Double-strand break repair and G2 block in Chinese hamster ovary cells and their radiosensitive mutants

Weibezahn, Karl-Friedrich; Lohrer, Horst D.; Herrlich, Peter

doi:10.1016/0167-8817(85)90025-2

Cited by 74 publications

(18 citation statements)

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“…Recently, it was shown that members of the fifth X-ray cross complementation group (XRCC5), which are defective in DNA DSB repair and V(D)J recombination, are mutated in the Ku86 gene (18, 21). Thus, DNA-PK has been unequivocally identified as an important mammalian DNA repair complex, and mutations in either DNA-PK cs or the 86-kDa subunit of Ku result in DNA DSB repair defects that manifest themselves as X-ray sensitivity and impaired V(D)J recombination.Interestingly, the survival of certain mammalian mutant cells sensitive to ionizing radiation has also been shown to depend upon cell cycle position at the time of X-ray exposure, with maximal sensitivity to irradiation occurring at the G 1 /S border (69,75,77). To see if this paradigm could be extended to DNA-PK in general and scid cells in particular, we set forth to test whether the activity of DNA-PK and the ionizing-radiation sensitivity of scid cells fluctuated during the cell cycle.…”

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“…Interestingly, the survival of certain mammalian mutant cells sensitive to ionizing radiation has also been shown to depend upon cell cycle position at the time of X-ray exposure, with maximal sensitivity to irradiation occurring at the G 1 /S border (69,75,77). To see if this paradigm could be extended to DNA-PK in general and scid cells in particular, we set forth to test whether the activity of DNA-PK and the ionizing-radiation sensitivity of scid cells fluctuated during the cell cycle.…”

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Evidence for DNA-PK-Dependent and -Independent DNA Double-Strand Break Repair Pathways in Mammalian Cells as a Function of the Cell Cycle

Lee

Mitchell

Cheng

et al. 1997

Molecular and Cellular Biology

204

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Mice homozygous for the scid (severe combined immune deficiency) mutation are defective in the repair of DNA double-strand breaks (DSBs) and are consequently very X-ray sensitive and defective in the lymphoid V(D)J recombination process. Recently, a strong candidate for the scid gene has been identified as the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex. Here, we show that the activity of the DNA-PK complex is regulated in a cell cycle-dependent manner, with peaks of activity found at the G 1 /early S phase and again at the G 2 phase in wild-type cells. Interestingly, only the deficit of the G 1 /early S phase DNA-PK activity correlated with an increased hypersensitivity to X-irradiation and a DNA DSB repair deficit in synchronized scid pre-B cells. Finally, we demonstrate that the DNA-PK activity found at the G 2 phase may be required for exit from a DNA damage-induced G 2 checkpoint arrest. These observations suggest the presence of two pathways (DNA-PK-dependent and -independent) of illegitimate mammalian DNA DSB repair and two distinct roles (DNA DSB repair and G 2 checkpoint traversal) for DNA-PK in the cellular response to ionizing radiation.Normal lymphoid development is marked by the somatic rearrangement of separate genetic elements to form functional immunoglobulin and T-cell receptor genes. This process, known as V(D)J recombination, is mediated by a site-specific DNA rearrangement mechanism that targets conserved signal sequences flanking the genetic elements (reviewed in reference 46). The initial steps of V(D)J recombination involve the recognition of the signal sequences and the introduction of DNA double-strand breaks (DSBs) adjacent to them. These steps are carried out by the products of two lymphoid-specific genes, RAG-1 and RAG-2 (20, 51).Several studies have suggested that V(D)J recombination events may be restricted during the cell cycle. Analysis of RAG-2 protein demonstrated that it accumulated in cells preferentially in the G 0 /G 1 phase of the cell cycle, declined more than 20-fold before entering the S phase, and remained low throughout the S, G 2 , and M phases (47). Interestingly, the levels of RAG-1 protein, which fluctuated to a much lesser extent, still declined fivefold in the S phase (48). Lastly, the double-strand signal sequence breaks associated with V(D)J recombination can be detected only in the G 0 /G 1 phase of the cell cycle (65). These data strongly imply that the initiation of V(D)J recombination is mostly, and perhaps entirely, restricted to the G 1 phase of cycling cells.While RAG-1 and RAG-2 control the initial steps of V(D)J recombination, the later steps appear to be controlled by genes involved in generalized DNA DSB repair. In particular, a complex, DNA-dependent protein kinase (DNA-PK), which has DNA-dependent serine-threonine protein kinase activity and which consists of at least three components, the 465-kDa catalytic subunit (DNA-PK cs ) and the heterodimeric Ku protein, has been shown to be intimately involved in generalized DNA D...

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confidence: 99%

mentioning

confidence: 99%

Evidence for DNA-PK-Dependent and -Independent DNA Double-Strand Break Repair Pathways in Mammalian Cells as a Function of the Cell Cycle

Lee

Mitchell

Cheng

et al. 1997

Molecular and Cellular Biology

204

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“…The xrs mutants were isolated from a Chinese hamster ovary (CHO) cell line by Jeggo and Kemp (10). These mutants lack the ability to recover from potentially lethal radiation damage (38) and show reduced repair of DNA double-strand breaks (11,43). The two mutants selected for this study, xrsl and xrs7, belong to the same complementation group (9) and differ only slightly in their responses to ionizing radiations (10,38), but show substantially different responses to other genotoxic agents (10).…”

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Gene recombination in X-ray-sensitive hamster cells.

