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.
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References
Cau P, Navarro C, Harhouri K, Roll P, Sigaudy S, Kaspi E, Perrin S, De Sandre-Giovannoli A, Levy N (2014) Nuclear matrix, nuclear envelope and premature aging syndromes in a translational research perspective. Semin Cell Dev Biol doi: 10.1016/j.semcdb.2014.03.022. [Epub ahead of print]
Oberdoerffer P, Sinclair DA (2007) The role of nuclear architecture in genomic instability and ageing. Nat Rev Mol Cell Biol 8:692–702
Mekhail K, Moazed D (2010) The nuclear envelope in genome organization, expression and stability. Nat Rev Mol Cell Biol 11:317–328
Capell BC, Collins FS (2006) Human laminopathies: nuclei gone genetically awry. Nat Rev Genet 7:940–952
Liu B, Wang J, Chan KM, Tjia WM, Deng W, Guan X, Huang JD, Li KM, Chau PY, Chen DJ, Pei D, Pendas AM, Cadinanos J, Lopez-Otin C, Tse HF, Hutchison C, Chen J, Cao Y, Cheah KS, Tryggvason K, Zhou Z (2005) Genomic instability in laminopathy-based premature aging. Nat Med 11:780–785
Liu Y, Wang Y, Rusinol AE, Sinensky MS, Liu J, Shell SM, Zou Y (2008) Involvement of xeroderma pigmentosum group A (XPA) in progeria arising from defective maturation of prelamin A. FASEB J 22:603–611
Liu GH, Barkho BZ, Ruiz S, Diep D, Qu J, Yang SL, Panopoulos AD, Suzuki K, Kurian L, Walsh C, Thompson J, Boue S, Fung HL, Sancho-Martinez I, Zhang K, Yates J 3rd, Izpisua Belmonte JC (2011) Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome. Nature 472:221–225
Constantinescu D, Csoka AB, Navara CS, Schatten GP (2010) Defective DSB repair correlates with abnormal nuclear morphology and is improved with FTI treatment in Hutchinson-Gilford progeria syndrome fibroblasts. Exp Cell Res 316:2747–2759
Pegoraro G, Kubben N, Wickert U, Gohler H, Hoffmann K, Misteli T (2009) Ageing-related chromatin defects through loss of the NURD complex. Nat Cell Biol 11:1261–1267
Krishnan V, Chow MZ, Wang Z, Zhang L, Liu B, Liu X, Zhou Z (2011) Histone H4 lysine 16 hypoacetylation is associated with defective DNA repair and premature senescence in Zmpste24-deficient mice. Proc Natl Acad Sci U S A 108:12325–12330
Liu B, Wang Z, Zhang L, Ghosh S, Zheng H, Zhou Z (2013) Depleting the methyltransferase Suv39h1 improves DNA repair and extends lifespan in a progeria mouse model. Nat Commun 4:1868
Gonzalez-Suarez I, Gonzalo S (2010) Nurturing the genome: A-type lamins preserve genomic stability. Nucleus 1:129–135
Gonzalez-Suarez I, Redwood AB, Gonzalo S (2009) Loss of A-type lamins and genomic instability. Cell Cycle 8:3860–3865
Gonzalez-Suarez I, Redwood AB, Grotsky DA, Neumann MA, Cheng EH, Stewart CL, Dusso A, Gonzalo S (2011) A new pathway that regulates 53BP1 stability implicates cathepsin L and vitamin D in DNA repair. EMBO J 30:3383–3396
Gonzalez-Suarez I, Redwood AB, Perkins SM, Vermolen B, Lichtensztejin D, Grotsky DA, Morgado-Palacin L, Gapud EJ, Sleckman BP, Sullivan T, Sage J, Stewart CL, Mai S, Gonzalo S (2009) Novel roles for A-type lamins in telomere biology and the DNA damage response pathway. EMBO J 28:2414–2427
Redwood AB, Gonzalez-Suarez I, Gonzalo S (2011) Regulating the levels of key factors in cell cycle and DNA repair: new pathways revealed by lamins. Cell Cycle 10:3652–3657
Redwood AB, Perkins SM, Vanderwaal RP, Feng Z, Biehl KJ, Gonzalez-Suarez I, Morgado-Palacin L, Shi W, Sage J, Roti-Roti JL, Stewart CL, Zhang J, Gonzalo S (2011) A dual role for A-type lamins in DNA double-strand break repair. Cell Cycle 10:2549–2560
Poon SS, Martens UM, Ward RK, Lansdorp PM (1999) Telomere length measurements using digital fluorescence microscopy. Cytometry 36:267–278
Grotsky DA, Gonzalez-Suarez I, Novell A, Neumann MA, Yaddanapudi SC, Croke M, Martinez-Alonso M, Redwood AB, Ortega-Martinez S, Feng Z, Lerma E, Ramon Y Cajal T, Zhang J, Matias-Guiu X, Dusso A, Gonzalo S (2013) BRCA1 loss activates cathepsin L-mediated degradation of 53BP1 in breast cancer cells. J Cell Biol 200:187–202
Acknowledgment
This work was supported by NIGMS Grant RO1 GM094513-01, DOD BCRP Idea Award BC110089, and Presidential Research Award from St Louis University. R.K. is recipient of the William S. Sly Fellowship in Biomedical Sciences. The authors declare no conflict of interest.
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Gonzalo, S., Kreienkamp, R. (2016). Methods to Monitor DNA Repair Defects and Genomic Instability in the Context of a Disrupted Nuclear Lamina. In: Shackleton, S., Collas, P., Schirmer, E. (eds) The Nuclear Envelope. Methods in Molecular Biology, vol 1411. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3530-7_26
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DOI: https://doi.org/10.1007/978-1-4939-3530-7_26
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