|Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
The human genome is under constant attack by (1) exogenous DNA damage such as double stranded breaks inflicted by ionizing irradiation, and (2) endogenous DNA damage originating in stalled or collapsed replication forks. It is still unknown how DNA lesions such as DNA double strand break are actually repaired in mammalian cells. The reason for this is lack of appropriate technology to induce DSB simultaneously at the defined chromosomal site. In yeast it is feasible to create a single chromosomal DSB by using expression of HO endonuclease under the regulation of strong inducible promoter.
In this study, we wished to develop such system using two technologies:(1) Recombinant endonuclease I-SceI tagged with membrane translocation sequence TAT, which is purifed from E.coli expression system. By adding this protein to culture medium, it is expected to translocate cell membrane and eventually be transported to the nucleus. (2) Stable expression of I-SceI fused with mutat
ed ligand-biding domain of estrogen receptor (Mer). In normal condition, this protein is maintained in cytoplasm, but upon addition of estrogen analog tamoxifen, the protein is expected to move rapidly into the nucleus. We used chicken DT40 cell line in which recombination substrate SCneo was knocked-in to OVA locus. The SCneo has 18-bp recognition sequence for I-SceI, and I-SceI induced DSB can be repaired through homologous recombination using upstream nonfunctional neo segment as a template, conversing the cell to reo-resistant.
(1) We purified TAT-I-SceI fusion protein from E.coli, however, it precipitated and probably because of this, it was nonfunctional, In our hands, we were not able to solve this problem.
(2) We prepared three kinds of constructs (Mer-I-SceI, Mer-I-EceI-Mer, I-SceI-Mer), and expressed each of them into DT40 harboring SCneo. We could show rapid translocation of the Mer-I-EceI-Mer protein from cytoplasm to nucleus using immunocytochemistry, however, appearance of neo-resistant colony was less than expected. In all of the fusion constructs the rate of HR repair (conversion to neo resistance) was low compared to transient transfection of I-SceI. Consistently, we were able to detect DSB by ligation-mediated PCR but not by Southern blotting.
We created a system that can induce chromosomal DSB by adding tamoxifen to culture media, however, its efficiency is not high enough to allow real time monitoring of DSB repair. Less