title: Methods to Monitor DNA Repair Defects and Genomic Instability in the Context of a Disrupted Nuclear Lamina.
abstract: The organization of the genome within the nuclear space is viewed as an additional level of regulation of genome function, as well as a means to ensure genome integrity. Structural proteins associated with the nuclear envelope, in particular lamins (A- and B-type) and lamin-associated proteins, play an important role in genome organization. Interestingly, there is a whole body of evidence that links disruptions of the nuclear lamina with DNA repair defects and genomic instability. Here, we describe a few standard techniques that have been successfully utilized to identify mechanisms behind DNA repair defects and genomic instability in cells with an altered nuclear lamina. In particular, we describe protocols to monitor changes in the expression of DNA repair factors (Western blot) and their recruitment to sites of DNA damage (immunofluorescence); kinetics of DNA double-strand break repair after ionizing radiation (neutral comet assays); frequency of chromosomal aberrations (FISH, fluorescence in situ hybridization); and alterations in telomere homeostasis (Quantitative-FISH). These techniques have allowed us to shed some light onto molecular mechanisms by which alterations in A-type lamins induce genomic instability, which could contribute to the pathophysiology of aging and aging-related diseases.
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, St. Louis, MO, 63104, USA. sgonzalo@slu.edu.
authors.forename: Susana
authors.initials: S
authors.lastname: Gonzalo
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, St. Louis, MO, 63104, USA.
authors.forename: Ray
authors.initials: R
authors.lastname: Kreienkamp
mesh_terms: D015153:Blotting, Western; D020552:Comet Assay; D004249:DNA Damage; D004260:DNA Repair; D042822:Genomic Instability; D023281:Genomics / Q000379:methods; D017404:In Situ Hybridization, Fluorescence; D008856:Microscopy, Fluorescence; D034881:Nuclear Lamina / Q000378:metabolism*
keywords: DNA damage response; DNA repair; Genomic instability; Immunofluorescence; Neutral comet assay; Nuclear lamina; Q-FISH; Western blot