Abstract
The mouse carcinoma cell line SX10 is a hypersensitive mutant to x-rays and bleomycin. An earlier complementation test suggests that SX10 would belong to x-ray-cross complementing group (XRCC) 4. However, in this study, a human XRCC4 expression vector failed to complement the SX10 phenotype. Consistent with the previous report, SX10 showed the same level of DNA-dependent protein kinase activity as the wild-type SR-1. We isolated and analyzed hybrids between SX10 and human diploid fibroblast cells and found that human chromosome 13 conferred the x-ray resistance to the hybrids, suggesting that a candidate gene would be located on this chromosome. Polymerase chain reaction analysis with these hybrids and x-ray-resistant transformants obtained by introducing human chromosomes into SX10 indicated that the mutant was likely to be defective in DNA ligase IV. Sequence analysis of the DNA ligase IV gene confirmed that a defect in SX10 was attributed to a transition of G to A at nucleotide position 1413 of the gene, leading to an amino acid substitution from Trp at residue 471 to a stop codon. Revertant clones (Rev1-3) derived from SX10 showed a restored x-ray resistance; Rev1 reverted to the original nucleotide G at position 1413, whereas Rev2 and Rev3 to C. Transfection of a mouse DNA ligase IV cDNA vector into SX10 restored the resistance to both x-rays and bleomycin. SX10 showed a reduced frequency of chromosomal integration of transfected DNA, but the revertants restored the frequency found in the wild-type cells. These results suggest a possible involvement of DNA ligase IV in the integration event of foreign DNA as well as a crucial role in DNA double-strand break repair.
Highlights
DNA double-strand breaks (DSBs)1 are caused by ionizing radiation and DNA-damaging agents [1] or occur as intermediates in certain cellular processes such as V(D)J recombina
SX10 Is Likely to Differ from XRCC1–9 in the Complementation Group —Sato et al [25] reported that SX10 would belong to the same complementation group as that of the M10 mouse leukemia line, which is hypersensitive to x-rays [26]
We measured DNA-PK activity in whole cell extracts by pull-down assays [35], no difference was found between SR-1 and SX10 cells, indicating that SX10 would fall into a complementation group distinct from those of XRCC5–7
Summary
Cells and Culture Methods—Mouse mammary carcinoma FM3A cell lines, wild-type SR-1, mutant SX10, and its 6-thioguanine- and ouabain-resistant derivative SX10TOR [25] were used; cells were grown in suspension in ES medium (Nissui Seiyaku Co., Tokyo) [28] supplemented with 4% bovine calf serum (HyClone). The cells were harvested, plated, and grown in 0.15% agarose-containing growth medium with 500 ng/ml bleomycin. 10 g of genomic DNA purified from cells were digested with DdeI, electrophoresed in a 1.0% agarose gel, and transferred to a nylon membrane (Hybond-Nϩ, Amersham Pharmacia Biotech). The cells were harvested, replated, and cultured in 0.15% agarosecontaining growth medium with 0.8 mg/ml active Geneticin (G418, Life Technologies, Inc.) for about 2 weeks. 2 g of the plasmid DNA was transfected into 4 ϫ 106 cells by electroporation, and the cells were diluted appropriately in growth medium and cultured for 24 h. The cells were collected, diluted, and replated at 1–5 ϫ 105 and 102 cells/60-mm dish into 0.15% agarosecontaining growth medium with and without active G418 (0.9 mg/ml), respectively They were cultured for 14 –18 days at 37 °C. The number of resulting colonies was scored, and the integration frequency was calculated by dividing the number of G418r colonies with surviving cells
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