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Professor TANAKA, Kiyoji, M.D., Ph.D. +81-6-6879-7971
Associate Prof. SAIJO, Masafumi, Ph.D. +81-6-6879-7974
Assistant Prof. HORIBATA, Katsuyoshi, Ph.D. +81-6-6879-7974
Assistant Prof. NARITA, Takashi, Ph.D. +81-6-6879-7974

FAX +81-6-6877-9136
Postal Mail Address Laboratories of Organismal Biosystems, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871 Japan

The aim of our research is to elucidate DNA repair network in mammals and molecular pathogenesis of genetic diseases that have a defect in DNA repair network. Particularly, we are interested in the analysis of molecular mechanisms of transcription-coupled DNA repair (TCR) which efficiently removes a variety of lesions from the transcribed strand of active genes, and of pathogenesis in TCR-deficient Cockayne syndrome and its related diseases with diverse clinical symptoms such as photosensitivity, severe mental retardation and developmental defects.
Fig. 1 Model of ranscription-coupled DNA repair    

1 Mechanism of transcription-coupled repair: Roles of Cockayne syndrome proteins

CSA and CSB proteins responsible for Cockayne syndrome are involved in TCR, but the exact functions of these proteins in TCR have been unknown. We found that CSA protein is rapidly translocated to nuclear matrix after UV irradiation depending on CSB function. On the other hand, CSA protein has five WD40 repeat motifs, suggesting that it is involved in protein complex formation. We therefore purify the CSA protein complex by epitope-tagged method and elucidate the molecular mechanisms of UV-induced translocation of CSA protein to nuclear matrix, detection of the damage in transcribed strand, modification of RNA polymerase II, signaling to checkpoint and recovery of RNA synthesis, that are relevant to TCR pathway, by analyzing structure and function of the CSA complex.

Fig. 2 Immunofluorescence staining of CSA protein in the CS3BESV (CS-A) cells expressing HA-tagged CSA cDNA.

(A) The cells were irradiated with 20 J/m2 of UV (+UV) or non-irradiated (-UV), and treated with the Triton-extraction buffer before fixation. The CSA protein was detected with anti-HA rat monoclonal antibody. DNA was shown by TO-PRO-3 staining. The UV-irradiated cells displayed staining of CSA protein that was resistant to the Triton-extraction, while the non-irradiated cells did not.
(B) The cells were irradiated with UV and treated with Triton-extraction buffer supplemented with or without 0.2 mg/ml DNase I before fixation. Nuclear pore complex (NPC) was detected with anti-NPC. These results indicate that the CSA protein is associated with nuclear matrix but not with DNA after UV-irradiation.

2 Mechanisms of transcription and transcription-coupled repair: Roles of XAB2 protein

We have identified XAB2 (XPA-binding protein 2) that interacts with XPA, a factor central to nucleotide excision repair and responsible for xeroderma pigmentosum group A. XAB2 interacts with CSA, CSB and RNA polymerase II as well as XPA. Furthermore, we found that XAB2 is involved in TCR and transcription. We examine molecular functions of XAB2 in transcription and TCR by means of in vitro transcription elongation system using template DNA with single lesion. On the other hand, DNA damage in the transcribed strand leads to a decreased fidelity of transcription. We examine the mechanism of decreased transcriptional fidelity and its biological significance.

3 Molecular cloning of a novel gene responsible for the disease with defect in transcription-coupled repair

We are trying to clone a gene responsible for human genetic disease with transcription-coupled repair deficiency by combined means of human genome data base and functional cloning.

4 Carcinogenesis and aging in model mice deficient in DNA repair network

We examine a pathogenesis of model mice with deficiency in DNA repair network. Particularly, we are interested in the analysis of the mechanism of singnaling of UV-induced DNA damage to cell cycle checkpoint/apoptosis in the course of transcription-coupled repair and of the consequences of its defect in carcinogenesis. We also examine the relationship between TCR-deficiency and transcription elongation and aging in the mice model.

Fig. 3 Histological abnormalities of cerebellum in XPA-/-CSB -/- mice.

Sagittal cerebellar sections of each genotype mice at P14 (A-D) and P20 (E, F). Some fissures become shallower in the XPA-/-CSB -/- mice cerebellum (D, black arrowheads), and the tenth lobule (white triangles) in the double knockout mice appears to be small compared with other genotypes. Scale bar: 1mm.

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