Hamilton¹,

Thacker²

1987

Mol. Cell. Biol.

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Recombination was measured in Chinese hamster ovary (CHO-Kl) cells and in the X-ray-sensitive mutants xrsl and xrs7, which show a defect in DNA double-strand break repair. To assay recombination, pairs of derivatives of the plasmid pSV2gpt were constructed with nonoverlapping deletions in the gpt gene region and cotransferred into the dilTerent cell types. Recombination efficiencies, measured as the transformation frequency with a pair of deletion plasmids relative to that with the complete pSV2gpt plasmid, were about 6% in both CHO-Kl and the xrs mutants for plasmids linearized at a site outside the gpt gene. However, these efficiencies were substantially enhanced by the introduction of a double-strand break into the homologous region of the gpt gene in one of a pair of deletion plasmids before cotransfer. This enhancement was apparently only about half as great for the xrs cells as for CHO-Kl, but variation In microbes, a genetic link has been established between ionizing-radiation resistance and recombination proficiency; prominent examples of the genes involved are found in the RecF pathway of Escherichia coli (41) and in the RAD52 group of Saccharomyces cerevisiae (14). Additionally, mutation of such genes often affects the ability to repair DNA double-strand breaks. Thus, it has been hypothesised that double-strand breaks induced by ionizing radiation are rejoined by a process of recombination between homologous chromosomal regions (24).To investigate the relationship between ionizing-radiation resistance and recombination in mammalian cells, we have examined the recombination proficiencies of wild-type and X-ray-sensitive (xrs) hamster cells. The xrs mutants were isolated from a Chinese hamster ovary (CHO) cell line by Jeggo and Kemp (10). These mutants lack the ability to recover from potentially lethal radiation damage (38) and show reduced repair of DNA double-strand breaks (11, 43). The two mutants selected for this study, xrsl and xrs7, belong to the same complementation group (9) and differ only slightly in their responses to ionizing radiations (10, 38), but show substantially different responses to other genotoxic agents (10).The study of recombination in mammalian cells has been facilitated in the last few years by the use of virus-and plasmid-based gene transfer methods. It has been shown that mammalian cells have the enzymatic processes necessary for both homologous and nonhomologous recombination of introduced DNA (6,12,23,27,31,32,34,39,40,44). We have attempted to apply these methods quantitatively by carefully controlling DNA transfer conditions to determine plasmid recombination efficiencies in the parent and mutant CHO cells. Additionally we have followed up the observation, made initially for plasmid-chromosome recombination in S. cerevisiae (21), that a double-strand break in regions of * Corresponding author. homology greatly stimulates recombination. While our experiments were in progress on plasmid-plasmid recombination in CHO cells, it was reported that double-strand breaks are...

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“…Complementation of the Defect in dsb Rejoining. The radiation sensitivity of the IR complementation group 5 mutants is associated with a defect in dsb repair (10,17,18). To investigate complementation of this phenotype, one hybrid (XR-RA3-5 Hi) and the parental mutant and normal cell lines (XR-VJSB and V79) were compared for their ability to rejoin radiation-induced DNA dsb by pulsed-field gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

“…One complementation group includes six mutants isolated from the CHO cell line xrsl-6 and one mutant (XR-VJ5B) derived from V79 cells (16,17). All these mutants exhibit high sensitivity to IR, an impaired ability to rejoin DNA dsb but little or no sensitivity to UV irradiation (10,18). The repair gene defective in these mutants has been designated XRCCS and the complementation group has been designated IR complementation group 5.…”

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Localization of a DNA repair gene (XRCC5) involved in double-strand-break rejoining to human chromosome 2.

Jeggo

Hafezparast

Thompson

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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Complementation of the repair defect in hamster xrs mutants has been achieved by transfer of human chromosome 2 using the method of microcell-mediated chromosome transfer. The xrs mutants belong to ionizing radiation complementation group 5, are highly sensitive to ionizing radiation, and have an impaired ability to rejoin radiation-induced DNA double-strand breaks. Both phenotypes were corrected by chromosome 2, although the correction of radiation sensitivity was only partial. Complementation was achieved in two members of this complementation group, xrs6 and XR-V15B, derived independently from the CHO and V79 cell lines, respectively. The presence of human chromosome 2 in complemented clones was examined cytogenetically and by PCR analysis with primers directed at a human-specific long interspersed repetitive sequence or chromosome 2-specific genes. Complementation was observed in 25/27 hybrids, one of which contained only the q arm of chromosome 2. The two noncomplementing hybrids were missing segments of chromosome 2. The use of a back-selection system enabled the isolation of clones that had lost the human chromosome and these regained radiation sensitivity. Transfer of several other human chromosomes did not result in complementation of the repair defect in XR-V15B. These data show that the gene defective in xrs cells, XRCC5, which is involved in double-strand break rejoining, is located on human chromosome 2q.

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Double-strand break repair and G2 block in Chinese hamster ovary cells and their radiosensitive mutants

Cited by 74 publications

References 10 publications

Evidence for DNA-PK-Dependent and -Independent DNA Double-Strand Break Repair Pathways in Mammalian Cells as a Function of the Cell Cycle

Evidence for DNA-PK-Dependent and -Independent DNA Double-Strand Break Repair Pathways in Mammalian Cells as a Function of the Cell Cycle

Gene recombination in X-ray-sensitive hamster cells.

Localization of a DNA repair gene (XRCC5) involved in double-strand-break rejoining to human chromosome 2.

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