Abstract
It is important to elucidate the fate of DNA double strand breaks (DSBs) occurred spontaneously or induced by ionizing irradiation. The DSBs are usually repaired in mammalian cells through either of two pathways: end-joining (EJ) or homologous recombination (HR). Although the former is predominant in mammalian cells, the relative contribution of each pathway is unclear. We introduced the restriction enzyme site I-SceI into the thymidine kinase (tk) gene of human lymphoblastoid cell line TK6 and established a system to investigate the fate of DSBs quantitatively and qualitatively. TK6 is heterozygous (tk+/-) for a point mutation in exon 4. We produced TSCE5 and TSCER2 cell lines from TK6 by gene targeting. We inserted into both cell lines a 31 bp DNA fragment containing an 18 bp I-SceI site in intron 4 of the tk + allele and, in TSCER2, a point mutation in exon 5, making it a compound heterozygote (tk-/-). When a DSB at the I-SceI site is repaired by EJ in TSCE5, it causes a deletion, permitting the cell to be isolated as a TK-deficient mutant. In TSCER2, on the other hand, when the DSB is repaired by HR between tk alleles, a tk + allele is generated and
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Honma, M;
Wong, W;
Sakuraba, M;
Tadokoro, S;
Hayashi, M;
[1]
Izumi, M;
Hanaoka, F;
Yatagai, F
[2]
- National Institute of Health Sciences, (Japan)
- The Institute of Physical and Chemical Research, (Japan). RIKEN
Citation Formats
Honma, M, Wong, W, Sakuraba, M, Tadokoro, S, Hayashi, M, Izumi, M, Hanaoka, F, and Yatagai, F.
Repair of DNA double strand breaks in human cells induced by restriction enzyme I-SceI.
Australia: N. p.,
2003.
Web.
Honma, M, Wong, W, Sakuraba, M, Tadokoro, S, Hayashi, M, Izumi, M, Hanaoka, F, & Yatagai, F.
Repair of DNA double strand breaks in human cells induced by restriction enzyme I-SceI.
Australia.
Honma, M, Wong, W, Sakuraba, M, Tadokoro, S, Hayashi, M, Izumi, M, Hanaoka, F, and Yatagai, F.
2003.
"Repair of DNA double strand breaks in human cells induced by restriction enzyme I-SceI."
Australia.
@misc{etde_20485639,
title = {Repair of DNA double strand breaks in human cells induced by restriction enzyme I-SceI}
author = {Honma, M, Wong, W, Sakuraba, M, Tadokoro, S, Hayashi, M, Izumi, M, Hanaoka, F, and Yatagai, F}
abstractNote = {It is important to elucidate the fate of DNA double strand breaks (DSBs) occurred spontaneously or induced by ionizing irradiation. The DSBs are usually repaired in mammalian cells through either of two pathways: end-joining (EJ) or homologous recombination (HR). Although the former is predominant in mammalian cells, the relative contribution of each pathway is unclear. We introduced the restriction enzyme site I-SceI into the thymidine kinase (tk) gene of human lymphoblastoid cell line TK6 and established a system to investigate the fate of DSBs quantitatively and qualitatively. TK6 is heterozygous (tk+/-) for a point mutation in exon 4. We produced TSCE5 and TSCER2 cell lines from TK6 by gene targeting. We inserted into both cell lines a 31 bp DNA fragment containing an 18 bp I-SceI site in intron 4 of the tk + allele and, in TSCER2, a point mutation in exon 5, making it a compound heterozygote (tk-/-). When a DSB at the I-SceI site is repaired by EJ in TSCE5, it causes a deletion, permitting the cell to be isolated as a TK-deficient mutant. In TSCER2, on the other hand, when the DSB is repaired by HR between tk alleles, a tk + allele is generated and the phenotype reverts. With introduction of a I-SceI expression vector, mutants from TSCE5 and revertants from TSCER2 appeared at the frequency of 10 -4 and 10 -6 , respectively, suggesting that EJ contributed to the repair of DSBs 100 times more frequently than HR. Analysis of the tk gene revealed that EJ mainly caused small deletions with a 1 or 8 bp microhomology at the joint. Moreover, almost all revertants generated by HR were produced by gene conversion. This system can be considered as a model for repair of a single DSB induced by low-dose irradiation. We are also making an effort to use this system as a tool to investigate the factors associated with DSB repair in mammalian cells.}
place = {Australia}
year = {2003}
month = {Jul}
}
title = {Repair of DNA double strand breaks in human cells induced by restriction enzyme I-SceI}
author = {Honma, M, Wong, W, Sakuraba, M, Tadokoro, S, Hayashi, M, Izumi, M, Hanaoka, F, and Yatagai, F}
abstractNote = {It is important to elucidate the fate of DNA double strand breaks (DSBs) occurred spontaneously or induced by ionizing irradiation. The DSBs are usually repaired in mammalian cells through either of two pathways: end-joining (EJ) or homologous recombination (HR). Although the former is predominant in mammalian cells, the relative contribution of each pathway is unclear. We introduced the restriction enzyme site I-SceI into the thymidine kinase (tk) gene of human lymphoblastoid cell line TK6 and established a system to investigate the fate of DSBs quantitatively and qualitatively. TK6 is heterozygous (tk+/-) for a point mutation in exon 4. We produced TSCE5 and TSCER2 cell lines from TK6 by gene targeting. We inserted into both cell lines a 31 bp DNA fragment containing an 18 bp I-SceI site in intron 4 of the tk + allele and, in TSCER2, a point mutation in exon 5, making it a compound heterozygote (tk-/-). When a DSB at the I-SceI site is repaired by EJ in TSCE5, it causes a deletion, permitting the cell to be isolated as a TK-deficient mutant. In TSCER2, on the other hand, when the DSB is repaired by HR between tk alleles, a tk + allele is generated and the phenotype reverts. With introduction of a I-SceI expression vector, mutants from TSCE5 and revertants from TSCER2 appeared at the frequency of 10 -4 and 10 -6 , respectively, suggesting that EJ contributed to the repair of DSBs 100 times more frequently than HR. Analysis of the tk gene revealed that EJ mainly caused small deletions with a 1 or 8 bp microhomology at the joint. Moreover, almost all revertants generated by HR were produced by gene conversion. This system can be considered as a model for repair of a single DSB induced by low-dose irradiation. We are also making an effort to use this system as a tool to investigate the factors associated with DSB repair in mammalian cells.}
place = {Australia}
year = {2003}
month = {Jul}
}