Human RAD18 is involved in S phase-specific single-strand break repair without PCNA monoubiquitination

Switching from a replicative to a translesion polymerase is an important step to further continue on replication at the site of DNA lesion. Recently, RAD18 (a ubiquitin ligase) was shown to monoubiquitinate proliferating cell nuclear antigen (PCNA) in cooperation with RAD6 (a ubiquitin-conjugating enzyme) at the replication-stalled sites, causing the polymerase switch. Analyzing RAD18-knockout (RAD18−/−) cells generated from human HCT116 cells, in addition to the polymerase switch, we found a new function of RAD18 for S phase-specific DNA single-strand break repair (SSBR). Unlike the case with polymerase switching, PCNA monoubiquitination was not necessary for the SSBR. When compared with wild-type HCT116 cells, RAD18−/− cells, defective in the repair of X-ray-induced chromosomal aberrations, were significantly hypersensitive to X-ray-irradiation and also to the topoisomerase I inhibitor camptothecin (CPT) capable of inducing single-strand breaks but were not so sensitive to the topoisomerase II inhibitor etoposide capable of inducing double-strand breaks. However, such hypersensitivity to CPT observed with RAD18−/− cells was limited to only the S phase due to the absence of the RAD18 S phase-specific function. Furthermore, the defective SSBR observed in S phase of RAD18−/− cells was also demonstrated by alkaline comet assay

Identification of the XPG region that causes the onset of Cockayne syndrome by using Xpg mutant mice

2nd UA-Japan DNA repair meetin

Generation of mismatch repair-profi cient cell lines from human HCT116 cells

We have used HCT116 as a parental cell line for targeted disruption of DNA repair related genes, because HCT116 has many advantages for gene-disruption studies (such as near dipoloid karyotype, stable chromosome number).However, HCT116 has a disadvantage that is the defect in mismatch repair (MMR) due to a point mutation (from serine [TCA] to stop [TAA] at amino acid residue 252) in both allele of one of MMR genes, hMLH1. In order to rescue the MMR-defect in HCT116 cells, we tried to replace the mutated portion of hMLH1 gene with normal hMLH1 fragment using the gene-replacement vector constructed with the normal hMLH1 fragment. The vector also has neo gene as a dominant selective marker. After introducing the vector DNA into HCT116 cells by electroporation, we selected G418-resistant clones. Out of these clones we selected the clones in which mutated portion of hMLH1 were replaced with normal sequence examining by PCR and Southern blotting.Furthermore normal size of hMLH1 protein was detected in these clones.Because MMR-deficient cells have been reported to show 10 times more resistance to the drug, 6-thioguanine (6TG), and also show more than 100 times higher spontaneous mutation frequency, we examined the hMLH1-proficient cells for the sensitivity to 6TG and spontaneous mutation frequencies at HGPRT locus. These cells showed normal sensitivity to 6TG and normal levels of spontaneous mutation frequency indicating that these cells were normal in MMR function.The 32nd Annual Meeting of the Molecular Biology Society of Japa

Generation and characterization of DNA double-strand break repair gene deficient human cell lines

DNA double strand breaks (DSBs) can arise from multiple sources including ionizing radiation (IR), and is the most serious DNA damage. Non-homologous end joining (NHEJ), which simply pieces together the broken DNA ends, can function in all phases of the cell cycle and is the predominant repair pathway in mammalian cells. In the current study, we carried out the generation and characterization of the NHEJ-related gene deficient human cell lines to clarify the biological role of NHEJ-related genes on DNA damage induced by IR. We have produced cells that bearing a disrupted NHEJ-related gene, such as XRCC4, Artemis and MDC1, by using gene-targeting technique in human colon tumor cell line (HCT116). For clonogenic survival experiments, cells were exposed to X-rays (~ 5 Gy), plated for colony formation assay immediately after irradiation, and processed for a visualization and count of colonies 2 weeks after plating. Enumerating discrete nuclear foci of g-H2AX visible by immunofluorescence was performed to evaluate the ability for repairing DSBs induced by IR (~ 2 Gy) in both wild and deficient cells. Proliferation rates were slightly slower in all deficient cell lines than that in wild type cells, although any morphological difference was not observed between the cell lines. The highest survival rate was exhibited in the wild type cells (D10 = 3.9 Gy) and the lowest was in XRCC4-/- cells (D10 = 1.2 Gy). Artemis-/- cells (D10 = 2.2 Gy) took a middle position between wild and XRCC4-/- cells. Formation of g-H2AX foci increased in a dose dependent manner of X-rays and peaked at 30 min after X-ray-exposure in all cell lines. A remarkable recovery from the DNA damage was observed in wild type cells and the number of g-H2AX foci returned to the basal level within 4 hr, whereas a slower disappearance of g-H2AX was shown in NHEJ-related gene deficient cells such as XRCC4-/- and MDC1-/- cells, indicating a delay of repairing DSBs induced by IR. These results suggest that deficient of NHEJ-related gene causes a deterioration of DNA DSB repair function and DNA repair deficient underlies a significant component of the radiosensitivity of these cells. NHEJ-related gene deficient human cell lines generated in this study could contribute to further development and understanding of basic research of DNA damage and repair in radiation-biology.The 13th International Congress of Radiation Researc