Shunichi Takeda

Homologous recombination and non‐homologous end‐joining pathways of DNA double‐strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells

Eukaryotic cells repair DNA double‐strand breaks (DSBs) by at least two pathways, homologous recombination (HR) and non‐homologous end‐joining (NHEJ). Rad54 participates in the first recombinational repair pathway while Ku proteins are involved in NHEJ. To investigate the distinctive as well as redundant roles of these two repair pathways, we analyzed the mutants RAD54−/−, KU70−/− and RAD54−/−/KU70−/−, generated from the chicken B‐cell line DT40. We found that the NHEJ pathway plays a dominant role in repairing γ‐radiation‐induced DSBs during G1–early S phase while recombinational repair is preferentially used in late S–G2 phase. RAD54−/−/KU70−/− cells were profoundly more sensitive to γ‐rays than either single mutant, indicating that the two repair pathways are complementary. Spontaneous chromosomal aberrations and cell death were observed in both RAD54−/− and RAD54−/−/KU70−/− cells, with RAD54−/−/KU70−/− cells exhibiting significantly higher levels of chromosomal aberrations than RAD54−/− cells. These observations provide the first genetic evidence that both repair pathways play a role in maintaining chromosomal DNA during the cell cycle.

Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death.

Yeast rad51 mutants are viable, but extremely sensitive to γ‐rays due to defective repair of double‐strand breaks. In contrast, disruption of the murine RAD51 homologue is lethal, indicating an essential role of Rad51 in vertebrate cells. We generated clones of the chicken B lymphocyte line DT40 carrying a human RAD51 transgene under the control of a repressible promoter and subsequently disrupted the endogenous RAD51 loci. Upon inhibition of the RAD51 transgene, Rad51− cells accumulated in the G2/M phase of the cell cycle before dying. Chromosome analysis revealed that most metaphase‐arrested Rad51− cells carried isochromatid‐type breaks. In conclusion, Rad51 fulfils an essential role in the repair of spontaneously occurring chromosome breaks in proliferating cells of higher eukaryotes.

Trapping of PARP1 and PARP2 by Clinical PARP Inhibitors

Small-molecule inhibitors of PARP are thought to mediate their antitumor effects as catalytic inhibitors that block repair of DNA single-strand breaks (SSB). However, the mechanism of action of PARP inhibitors with regard to their effects in cancer cells is not fully understood. In this study, we show that PARP inhibitors trap the PARP1 and PARP2 enzymes at damaged DNA. Trapped PARP-DNA complexes were more cytotoxic than unrepaired SSBs caused by PARP inactivation, arguing that PARP inhibitors act in part as poisons that trap PARP enzyme on DNA. Moreover, the potency in trapping PARP differed markedly among inhibitors with niraparib (MK-4827) > olaparib (AZD-2281) >> veliparib (ABT-888), a pattern not correlated with the catalytic inhibitory properties for each drug. We also analyzed repair pathways for PARP-DNA complexes using 30 genetically altered avian DT40 cell lines with preestablished deletions in specific DNA repair genes. This analysis revealed that, in addition to homologous recombination, postreplication repair, the Fanconi anemia pathway, polymerase β, and FEN1 are critical for repairing trapped PARP-DNA complexes. In summary, our study provides a new mechanistic foundation for the rational application of PARP inhibitors in cancer therapy.

Increased ratio of targeted to random integration after transfection of chicken B cell lines

Constructs of four different genetic loci were transfected into the avian leukosis virus-induced chicken B cell line DT40, which continues diversification of its rearranged light chain immunoglobulin gene by gene conversion. Analysis of stable transfectants revealed an unexpectedly high frequency of targeted integration into the homologous gene loci of DT40. Transcriptional activity of the target gene locus is not required, since a construct of the untranscribed ovalbumin gene also integrated predominantly by homologous recombination. A construct derived from the beta-actin locus was transfected into other chicken cell lines to determine the cell type specificity of the phenomenon. Targeted integration still occurred at high frequency in two other B cell lines that do not have the gene conversion activity. However, the ratios of targeted to random integration were reduced by at least one order of magnitude in three non-B cell lines.

Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs

ABSTRACT The Rad51 protein, a eukaryotic homologue of Escherichia coli RecA, plays a central role in both mitotic and meiotic homologous DNA recombination (HR) in Saccharomyces cerevisiae and is essential for the proliferation of vertebrate cells. Five vertebrate genes, RAD51B, -C, and -D and XRCC2 and -3, are implicated in HR on the basis of their sequence similarity to Rad51 (Rad51 paralogs). We generated mutants deficient in each of these proteins in the chicken B-lymphocyte DT40 cell line and report here the comparison of four new mutants and their complemented derivatives with our previously reported rad51b mutant. The Rad51 paralog mutations all impair HR, as measured by targeted integration and sister chromatid exchange. Remarkably, the mutant cell lines all exhibit very similar phenotypes: spontaneous chromosomal aberrations, high sensitivity to killing by cross-linking agents (mitomycin C and cisplatin), mild sensitivity to gamma rays, and significantly attenuated Rad51 focus formation during recombinational repair after exposure to gamma rays. Moreover, all mutants show partial correction of resistance to DNA damage by overexpression of human Rad51. We conclude that the Rad51 paralogs participate in repair as a functional unit that facilitates the action of Rad51 in HR.

