Spatiotemporal dynamics of early DNA damage response proteins on complex DNA lesions
The response of cells to ionizing radiation-induced DNA double-strand breaks (DSB) is determined by the activation of multiple pathways aimed at repairing the injury and maintaining genomic integrity. Densely ionizing radiation induces complex damage consisting of different types of DNA lesions in c...
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| Veröffentlicht in: | PloS one 2013-02, Vol.8 (2), p.e57953 |
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| description | The response of cells to ionizing radiation-induced DNA double-strand breaks (DSB) is determined by the activation of multiple pathways aimed at repairing the injury and maintaining genomic integrity. Densely ionizing radiation induces complex damage consisting of different types of DNA lesions in close proximity that are difficult to repair and may promote carcinogenesis. Little is known about the dynamic behavior of repair proteins on complex lesions. In this study we use live-cell imaging for the spatio-temporal characterization of early protein interactions at damage sites of increasing complexity. Beamline microscopy was used to image living cells expressing fluorescently-tagged proteins during and immediately after charged particle irradiation to reveal protein accumulation at damaged sites in real time. Information on the mobility and binding rates of the recruited proteins was obtained from fluorescence recovery after photobleaching (FRAP). Recruitment of the DNA damage sensor protein NBS1 accelerates with increasing lesion density and saturates at very high damage levels. FRAP measurements revealed two different binding modalities of NBS1 to damage sites and a direct impact of lesion complexity on the binding. Faster recruitment with increasing lesion complexity was also observed for the mediator MDC1, but mobility was limited at very high damage densities due to nuclear-wide binding. We constructed a minimal computer model of the initial response to DSB based on known protein interactions only. By fitting all measured data using the same set of parameters, we can reproduce the experimentally characterized steps of the DNA damage response over a wide range of damage densities. The model suggests that the influence of increasing lesion density accelerating NBS1 recruitment is only dependent on the different binding modes of NBS1, directly to DSB and to the surrounding chromatin via MDC1. This elucidates an impact of damage clustering on repair without the need of invoking extra processing steps. |
| doi_str_mv | 10.1371/journal.pone.0057953 |
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Densely ionizing radiation induces complex damage consisting of different types of DNA lesions in close proximity that are difficult to repair and may promote carcinogenesis. Little is known about the dynamic behavior of repair proteins on complex lesions. In this study we use live-cell imaging for the spatio-temporal characterization of early protein interactions at damage sites of increasing complexity. Beamline microscopy was used to image living cells expressing fluorescently-tagged proteins during and immediately after charged particle irradiation to reveal protein accumulation at damaged sites in real time. Information on the mobility and binding rates of the recruited proteins was obtained from fluorescence recovery after photobleaching (FRAP). Recruitment of the DNA damage sensor protein NBS1 accelerates with increasing lesion density and saturates at very high damage levels. FRAP measurements revealed two different binding modalities of NBS1 to damage sites and a direct impact of lesion complexity on the binding. Faster recruitment with increasing lesion complexity was also observed for the mediator MDC1, but mobility was limited at very high damage densities due to nuclear-wide binding. We constructed a minimal computer model of the initial response to DSB based on known protein interactions only. By fitting all measured data using the same set of parameters, we can reproduce the experimentally characterized steps of the DNA damage response over a wide range of damage densities. The model suggests that the influence of increasing lesion density accelerating NBS1 recruitment is only dependent on the different binding modes of NBS1, directly to DSB and to the surrounding chromatin via MDC1. This elucidates an impact of damage clustering on repair without the need of invoking extra processing steps.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0057953</identifier><identifier>PMID: 23469115</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Binding ; Biology ; Carcinogenesis ; Carcinogens ; Cell cycle ; Cell Cycle Proteins - metabolism ; Cell Line, Tumor ; Charged particles ; Chromatin ; Chromatin - genetics ; Chromatin - metabolism ; Clustering ; Complexity ; Damage accumulation ; Deoxyribonucleic acid ; DNA ; DNA Damage ; DNA Repair ; Double-strand break repair ; Fluorescence ; Fluorescence recovery after photobleaching ; Humans ; Impact damage ; Ionizing radiation ; Irradiation ; Kinases ; Lesions ; Linear Energy Transfer ; Maintenance ; Microscopy ; Mobility ; Models, Biological ; Nuclear Proteins - metabolism ; Phosphorylation ; Photobleaching ; Physics ; Protein interaction ; Protein Transport ; Proteins ; Radiation ; Radiation damage ; Radiation effects ; Radiation injuries ; Recovery (Medical) ; Recruitment ; Repair ; Spatio-Temporal Analysis ; Trans-Activators - metabolism</subject><ispartof>PloS one, 2013-02, Vol.8 (2), p.e57953</ispartof><rights>2013 Tobias et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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Densely ionizing radiation induces complex damage consisting of different types of DNA lesions in close proximity that are difficult to repair and may promote carcinogenesis. Little is known about the dynamic behavior of repair proteins on complex lesions. In this study we use live-cell imaging for the spatio-temporal characterization of early protein interactions at damage sites of increasing complexity. Beamline microscopy was used to image living cells expressing fluorescently-tagged proteins during and immediately after charged particle irradiation to reveal protein accumulation at damaged sites in real time. Information on the mobility and binding rates of the recruited proteins was obtained from fluorescence recovery after photobleaching (FRAP). Recruitment of the DNA damage sensor protein NBS1 accelerates with increasing lesion density and saturates at very high damage levels. 