Kiener et al, 1989) and with substrates ranging in... 

The Drosophila and Xenopus (6-4) photolyases appear to bind DNA containing a (6-4) photoproduct by three-dimensional diffusion and to make contacts around the lesion quite similar to the contacts made by photolyase with DNA containing a cyclobutane pyrimidine dimer (Hitomi et al, 1997 Zhao et al, 1997). The (6-4) photolyase, like the cyclobutane photolyase, binds to its cognate lesion in ssDNA and dsDNA with essentially equal affinities (Zhao et al, 1997). When bound to a dsDNA substrate, the enzyme confers single-strandedness to a 4-bp region around the lesion, and the presence of a mismatch across the (6—4) photoproduct increases the affinity of the enzyme for the substrate (Zhao et al, 1997). These three features of binding, that is, binding to substrate in ssDNA with... 

Fig. 8. Reaction mechanism of (6-4) photolyase. The enzyme binds to DNA containing a (6-4) photoproduct and flips out the dinucleotide adduct into the active site cavity, where the open form of the photoproduct is converted to the oxetane intermediate by a light-independent general acid-base mechanism. Catalysis is initiated by light MTHF absorbs a photon and transfers energy to FADH , which then transfers an electron to the oxetane intermediate bond rearrangement in the oxetane radical regenerates two canonical pyrimidines, and back-electron transfer restores the flavin radical to catalytically competent FADH form. The repaired dipyrimidine flips back into the DNA duplex, and the enzyme is dissociated from the substrate.

Clearly, all indications are that (6—4) photolyase binds DNA and repairs its substrate by a mechanism quite similar to that of classical photolyase. However, there appears to be a fundamental difference in the photochemical reaction catalyzed by the two enzymes. The quantum yield of repair by excited singlet-state flavin by classical photolyase is near unity, whereas the quantum yield of repair by excited flavin in (6-4) photolyase is 0.05-0.10. Whether this low quantum yield of repair by (6—4) photolyase is a result of the low efficiency of formation of the oxetane intermediate thermally, low efficiency of electron transfer from the flavin to the photoproduct, or low efficiency splitting of the oxetane anion coupled with high rate of back electron transfer is not known at present. Furthermore, it was found that (6-4) photolyase can photorepair the Dewar valence isomer of the (6-4) photoproduct (Taylor, 1994) that cannot form an oxetane intermediate, casting some doubt about the basic premise of the retro [2+2] reaction. However, the Dewar isomer is repaired with 300-400 lower quantum yield than the (6-4) photoproduct, and it has been proposed (Zhao et ai, 1997) that the Dewar isomer may be repaired by the enzyme through a two-photon reaction in which the first photon converts the Dewar isomer to the Kekule form and a second electron transfer reaction initiated by the second photon promotes the retro [2+2] reaction. 

Husain, I., and Sancar, A. (1987). Binding of E. coli DNA photolyase to defined substrate containing a single ToT dimer. Nucleic Adds Bes. 15, 1109—1120. 

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