Topological Analysis of γH2AX and MRE11 Clusters Detected by Localization Microscopy during X-ray-Induced DNA Double-Strand Break Repair

DNA double-strand breaks (DSBs), known as the most severe damage in chromatin, were induced in breast cancer cells and normal skin fibroblasts by 2 Gy ionizing photon radiation. In response to DSB induction, phosphorylation of the histone variant H2AX to γH2AX was observed in the form of foci visual...

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Autores principales:	Hannes Hahn, Charlotte Neitzel, Olga Kopečná, Dieter W. Heermann, Martin Falk, Michael Hausmann
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	DNA double-strand breaks topology of γH2AX clusters topology of MRE11 clusters persistent homology single-molecule localization microscopy ionizing photon irradiation Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282
Acceso en línea:	https://doaj.org/article/6e2f640b0aaf4e23ae74e77077dccded
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spelling	oai:doaj.org-article:6e2f640b0aaf4e23ae74e77077dccded2021-11-11T15:35:07ZTopological Analysis of γH2AX and MRE11 Clusters Detected by Localization Microscopy during X-ray-Induced DNA Double-Strand Break Repair10.3390/cancers132155612072-6694https://doaj.org/article/6e2f640b0aaf4e23ae74e77077dccded2021-11-01T00:00:00Zhttps://www.mdpi.com/2072-6694/13/21/5561https://doaj.org/toc/2072-6694DNA double-strand breaks (DSBs), known as the most severe damage in chromatin, were induced in breast cancer cells and normal skin fibroblasts by 2 Gy ionizing photon radiation. In response to DSB induction, phosphorylation of the histone variant H2AX to γH2AX was observed in the form of foci visualized by specific antibodies. By means of super-resolution single-molecule localization microscopy (SMLM), it has been recently shown in a first article about these data that these foci can be separated into clusters of about the same size (diameter ~400 nm). The number of clusters increased with the dose applied and decreased with the repair time. It has also been shown that during the repair period, antibody-labeled MRE11 clusters of about half of the γH2AX cluster diameter were formed inside several γH2AX clusters. MRE11 is part of the MRE11–RAD50–NBS1 (MRN) complex, which is known as a DNA strand resection and broken-end bridging component in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). This article is a follow-up of the former ones applying novel procedures of mathematics (topology) and similarity measurements on the data set: to obtain a measure for cluster shape and shape similarities, topological quantifications employing persistent homology were calculated and compared. In addition, based on our findings that γH2AX clusters associated with heterochromatin show a high degree of similarity independently of dose and repair time, these earlier published topological analyses and similarity calculations comparing repair foci within individual cells were extended by topological data averaging (2nd-generation heatmaps) over all cells analyzed at a given repair time point; thereby, the two dimensions (0 and 1) expressed by components and holes were studied separately. Finally, these mean value heatmaps were averaged, in addition. For γH2AX clusters, in both normal fibroblast and MCF-7 cancer cell lines, an increased similarity was found at early time points (up to 60 min) after irradiation for both components and holes of clusters. In contrast, for MRE11, the peak in similarity was found at later time points (2 h up to 48 h) after irradiation. In general, the normal fibroblasts showed quicker phosphorylation of H2AX and recruitment of MRE11 to γH2AX clusters compared to breast cancer cells and a shorter time interval of increased similarity for γH2AX clusters. γH2AX foci and randomly distributed MRE11 molecules naturally occurring in non-irradiated control cells did not show any significant topological similarity.Hannes HahnCharlotte NeitzelOlga KopečnáDieter W. HeermannMartin FalkMichael HausmannMDPI AGarticleDNA double-strand breakstopology of γH2AX clusterstopology of MRE11 clusterspersistent homologysingle-molecule localization microscopyionizing photon irradiationNeoplasms. Tumors. Oncology. Including cancer and carcinogensRC254-282ENCancers, Vol 13, Iss 5561, p 5561 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	DNA double-strand breaks topology of γH2AX clusters topology of MRE11 clusters persistent homology single-molecule localization microscopy ionizing photon irradiation Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282
