DNA damaging agents such as ionizing irradiation induce lesions in the DNA such as double strand breaks (DSBs). Depending on cell type, 10-25% of these DSBs are induced in heterochromatin. Heterochromatic DSBs are resolved with slow kinetics (compared to DSBs in euchromatin) and require ATM activity for repair. Investigating the underlying causes of the slow component of DSB repair and the role of individual response factors in this process provides insight into DSB response pathways and will further the understanding of diseases where such pathways are dysfunctional due to mutation. Here, we describe a method to detect DSB repair foci in the heterochromatin of human cells. We provide a detailed protocol for cell culture preparation, immunofluorescence microscopy, and a computer-assisted approach to analyze overlap between DSB foci and heterochromatin.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1007/978-1-4939-6955-5_22 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
NGS Detects Extensive Genomic Alterations in Survivors of Irradiated Normal Human Fibroblast Cells.
Radiat Res
December 2024
Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Essen, Germany.
It is thought that cells surviving ionizing radiation exposure repair DNA double-strand breaks (DSBs) and restore their genomes. However, the recent biochemical and genetic characterization of DSB repair pathways reveals that only homologous recombination (HR) can function in an error-free manner and that the non-homologous end joining (NHEJ) pathways canonical NHEJ (c-NHEJ), alternative end joining (alt-EJ), and single-strand annealing (SSA) are error-prone, and potentially leave behind genomic scars and altered genomes. The strong cell cycle restriction of HR to S/G2 phases and the unparalleled efficiency of c-NHEJ throughout the cell cycle, raise the intriguing question as to how far a surviving cell "reaches" after repairing the genome back to its pre-irradiation state.
View Article and Find Full Text PDFRegulation of pathway choice in DNA repair after double-strand breaks.
Curr Opin Pharmacol
December 2024
Biotechnology Research and Innovation Council - National Institute of Immunology (BRIC-NII), Aruna Asaf Ali Marg, New Delhi 110067, India; Biotechnology Research and Innovation Council - National Institute of Biomedical Genomics (BRIC-NIBMG), Kalyani 741251, India. Electronic address:
DNA damage signaling is a highly coordinated cellular process which is required for the removal of DNA lesions. Amongst the different types of DNA damage, double-strand breaks (DSBs) are the most harmful type of lesion that attenuates cellular proliferation. DSBs are repaired by two major pathways-homologous recombination (HR), and non-homologous end-joining (NHEJ) and in some cases by microhomology-mediated end-joining (MMEJ).
View Article and Find Full Text PDFPrediction of key biological processes from intercellular DNA damage differences through model-based fitting.
iScience
December 2024
Division of Molecular Oncological Pharmacy, Faculty of Pharmacy, Keio University, 1-5-30, Shibakoen, Minato-ku, Tokyo 105-8512, Japan.
DNA double-strand breaks (DSBs) occurring within the genomic DNA of mammalian cells significantly impact cell survival, depending upon their repair capacity. This study presents a mathematical model to fit fibroblast survival rates with a sequence-specific DSB burden induced by the restriction enzyme AsiSI. When cells had a sporadic DSB burden under mixed culture, cell growth showed a good fit to the Lotka-Volterra competitive equation, predicting the presence of modifying factors acting as competitive cell-to-cell interactions compared to monocultures.
View Article and Find Full Text PDFThe Mre11 complex comprises Mre11, Rad50 and Nbs1 (Xrs2 in ). The core components, Mre11 and Rad50 are highly conserved, with readily identifiable orthologs in all clades of life, whereas Nbs1/Xrs2 are present only in eukaryotes. In eukaryotes, the complex is integral to the DNA damage response, acting in DNA double strand break (DSB) detection and repair, and the activation of DNA damage signaling.
View Article and Find Full Text PDF[How to Shift the Equilibrium of DNA Break Repair in Favor of Homologous Recombination].
Mol Biol (Mosk)
December 2024
Institute of Functional Genomics, Moscow State University, Moscow, 119991 Russia.
The CRISPR/Cas technology of targeted genome editing made it possible to carry out genetic engineering manipulations with eukaryotic genomes with a high efficiency. Targeted induction of site-specific DNA breaks is one of the key stages of the technology. The cell repairs the breaks via one of the two pathways, nonhomologous end joining (NHEJ) and homology-driven repair (HDR).
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!