Binding and repair of UV-damaged DNA by photolyase

Ultraviolet radiation, usually from the sun, can damage DNA severely enough to cause cancer. Although there are numerous and varied forms of UV damage to DNA, the predominant structural defects are cyclobutylpyrimidine dimers (CPDs). CPDs are generated by cross-linking adjacent pyrimidines to form a bridging cyclobutane ring. The resulting dimerized structure is not recognizable as a nucleotide by cellular proteins which are responsible for transcription and replication. A single CPD is sufficient to cause apoptosis, and several of them, if properly placed, can induce a cell to become cancerous. Fortunately there are mechanisms in place to repair the damage. One of these mechanisms involves DNA photolyase, which is a monomeric flavoprotein that repairs CPDs in situ by means of light-driven catalysis.; Our accomplishments fall into two broad categories of substrate binding and electron transport. We show that photolyase binds CPDs without conformational change to the enzyme, but with significant distortion of the DNA helix. The latter effect is known as “base flipping,” and had been postulated for some time but never previously observed. Another good example of getting in there and measuring it is the ultraviolet transient absorption experiment. By probing at the wavelength where the substrate absorbs when repaired but not when damaged, it was possible to measure what was happening to the DNA, instead of what was happening to the protein. This experiment resolved a decade of obscuring complications and revealed that previous estimates of the electron transfer rate were based on data that included multiple simultaneous processes. This same experiment revealed that reconstitution of the enzymes active oxidation state occurs via photoreduction by a secondary photon and not by direct back electron transfer; contravening 15 years of photolyase theory. Transient absorption experiments performed on oxidized photolyase raise the intriguing possibility that the enzyme may have evolved to use the dimer's dipolar electrostatic field to facilitate electron transport. Our measurement and modeling of the CPD's electric field on the absorption spectrum of FAD raises the possibility that this motif is widely employed, as many redox-active substrates have a measurable dipole moment.