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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 |
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| 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.
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| 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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