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This elucidates an impact of damage clustering on repair without the need of invoking extra processing steps.</description><subject>Binding</subject><subject>Biology</subject><subject>Carcinogenesis</subject><subject>Carcinogens</subject><subject>Cell cycle</subject><subject>Cell Cycle Proteins - metabolism</subject><subject>Cell Line, Tumor</subject><subject>Charged particles</subject><subject>Chromatin</subject><subject>Chromatin - genetics</subject><subject>Chromatin - metabolism</subject><subject>Clustering</subject><subject>Complexity</subject><subject>Damage accumulation</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA Damage</subject><subject>DNA Repair</subject><subject>Double-strand break repair</subject><subject>Fluorescence</subject><subject>Fluorescence recovery after photobleaching</subject><subject>Humans</subject><subject>Impact damage</subject><subject>Ionizing radiation</subject><subject>Irradiation</subject><subject>Kinases</subject><subject>Lesions</subject><subject>Linear Energy Transfer</subject><subject>Maintenance</subject><subject>Microscopy</subject><subject>Mobility</subject><subject>Models, Biological</subject><subject>Nuclear Proteins - 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metabolism</topic><topic>Cell Line, Tumor</topic><topic>Charged particles</topic><topic>Chromatin</topic><topic>Chromatin - genetics</topic><topic>Chromatin - metabolism</topic><topic>Clustering</topic><topic>Complexity</topic><topic>Damage accumulation</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA Damage</topic><topic>DNA Repair</topic><topic>Double-strand break repair</topic><topic>Fluorescence</topic><topic>Fluorescence recovery after photobleaching</topic><topic>Humans</topic><topic>Impact damage</topic><topic>Ionizing radiation</topic><topic>Irradiation</topic><topic>Kinases</topic><topic>Lesions</topic><topic>Linear Energy Transfer</topic><topic>Maintenance</topic><topic>Microscopy</topic><topic>Mobility</topic><topic>Models, Biological</topic><topic>Nuclear Proteins - metabolism</topic><topic>Phosphorylation</topic><topic>Photobleaching</topic><topic>Physics</topic><topic>Protein interaction</topic><topic>Protein Transport</topic><topic>Proteins</topic><topic>Radiation</topic><topic>Radiation damage</topic><topic>Radiation effects</topic><topic>Radiation injuries</topic><topic>Recovery (Medical)</topic><topic>Recruitment</topic><topic>Repair</topic><topic>Spatio-Temporal Analysis</topic><topic>Trans-Activators - 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Densely ionizing radiation induces complex damage consisting of different types of DNA lesions in close proximity that are difficult to repair and may promote carcinogenesis. Little is known about the dynamic behavior of repair proteins on complex lesions. In this study we use live-cell imaging for the spatio-temporal characterization of early protein interactions at damage sites of increasing complexity. Beamline microscopy was used to image living cells expressing fluorescently-tagged proteins during and immediately after charged particle irradiation to reveal protein accumulation at damaged sites in real time. Information on the mobility and binding rates of the recruited proteins was obtained from fluorescence recovery after photobleaching (FRAP). Recruitment of the DNA damage sensor protein NBS1 accelerates with increasing lesion density and saturates at very high damage levels. FRAP measurements revealed two different binding modalities of NBS1 to damage sites and a direct impact of lesion complexity on the binding. Faster recruitment with increasing lesion complexity was also observed for the mediator MDC1, but mobility was limited at very high damage densities due to nuclear-wide binding. We constructed a minimal computer model of the initial response to DSB based on known protein interactions only. By fitting all measured data using the same set of parameters, we can reproduce the experimentally characterized steps of the DNA damage response over a wide range of damage densities. The model suggests that the influence of increasing lesion density accelerating NBS1 recruitment is only dependent on the different binding modes of NBS1, directly to DSB and to the surrounding chromatin via MDC1. This elucidates an impact of damage clustering on repair without the need of invoking extra processing steps.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>23469115</pmid><doi>10.1371/journal.pone.0057953</doi><oa>free_for_read</oa></addata></record> |
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| subjects | Binding Biology Carcinogenesis Carcinogens Cell cycle Cell Cycle Proteins - metabolism Cell Line, Tumor Charged particles Chromatin Chromatin - genetics Chromatin - metabolism Clustering Complexity Damage accumulation Deoxyribonucleic acid DNA DNA Damage DNA Repair Double-strand break repair Fluorescence Fluorescence recovery after photobleaching Humans Impact damage Ionizing radiation Irradiation Kinases Lesions Linear Energy Transfer Maintenance Microscopy Mobility Models, Biological Nuclear Proteins - metabolism Phosphorylation Photobleaching Physics Protein interaction Protein Transport Proteins Radiation Radiation damage Radiation effects Radiation injuries Recovery (Medical) Recruitment Repair Spatio-Temporal Analysis Trans-Activators - metabolism |
| title | Spatiotemporal dynamics of early DNA damage response proteins on complex DNA lesions |
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