Hitoshi Ayaki

Similarity Among the Drosophila (6-4)Photolyase, a Human Photolyase Homolog, and the DNA Photolyase-Blue-Light Photoreceptor Family

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 was 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.

Correlation between DNA methylation and expression of O6-methylguanine-DNA methyltransferase gene in cultured human tumor cells.

Approximately 20% of human tumor cell strains are deficient in a DNA repair protein, O6-methylguanine-DNA methyltransferase (MGMT), and are called Mer- strains. In an attempt to determine the molecular basis for the extinction of MGMT expression in Mer- human cells, the distribution of DNA methylation sites in and around the exon sequences of the repair gene was compared in 6 Mer+ (repair-proficient) and 12 Mer- cell lines. Southern blot analysis of the genomic DNA digested with isoschizomeric restriction endonucleases MspI and HpaII to detect 5-methylcytosine in CCGG sequences indicated that the DNA of all the Mer+ cells but of none of the Mer- cells is heavily methylated in the exon-containing regions. The methylation pattern contradicts the general belief that inactive genes are hypermethylated compared to hypomethylation of transcriptionally active genes. It appears that the regulation of the MGMT gene in human cells is much more complex than simply dictated by its methylation level.

Expression of a mammalian DNA photolyase confers light-dependent repair activity and reduces mutations of UV-irradiated shuttle vectors in xeroderma pigmentosum cells

Photoreactivation is one of the DNA repair mechanisms to remove UV lesions from cellular DNA with a function of the DNA photolyase and visible light. Two types of photolyase specific for cyclobutane pyrimidine dimers (CPD) and for pyrimidine (6-4) pyrimidones (6-4PD) are found in nature, but neither is present in cells from placental mammals. To investigate the effect of the CPD-specific photolyase on killing and mutations induced by UV, we expressed a marsupial DNA photolyase in DNA repair-deficient group A xeroderma pigmentosum (XP-A) cells. Expression of the photolyase and visible light irradiation removed CPD from cellular DNA and elevated survival of the UV-irradiated XP-A cells, and also reduced mutation frequencies of UV-irradiated shuttle vector plasmids replicating in XP-A cells. The survival of UV-irradiated cells and mutation frequencies of UV-irradiated plasmids were not completely restored to the unirradiated levels by the removal of CPD. These results suggest that both CPD and other UV damage, probably 6-4PD, can lead to cell killing and mutations.