Respective roles of cyclobutane pyrimidine dimers, (6-4)photoproducts, and minor photoproducts in ultraviolet mutagenesis of repair-deficient xeroderma pigmentosum A cells.

The role of UV light-induced photoproducts in initiating base substitution mutation in human cells was examined by determining the frequency and spectrum of mutation in a supF tRNA gene in a shuttle vector plasmid transfected into DNA repair deficient cells (xeroderma pigmentosum complementation group A). To compare the role of two major UV-induced photoproducts, cis-syn cyclobutane-type pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), each photoproduct was removed from UV-irradiated plasmid by photoreactivation before transfection. Removal of either CPDs or 6-4PPs by in vitro photoreactivation reduced the mutation frequency while keeping the mutation distribution and the predominance of G:C-A:T transitions as UV-irradiated plasmid without photoreactivation, indicating that both cytosine-containing CPDs and 6-4PPs were premutagenic lesions for G:C-A:T transitions. On the other hand, A:T-G:C transitions were not recovered from plasmids after the removal of 6-4PPs, whereas this type of mutation occurred at a significant level (11%) after the removal of CPDs. Thus, the premutagenic lesions for the A:T-G:C transition are 6-4PPs. Removal of both CPDs and 6-4PPs resulted in the disappearance of mutational hot spots and random distribution of mutation as observed in unirradiated control plasmids. However, the mutational spectrum of photoreactivated plasmids differed significantly from that of unirradiated plasmids. A characteristic feature is a high portion of A:T-T:A transversions (11%) in the photoreactivated plasmid. This mutation is due to nondipyrimidinic "minor" photoproducts, and the mutation spectrum suggests that TA*, the major photoproduct of thymidylyl-(3'-5')-deoxyadenosine, is the premutagenic lesion for this mutation. This is the first report revealing the distinct mutagenic roles of the major UV photoproducts and "minor" photoproducts by the use of (6-4)photolyase.

Cloning and characterization of a gene (UVR3) required for photorepair of 6-4 photoproducts in Arabidopsis thaliana.

UV radiation induces two major classes of pyrimidine dimers: the pyrimidine [6-4] pyrimidone photoproduct (6-4 product) and the cyclobutane pyrimidine dimer (CPD). Many organisms produce enzymes, termed photolyases, that specifically bind to these damage products and split them via a UV-A/blue light-dependent mechanism, thereby reversing the damage. These photolyases are specific for either CPDs or 6-4 products. A gene that expresses a protein with 6-4 photolyase activity in vitro was recently cloned from Drosophila melanogaster and Xenopus laevis. We report here the isolation of a homolog of this gene, cloned on the basis of sequence similarity, from the higher plant Arabidopsis thaliana. This cloned gene produces a protein with 6-4 photolyase activity when expressed in Escherichia coli. We also find that a previously described mutant of Arabidopsis (uvr3) that is defective in photoreactivation of 6-4 products carries a nonsense mutation in this 6-4 photolyase homolog. We have therefore termed this gene UVR3. Although homologs of this gene have previously been shown to produce a functional 6-4 photolyase when expressed in heterologous systems, this is the first demonstration of a requirement for this gene for photoreactivation of 6-4 products in vivo.

Characterization of a human homolog of (6-4) photolyase.

(6-4)Photolyase catalyzes light-dependent repair of UV-induced pyrimidine (6-4) pyrimidone photoproducts. A human cDNA clone which has high sequence homology to the (6-4)photolyase gene (H64PRH gene) was identified. In this paper we also isolated a genomic clone corresponding to the H64PRH cDNA and mapped it to chromosome 12q24.1 by fluorescence in situ hybridization (FISH). Northern-blot analysis revealed transcription of this gene in all human tissues examined. The H64PRH protein was overproduced in E. coli, partially purified and characterized. Like (6-4)photolyase, the enzyme contains two chromophores, one of which is FAD. However, the enzyme does not show any detectable photolyase activity.

Flavin adenine dinucleotide as a chromophore of the Xenopus (6-4)photolyase.

Two types of enzyme utilizing light from the blue and near-UV spectral range (320-520 nm) are known to have related primary structures: DNA photolyase, which repairs UV-induced DNA damage in a light-dependent manner, and the blue light photoreceptor of plants, which mediates light-dependent regulation of seedling development. Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)photoproducts] are the two major photoproducts produced in DNA by UV irradiation. Two types of photolyases have been identified, one specific for CPDs (CPD photolyase) and another specific for (6-4)photoproducts [(6-4)photolyase]. (6-4)Photolyase activity was first found in Drosophila melanogaster and to date this gene has been cloned only from this organism. The deduced amino acid sequence of the cloned gene shows that (6-4)photolyase is a member of the CPD photolyase/blue light photoreceptor family. Both CPD photolyase and blue light photoreceptor are flavoproteins and bound flavin adenine dinucleotides (FADs) are essential for their catalytic activity. Here we report isolation of a Xenopus laevis(6-4)photolyase gene and show that the (6-4)photolyase binds non- covalently to stoichiometric amounts of FAD. This is the first indication of FAD as the chromophore of (6-4)photolyase.

Binding and catalytic properties of Xenopus (6-4) photolyase.

Xenopus (6-4) photolyase binds with high affinity to DNA bearing a (6-4) photoproduct and repairs it in a light-dependent reaction. To clarify its repair mechanism of (6-4) photolyase, we determined its binding and catalytic properties using synthetic DNA substrate which carries a photoproduct at a single location. The (6-4) photolyase binds to T[6-4]T in double-stranded DNA with high affinity (KD = 10(-9)) and to T[6-4]T in single-stranded DNA and T[Dewar]T in double- and single-stranded DNA although with slightly lower affinity (KD = approximately 2 x 10(-8)). Majority of the T[6-4]T-(6-4) photolyase complex dissociates very slowly (koff = 2.9 x 10(-5) s-1). Its absolute action spectrum without a second chromophore in the 350-600 nm region closely matches the absorption spectrum of the enzyme. The quantum yield (phi) of repair is approximately 0.11. The fully reduced form (E-FADH-) of (6-4) photolyase is catalytically active. Direct analysis of the photoreactivated product showed that (6-4) photolyase restores the original pyrimidines. These findings demonstrate that cis, syn-cyclobutane pyrimidine dimer photolyase and (6-4) photolyase are quite similar, but they are different with regard to the binding properties.