Ultraviolet light (UV)-induced DNA damage can be repaired by DNA photolyase in a light-dependent manner. Two types of photolyase are known, one specific for cyclobutane pyrimidine dimers (CPD photolyase) and another specific for pyrimidine (6-4) pyrimidone photoproducts[(6-4)photolyase]. In contrast to the CPD photolyase, which has been detected in a wide variety of organisms, the (6-4)photolyase has been found only in Drosophila melanogaster. In the present study a gene encoding the Drosophila(6-4)photolyase ws cloned, and the deduced amino acid sequence of the product was found to be similar to the CPD photolyase and to the blue-light photoreceptor of plants. A homolog of the Drosophila (6-4)photolyase gene was also cloned from human cells.
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 ؊ ) 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.The ultraviolet component of sunlight has mutagenic, carcinogenic, and lethal effects (1). The main products in DNA of UVA and UVB radiation are the cis,syn-cyclobutane pyrimidine dimer (CPD) 1 and pyrimidine-pyrimidone (6-4) photoproduct ((6-4) photoproduct) produced at the di-pyrimidine site on DNA, which comprise 70 -80 and 20 -30%, respectively, of the total photoproducts (2, 3). Cells protect themselves against these lesions by eliminating the photoproducts from their genomes by excision repair or by photoreactivation. The phenomenon of photoreactivation (the reduction of lethal and mutagenic effects of UV radiation by simultaneous or subsequent irradiation with near UV or visible light) has been identified in a variety of organisms, and the enzyme responsible has been designated photoreactivating enzyme (DNA photolyase) (4). DNA photolyase binds to the photoproducts on DNA in a lightindependent manner, then utilizes light energy to convert them to the original two pyrimidines, and dissociates from the repaired DNA. Two types of DNA photolyase are known, one specific for CPD (CPD photolyase) (5) and the other specific for (6-4) photoproduct ((6-4) photolyase) (6).Gene coding for CPD photolyase is widely distributed among species, being isolated from more than 16 organisms, and their enzymatic nature has been characterized in detail (4,5,7,8). CPD photolyase has stoichiometric amounts of two noncovalent chromophores. One of these is FADH Ϫ , and the other is either methenyltetrahydrofolate or 8-hydroxy-5-deazaflavin (5). The proposed repair mechanism is as follows. The enzyme binds the DNA substrate, the second chromophore of the bound enzyme absorbs a UV-visible photon, and by dipole-dipole interaction transfers energy to FADH Ϫ which, in turn, transfers an electron to the CPD in the DNA. The CPD splits and back electron transfer restores the dipyrimidine and the functional form of flavin ready for a new cycle of catalysis (5).(6-4) photolyase activity has been found in Drosophila melanogaster, Xenopus laevis, Crotalus atrox, and Arabidopsis thaliana (6, 9, 10), the gene having been isolated from Drosophila (11) and Xenopus (12). Its enzymatic properties and reaction mechanism have yet to be well characterized. Isolation of the gene showed that (6-4) photolyase has an amino acid sequence similar to CPD photolyase and that the purified protein has two chromo...
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.
We examined loss of heterozygosity at 13 loci on 5 chromosomes in hepatocellular carcinomas (HCCs) from 56 patients. In 42 of these cases, regenerative nodules of liver cirrhosis were also analyzed. High frequencies of allelic losses were detected on chromosomes 13q (47%), 16q (40%) and 17p (64%), whereas losses on chromosome 4p and 11p were observed in less than 22% of cases in HCCs. In contrast, LOH was not detected on any loci in cirrhotic nodules. On chromosome 13q, the common region of allelic loss was mapped to the region including the retinoblastoma (RB) locus, by using 8 polymorphic probes. Furthermore, one case with 13q loss had an interstitial deletion of the RB gene, indicating the involvement of inactivation of the RB gene in hepatotumorigenesis. Losses were associated with portal-vein thrombosis or intrahepatic metastasis, increased tumor size, a poorly differentiated phenotype and clinical stage. Losses occurring together on 13q, 16q and 17p were significantly higher in patients in clinical stage IV or histologically poorly differentiated tumors, suggesting that the accumulation of allelic loss occurs in advanced tumors and that patients with multiple allelic losses may have a worse prognosis than those with a single loss.
Successive loss of function of both alleles of the retinoblastoma susceptibility gene (RB) on human chromosome 13 seems to be critical in the development of retinoblastoma and osteosarcoma. In cases where the tumour is familial and susceptibility is inherited, a mutation in one of the alleles is carried in the germline. We have recently shown that cytogenetically visible germline mutations are usually in the paternally derived gene. Such a bias would not be expected for sporadic (non-familial) tumours, where both mutations occur in somatic tissue, but there has been some indication of a bias towards initial somatic mutation in the paternally derived gene on chromosome 11 in sporadic Wilms tumour. We have now examined 13 sporadic osteosarcomas and find evidence which indicates that in 12 cases the initial mutation was in the paternal gene, suggesting the involvement of germinal imprinting in producing the differential susceptibility of the two genes to mutation.
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