spellingShingle	DNA double-strand breaks topology of γH2AX clusters topology of MRE11 clusters persistent homology single-molecule localization microscopy ionizing photon irradiation Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282 Hannes Hahn Charlotte Neitzel Olga Kopečná Dieter W. Heermann Martin Falk Michael Hausmann Topological Analysis of γH2AX and MRE11 Clusters Detected by Localization Microscopy during X-ray-Induced DNA Double-Strand Break Repair
description	DNA double-strand breaks (DSBs), known as the most severe damage in chromatin, were induced in breast cancer cells and normal skin fibroblasts by 2 Gy ionizing photon radiation. In response to DSB induction, phosphorylation of the histone variant H2AX to γH2AX was observed in the form of foci visualized by specific antibodies. By means of super-resolution single-molecule localization microscopy (SMLM), it has been recently shown in a first article about these data that these foci can be separated into clusters of about the same size (diameter ~400 nm). The number of clusters increased with the dose applied and decreased with the repair time. It has also been shown that during the repair period, antibody-labeled MRE11 clusters of about half of the γH2AX cluster diameter were formed inside several γH2AX clusters. MRE11 is part of the MRE11–RAD50–NBS1 (MRN) complex, which is known as a DNA strand resection and broken-end bridging component in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). This article is a follow-up of the former ones applying novel procedures of mathematics (topology) and similarity measurements on the data set: to obtain a measure for cluster shape and shape similarities, topological quantifications employing persistent homology were calculated and compared. In addition, based on our findings that γH2AX clusters associated with heterochromatin show a high degree of similarity independently of dose and repair time, these earlier published topological analyses and similarity calculations comparing repair foci within individual cells were extended by topological data averaging (2nd-generation heatmaps) over all cells analyzed at a given repair time point; thereby, the two dimensions (0 and 1) expressed by components and holes were studied separately. Finally, these mean value heatmaps were averaged, in addition. For γH2AX clusters, in both normal fibroblast and MCF-7 cancer cell lines, an increased similarity was found at early time points (up to 60 min) after irradiation for both components and holes of clusters. In contrast, for MRE11, the peak in similarity was found at later time points (2 h up to 48 h) after irradiation. In general, the normal fibroblasts showed quicker phosphorylation of H2AX and recruitment of MRE11 to γH2AX clusters compared to breast cancer cells and a shorter time interval of increased similarity for γH2AX clusters. γH2AX foci and randomly distributed MRE11 molecules naturally occurring in non-irradiated control cells did not show any significant topological similarity.
format	article
author	Hannes Hahn Charlotte Neitzel Olga Kopečná Dieter W. Heermann Martin Falk Michael Hausmann
author_facet	Hannes Hahn Charlotte Neitzel Olga Kopečná Dieter W. Heermann Martin Falk Michael Hausmann
author_sort	Hannes Hahn
title	Topological Analysis of γH2AX and MRE11 Clusters Detected by Localization Microscopy during X-ray-Induced DNA Double-Strand Break Repair
title_short	Topological Analysis of γH2AX and MRE11 Clusters Detected by Localization Microscopy during X-ray-Induced DNA Double-Strand Break Repair
title_full	Topological Analysis of γH2AX and MRE11 Clusters Detected by Localization Microscopy during X-ray-Induced DNA Double-Strand Break Repair
title_fullStr	Topological Analysis of γH2AX and MRE11 Clusters Detected by Localization Microscopy during X-ray-Induced DNA Double-Strand Break Repair
title_full_unstemmed	Topological Analysis of γH2AX and MRE11 Clusters Detected by Localization Microscopy during X-ray-Induced DNA Double-Strand Break Repair
title_sort	topological analysis of γh2ax and mre11 clusters detected by localization microscopy during x-ray-induced dna double-strand break repair
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/6e2f640b0aaf4e23ae74e77077dccded
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Topological Analysis of γH2AX and MRE11 Clusters Detected by Localization Microscopy during X-ray-Induced DNA Double-Strand Break Repair

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