Evidence for Dinucleotide Flipping by DNA Photolyase

Abstract

DNA photolyases repair pyrimidine dimers via a reaction in which light energy drives electron donation from a catalytic chromophore, FADH ؊ , to the dimer. The crystal structure of Escherichia coli photolyase suggested that the pyrimidine dimer is flipped out of the DNA helix and into a cavity that leads from the surface of the enzyme to FADH ؊ . We have tested this model using the Saccharomyces cerevisiae Phr1 photolyase which is >50% identical to E. coli photolyase over the region comprising the DNA binding domain. By using the bacterial photolyase as a starting point, we modeled the region encompassing amino acids 383-530 of the yeast enzyme. The model retained the cavity leading to FADH ؊ as well as the band of positive electrostatic potential which defines the DNA binding surface. We found that alanine substitution mutations at sites within the cavity reduced both substrate binding and discrimination, providing direct support for the dinucleotide flip model. The roles of three residues predicted to interact with DNA flanking the dimer were also tested. Arg 452 was found to be particularly critical to substrate binding, discrimination, and photolysis, suggesting a role in establishing or maintaining the dimer in the flipped state. A structural model for photolyase-dimer interaction is presented.Pyrimidine bases absorb strongly in the UV region and are highly susceptible to photochemical reactions that alter their structures. In DNA, cyclobutane pyrimidine dimers (CPDs) 1 and pyrimidine-pyrimidone (6-4) photoproducts are the most frequent and biologically significant products of these reactions. These lesions are lethal and mutagenic and must be repaired to ensure cell survival and genetic stability. DNA photolyases repair CPDs and (6-4) photoproducts via reactions in which near UV or visible light provides the energy for bond rearrangements which restore the pyrimidines to their undamaged state. Two types of DNA photolyases have been recognized with regard to substrate specificity as follows: cyclobutane dipyrimidine photolyases and (6-4) photolyases (see Ref. 1 for a recent review). Each type of enzyme recognizes and efficiently repairs a single type of damage (2, 3). The cyclobutane dipyrimidine photolyases, hereafter referred to as CPD photolyases, are the subject of this report.Understanding how the CPD photolyases efficiently repair pyrimidine dimers in DNA entails answering the following two questions: how do the enzymes recognize pyrimidine dimers specifically in the midst of a vast excess of nondamaged bases, and how do the enzymes catalyze dimer photolysis? Photolysis involves two noncovalently bound chromophores, reduced FAD and a second chromophore which, depending upon the source of the enzyme, is either folate or deazaflavin (4). Absorbance of a photon of photoreactivating light subsequent to substrate binding initiates electron donation by enzyme-bound FADH Ϫ to one of the bases in the dimer (4, 5). The photon may be absorbed either directly by FADH Ϫ or, more often, by the second chromophore w...

The Extent of DNA Deformation in DNA Photolyase– Substrate Complexes: A Solution State Fluorescence Study

“…It had been hypothesized that the CPD has to be flipped out of the double helix into the active site of PL for efficient electron transfer leading to repair (6). Many results support this hypothesis (6,9,25–31). A recent crystal structure (9) showed that PL binds a flipped CPD‐substrate analog leaving the opposing adenine bases unpaired.…”

Light‐induced reactions of Escherichia coli DNA photolyase monitored by Fourier transform infrared spectroscopy

“…5). If thymidine dimer repair occurs, the conformation of the backbone should, on the other hand, change while the enzyme–product complex decays (or the enzyme–substrate complex is formed), because the chemical environment of the backbone phosphate is altered: In an enzyme–DNA complex, electrostatic interaction of backbone phosphate and basic residues of the DNA photolyase significantly contribute to the DNA binding of the enzyme [41–44]. Therefore, an additional difference band at 1244/1224 cm −1 can be detected in Fig.…”

Mechanistic studies of the radical SAM enzyme spore photoproduct lyase (SPL)

“…80 For instance, the T<>T repair by photolyase as well as the DNA alkylation repair by iron-containing enzymes like AlkB are all conducted under this mechanism. 81–83 The phosphodiester linker in the dinucleotide SP is more likely to function as the conformational holder as that in T<>T, as reflected by the slightly decreased enzyme reaction rate using SP with a formacetal linker as the substrate 68 and the much reduced rate when the linkage is absent. 61,63–64 …”