Sister Chromatid Exchanges Are Mediated by Homologous Recombination in Vertebrate Cells

ABSTRACT Sister chromatid exchange (SCE) frequency is a commonly used index of chromosomal stability in response to environmental or genetic mutagens. However, the mechanism generating cytologically detectable SCEs and, therefore, their prognostic value for chromosomal stability in mitotic cells remain unclear. We examined the role of the highly conserved homologous recombination (HR) pathway in SCE by measuring SCE levels in HR-defective vertebrate cells. Spontaneous and mitomycin C-induced SCE levels were significantly reduced for chicken DT40 B cells lacking the key HR genes RAD51 and RAD54but not for nonhomologous DNA end-joining (NHEJ)-defectiveKU70−/− cells. As measured by targeted integration efficiency, reconstitution of HR activity by expression of a human RAD51 transgene restored SCE levels to normal, confirming that HR is the mechanism responsible for SCE. Our findings show that HR uses the nascent sister chromatid to repair potentially lethal DNA lesions accompanying replication, which might explain the lethality or tumorigenic potential associated with defects in HR or HR-associated proteins.

MHC Class II Molecules Are Not Required for Survival of Newly Generated CD4+ T Cells, but Affect Their Long-Term Life Span

We grafted fetal thymi from wild-type mice into immunodeficient RAG-2-/- or class II-/-RAG-2-/- (class II MHC-) recipients and followed the fate of naive CD4+ T cells derived from the grafts. In both types of recipients, newly generated CD4+ T cells proliferated to the same extent in the periphery and rapidly filled the empty T cell compartment. However, CD4+ T cells in class II- recipients gradually decreased in number over 6 months. These results show that interactions between the TCR and class II molecules are not required for newly generated CD4+ T cells to survive and proliferate, but are necessary to maintain the size of the peripheral T cell pool for extended periods.

DNA damage-dependent acetylation and ubiquitination of H2AX enhances chromatin dynamics

ABSTRACT Chromatin reorganization plays an important role in DNA repair, apoptosis, and cell cycle checkpoints. Among proteins involved in chromatin reorganization, TIP60 histone acetyltransferase has been shown to play a role in DNA repair and apoptosis. However, how TIP60 regulates chromatin reorganization in the response of human cells to DNA damage is largely unknown. Here, we show that ionizing irradiation induces TIP60 acetylation of histone H2AX, a variant form of H2A known to be phosphorylated following DNA damage. Furthermore, TIP60 regulates the ubiquitination of H2AX via the ubiquitin-conjugating enzyme UBC13, which is induced by DNA damage. This ubiquitination of H2AX requires its prior acetylation. We also demonstrate that acetylation-dependent ubiquitination by the TIP60-UBC13 complex leads to the release of H2AX from damaged chromatin. We conclude that the sequential acetylation and ubiquitination of H2AX by TIP60-UBC13 promote enhanced histone dynamics, which in turn stimulate a DNA damage response.

The controlling role of ATM in homologous recombinational repair of DNA damage

The human genetic disorder ataxia telangiectasia (A‐T), caused by mutation in the ATM gene, is characterized by chromosomal instability, radiosensitivity and defective cell cycle checkpoint activation. DNA double‐strand breaks (dsbs) persist in A‐T cells after irradiation, but the underlying defect is unclear. To investigate ATMs interactions with dsb repair pathways, we disrupted ATM along with other genes involved in the principal, complementary dsb repair pathways of homologous recombination (HR) or non‐homologous end‐joining (NHEJ) in chicken DT40 cells. ATM−/− cells show altered kinetics of radiation‐induced Rad51 and Rad54 focus formation. Ku70‐deficient (NHEJ−) ATM−/− chicken DT40 cells show radiosensitivity and high radiation‐induced chromosomal aberration frequencies, while Rad54‐defective (HR−) ATM−/− cells show only slightly elevated aberration levels after irradiation, placing ATM and HR on the same pathway. These results reveal that ATM defects impair HR‐mediated dsb repair and may link cell cycle checkpoints to HR activation.

Construction of chimaeric processed immunoglobulin genes containing mouse variable and human constant region sequences

The specificity of monoclonal antibodies provides a powerful diagnostic and therapeutic tool in investigating human neoplasia. Radiological scanning and immunotherapy with mouse tumour-specific monoclonal antibodies have been applied to patients with some success1–3, but a major problem is the neutralization of the mouse antibody induced by repeated administration of heterologous antibodies. To avoid or reduce such immune reactions, chimaeric immunoglobulins consisting of mouse variable (V) and human constant (C) regions can be synthesized. We have constructed a recombinant retrovirus DNA carrying genomic heavy-chain (H) variable–diversity joining (VH−D−JH) and Cγ1 genes from different species and show here that the chimaeric intervening sequences are spliced out precisely. This procedure provides a useful method to construct the chimaeric mouse–human immunoglobulin gene to be expressed in Escherichia coli, yeast and animal cells. Unexpectedly, a hidden splice donor site in the 5′-flanking region of a human VH gene is used in place of the donor site of the leader sequence exon, resulting in the formation of the V region without the leader sequence.