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203
Pathways of DNA double-strand break repair during the mammalian cell cycle
- Mol. Cell. Biol
, 2003
"... Little is known about the quantitative contributions of nonhomologous end joining (NHEJ) and homologous recombination (HR) to DNA double-strand break (DSB) repair in different cell cycle phases after physiologically relevant doses of ionizing radiation. Using immunofluorescence detection of �-H2AX n ..."
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Little is known about the quantitative contributions of nonhomologous end joining (NHEJ) and homologous recombination (HR) to DNA double-strand break (DSB) repair in different cell cycle phases after physiologically relevant doses of ionizing radiation. Using immunofluorescence detection of �-H2AX nuclear foci as a novel approach for monitoring the repair of DSBs, we show here that NHEJ-defective hamster cells (CHO mutant V3 cells) have strongly reduced repair in all cell cycle phases after 1 Gy of irradiation. In contrast, HR-defective CHO irs1SF cells have a minor repair defect in G 1, greater impairment in S, and a substantial defect in late S/G 2. Furthermore, the radiosensitivity of irs1SF cells is slight in G 1 but dramatically higher in late S/G 2, while V3 cells show high sensitivity throughout the cell cycle. These findings show that NHEJ is important in all cell cycle phases, while HR is particularly important in late S/G 2, where both pathways contribute to repair and radioresistance. In contrast to DSBs produced by ionizing radiation, DSBs produced by the replication inhibitor aphidicolin are repaired entirely by HR. irs1SF, but not V3, cells show hypersensitivity to aphidicolin treatment. These data provide the first evaluation of the cell cycle-specific contributions of NHEJ and HR to the repair of radiation-induced versus replication-associated DSBs. DNA double-strand breaks (DSBs) are considered the most biologically damaging lesions produced by ionizing radiation
p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA doublestrand breaks
- J Cell Biol
, 2000
"... Abstract. p53 binding protein 1 (53BP1), a protein proposed to function as a transcriptional coactivator of the p53 tumor suppressor, has BRCT domains with high homology to the Saccharomyces cerevisiae Rad9p DNA damage checkpoint protein. To examine whether 53BP1 has a role in the cellular response ..."
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Cited by 85 (2 self)
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Abstract. p53 binding protein 1 (53BP1), a protein proposed to function as a transcriptional coactivator of the p53 tumor suppressor, has BRCT domains with high homology to the Saccharomyces cerevisiae Rad9p DNA damage checkpoint protein. To examine whether 53BP1 has a role in the cellular response to DNA damage, we probed its intracellular localization by immunofluorescence. In untreated primary cells and U2OS osteosarcoma cells, 53BP1 exhibited diffuse nuclear staining; whereas, within 5–15 min after exposure to ionizing radiation (IR), 53BP1 localized at discreet nuclear foci. We propose that these foci represent sites of processing of DNA double-strand breaks (DSBs), because they were induced by IR and chemicals that cause DSBs, but not by ultraviolet light; their peak number approximated the number of DSBs induced by IR and decreased over time with kinetics that parallel the rate of DNA repair; and they colocalized with IRinduced Mre11/NBS and �-H2AX foci, which have been previously shown to localize at sites of DSBs. Formation of 53BP1 foci after irradiation was not dependent on ataxia-telangiectasia mutated (ATM), Nijmegen breakage syndrome (NBS1), or wild-type p53. Thus, the fast kinetics of 53BP1 focus formation after irradiation and the lack of dependency on ATM and NBS1 suggest that 53BP1 functions early in the cellular response to DNA DSBs. Key words: 53BP1 • p53 • DNA damage • ionizing radiation • cancer
Phosphorylation and rapid relocalization of 53BP1 to nuclear foci upon DNA damage. Mol Cell Biol 2001;21:1719–29
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An improved zinc-finger nuclease architecture for highly specific genome editing.
- Nat. Biotechnol.
, 2007
"... Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalyti ..."
