Investigation of the Cyclobutane Pyrimidine Dimer (CPD) Photolyase DNA Recognition Mechanism by NMR Analyses

Abstract

The cyclobutane pyrimidine dimer (CPD) is one of the major forms of DNA damage caused by irradiation with ultraviolet (UV) light. CPD photolyases recognize and repair UV-damaged DNA. The DNA recognition mechanism of the CPD photolyase has remained obscure because of a lack of structural information about DNA-CPD photolyase complexes. In order to elucidate the CPD photolyase DNA binding mode, we performed NMR analyses of the DNA-CPD photolyase complex. Based upon results from 31 P NMR measurements, in combination with site-directed mutagenesis, we have demonstrated the orientation of CPD-containing single-stranded DNA (ssDNA) on the CPD photolyase. In addition, chemical shift perturbation analyses, using stable isotope-labeled DNA, revealed that the CPD is buried in a cavity within CPD photolyase. Finally, NMR analyses of a double-stranded DNA (dsDNA)-CPD photolyase complex indicated that the CPD is flipped out of the dsDNA by the enzyme, to gain access to the active site.Irradiation of DNA with ultraviolet (UV) light produces various damaged bases, leading to cellular transformation and cell death (1-3). One of the major products formed by UV irradiation is the cyclobutane pyrimidine dimer (CPD) 1 (Fig. 1), from the photo [2 ϩ 2] cycloaddition of the 5,6-double bond of two adjacent pyrimidine nucleotides. Organisms have a variety of enzymes playing crucial roles in repair-systems for damaged DNA, including nucleoside excision repair systems and photoreactivation (4). CPD photolyases, which function as members of the DNA repair systems, restore the CPD to normal pyrimidines by photoreactivation. To elucidate the mechanism of DNA repair by CPD photolyase, various biological and spectroscopic experiments have been performed. This enzyme has a flavin adenine dinucleotide (FAD) as an essential cofactor (5).The FAD is excited by light, and then transfers one electron to the CPD bases (6 -8). After the electron transfer, the cyclobutane ring splits and then one electron is transferred back to the FAD (9, 10). In order to understand the mechanism based on structural analyses, the crystal structures of the CPD photolyases from Escherichia coli, Anacystis nidulans, and Thermus thermophilus have been solved without the substrates, i.e. CPD-containing DNAs (11-13). The x-ray studies revealed that the enzymes share a similar global fold, which consists of an ␣/␤ domain and a helical domain. The helical domain is composed of clusters I and II, and a cavity is formed between the clusters, where the FAD is deeply buried. It has been suggested that the cavity is used for the CPD binding, because the asymmetric polarity of the cavity fits well with that of the CPD (11-15). Based upon the crystal structures without the substrates, two research groups have proposed computer models of the DNA-CPD photolyase complex, in which the relative orientations of the DNA chain are different from each other (11-15). However, no crystallographic or NMR structure information on the complexes is presently available.Here, we report NMR analyses of...

NMR Study of Repair Mechanism of DNA Photolyase by FAD-induced Paramagnetic Relaxation Enhancement

“…3). In addition to the conformation of the CPD, the aromatic groups in the CPDbinding site affect the chemical shifts of the resonances from CPD (20). Therefore, the results suggest that the conformation of the CPD-binding site is not significantly perturbed by the oxidation state.…”

“…For further insight into the CPD-repair mechanism, we constructed a model of the photolyase-DNA complex based on the NMR analysis results, as follows: (i) the CPD base is 16 Å away from FAD; (ii) the CPD bases are closer to FAD than the deoxyribose; and (iii) the 5Ј and 3Ј sides of the DNA bind to clusters I and II of the enzyme, respectively (20) (Fig. 6).…”

“…Details of the purification procedure will be published elsewhere. DNA oligomers containing CPD were prepared as described previously (20).…”

“…Our previous NMR analysis revealed the orientation of a CPD-containing ssDNA on CPD photolyase (20). We also proposed that the CPD lesion is flipped out of the dsDNA in the CPD photolyase-dsDNA complex.…”

Functional motifs in the (6-4) photolyase crystal structure make a comparative framework for DNA repair photolyases and clock cryptochromes

“…In contrast, structures representative (Fig. S1) of plant-specific (32) and DASH CRYs (33), class I CPD PHRs (34)(35)(36)(37) ,and other DNA base repair enzymes (38) have aided functional investigations. In the (6-4) PHR/clock CRY (6-4/clock) cluster, sequence analyses alone have failed to distinguish protein functions, so 3D structural analyses are needed to advance understanding.…”