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Cited by 61 (4 self)
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Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents. Zinc-finger nucleases (ZFNs) are rapidly emerging as versatile reagents for gene modification. These hybrid restriction enzymes, which link the cleavage domain of FokI to a designed zinc-finger protein (ZFP) 1,2 , may be used to introduce a variety of custom alterations into the genomes of eukaryotic cells. Examples range from precise sequence edits 3-6 to the targeted integration of entire genes 7 . ZFNs initiate these events by introducing a double-strand break at the site chosen for modification. If an exogenous repair template is also supplied then sequence alterations encoded in this donor may be incorporated into the genome by homology-directed repair (HDR) 8 . Targeting of the cleavage event, which is central to ZFN specificity and versatility, is mediated by the ZFP domain. This DNA-binding domain has been characterized in great detail 9-11 and may be engineered to recognize a wide variety of chosen DNA sequences ZFNs may offer a general method for engineering the genomes of diverse species as the requisite DNA repair pathways are highly conserved 17 . ZFN-stimulated gene modification has been demonstrated in plants An important feature of the current ZFN architecture is that DNA cleavage requires nuclease dimerization. ZFNs, as well as wild-type FokI, interact through their cleavage domains and are inactive as monomers Although the requirement for dimerization opens up the possibility of restricting cleavage to very long and rare sequences, it also introduces a problem arising from the fact that protein-protein interactions mediated by the wild-type FokI cleavage domain are not themselves selective for the heterodimer species
DNA replication-dependent nuclear dynamics
- of the Mre11 complex. Mol Cancer Res 2003;1:207–18
"... The Mre11 complex undergoes dramatic relocalization in the nuclei of;-irradiated and replicating human cells. In this study, we examined Mre11 complex localization and chromatin association in synchronous cultures to examine the molecular determinants of relocalization. The data indicate that the co ..."
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Cited by 41 (1 self)
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The Mre11 complex undergoes dramatic relocalization in the nuclei of;-irradiated and replicating human cells. In this study, we examined Mre11 complex localization and chromatin association in synchronous cultures to examine the molecular determinants of relocalization. The data indicate that the complex is deposited on chromatin in an S phase-specific manner. Mre11 complex chromatin association in S phase was resistant to detergent extraction, in contrast to that in;-irradiated cells. The complex exhibits extensive colocalization with proliferating cell nuclear antigen throughout S phase, and chromatin loading is enhanced by replication fork stalling, suggesting that the replication fork is a site of Mre11 complex chromatin loading. This is supported by the observation that the complex localized to single-stranded DNA arising in hydroxyurea-treated cells. Although the Mre11 complex appears to function as a DNA damage sensor, limited colocalization with Brca1 or;-H2AX was observed, arguing that neither DNA damage nor;-H2AX is required for Mre11 complex chromatin loading. These data provide a potential molecular basis for promotion of sister chromatid association and recombination by the Mre11 complex as well as for ATM-Mre11 complex-dependent activation of cell cycle checkpoints.
A doublestrand break repair defect in ATM-deficient cells contributes to radiosensitivity. Cancer Res 64: 500–508
, 2004
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Cited by 28 (3 self)
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Phosphorylation of the Bloom’s syndrome helicase and its role in recovery from S-phase arrest
, 2004
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DJ: Cellular responses to DNA double-strand breaks after low-dose {gamma}-irradiation. Nucleic Acids Res 2009
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Werner protein protects nonproliferating cells from oxidative DNA damage
- Mol. Cell Biol
, 2005
"... This article cites 61 articles, 33 of which can be accessed free ..."
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Cited by 19 (0 self)
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This article cites 61 articles, 33 of which can be accessed free
Retention but not recruitment of Crb2 at double-strand breaks requires Rad1 and Rad3 complexes
- Mol. Cell. Biol
, 2003
"... The fission yeast checkpoint protein Crb2, related to budding yeast Rad9 and human 53BP1 and BRCA1, has been suggested to act as an adapter protein facilitating the phosphorylation of specific substrates by Rad3-Rad26 kinase. To further understand its role in checkpoint signaling, we examined its lo ..."
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Cited by 18 (7 self)
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The fission yeast checkpoint protein Crb2, related to budding yeast Rad9 and human 53BP1 and BRCA1, has been suggested to act as an adapter protein facilitating the phosphorylation of specific substrates by Rad3-Rad26 kinase. To further understand its role in checkpoint signaling, we examined its localization in live cells by using fluorescence microscopy. In response to DNA damage, Crb2 localizes to distinct nuclear foci, which represent sites of DNA double-strand breaks (DSBs). Crb2 colocalizes with Rad22 at persistent foci, suggesting that Crb2 is retained at sites of DNA damage during repair. Damage-induced Crb2 foci still form in cells defective in Rad1, Rad3, and Rad17 complexes, but these foci do not persist as long as in wild-type cells. Our results suggest that Crb2 functions at the sites of DNA damage, and its regulated persistent localization at damage sites may be involved in facilitating DNA repair and/or maintaining the checkpoint arrest while DNA repair is under way. Eukaryotic cells respond to DNA damage by activating checkpoint signaling pathways conserved from yeast to humans (26, 51). Current models of DNA damage checkpoint signaling envision distinct groups of sensor, adapter, and effector pro-teins acting in a sequential manner to effect checkpoint re-
