Abstract
Fluorescence microscopy of the DNA damage response in living cells stands out from many other DNA repair assays by its ability to monitor the response to individual DNA lesions in single cells. This is particularly true in yeast, where the frequency of spontaneous DNA lesions is relatively low compared to organisms with much larger genomes such as mammalian cells. Single cell analysis of individual DNA lesions allows specific events in the DNA damage response to be correlated with cell morphology, cell cycle phase, and other specific characteristics of a particular cell. Moreover, fluorescence live cell imaging allows for multiple cellular markers to be monitored over several hours. This chapter reviews useful fluorescent markers and genotoxic agents for studying the DNA damage response in living cells and provides protocols for live cell imaging, time-lapse microscopy, and for induction of site-specific DNA lesions.
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References
Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909
Remington SJ (2006) Fluorescent proteins: maturation, photochemistry and photophysics. Curr Opin Struct Biol 16:714–721
Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273:1392–1395
Heim R, Tsien RY (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6:178–182
Keppler-Ross S, Noffz C, Dean N (2008) A new purple fluorescent color marker for genetic studies in Saccharomyces cerevisiae and Candida albicans. Genetics 179:705–710
Lisby M, Rothstein R, Mortensen UH (2001) Rad52 forms DNA repair and recombination centers during S phase. Proc Natl Acad Sci U S A 98:8276–8282
Alvaro D, Sunjevaric I, Reid RJ, Lisby M, Stillman DJ, Rothstein R (2006) Systematic hybrid LOH: a new method to reduce false positives and negatives during screening of yeast gene deletion libraries. Yeast 23:1097–1106
Torres-Rosell J, Sunjevaric I, De Piccoli G, Sacher M, Eckert-Boulet N, Reid R, Jentsch S, Rothstein R, Aragon L, Lisby M (2007) The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus. Nat Cell Biol 9:923–931
Andreson BL, Gupta A, Georgieva BP, Rothstein R (2010) The ribonucleotide reductase inhibitor, Sml1, is sequentially phosphorylated, ubiquitylated and degraded in response to DNA damage. Nucleic Acids Res 38:6490–6501
Germann SM, Oestergaard VH, Haas C, Salis P, Motegi A, Lisby M (2011) Dpb11/TopBP1 plays distinct roles in DNA replication, checkpoint response and homologous recombination. DNA Repair (Amst) 10:210–224
Reid R, Lisby M, Rothstein R (2002) Cloning-free genome alterations in Saccharomyce cerevisiae using adaptamer-mediated PCR. Methods Enzymol 350:258–277
Zhao X, Muller EG, Rothstein R (1998) A suppressor of two essential checkpoint genes identifies a novel protein that negatively affects dNTP pools. Mol Cell 2:329–340
Eckert-Boulet N, Rothstein R, Lisby M (2011) Cell biology of homologous recombination in yeast. Methods Mol Biol 745:523–536
Gordon A, Colman-Lerner A, Chin TE, Benjamin KR, Yu RC, Brent R (2007) Single-cell quantification of molecules and rates using open-source microscope-based cytometry. Nat Methods 4:175–181
Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572
Lim CR, Kimata Y, Oka M, Nomaguchi K, Kohno K (1995) Thermosensitivity of green fluorescent protein fluorescence utilized to reveal novel nuclear-like compartments in a mutant nucleoporin NSP1. J Biochem (Tokyo) 118:13–17
Lisby M, Barlow JH, Burgess RC, Rothstein R (2004) Choreography of the DNA damage response; spatiotemporal relationships among checkpoint and repair proteins. Cell 118:699–713
Sherman F, Fink GR, Hicks JB (1986) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Reichard P (1988) Interactions between deoxyribonucleotide and DNA synthesis. Annu Rev Biochem 57:349–374
Lopes M, Cotta-Ramusino C, Pellicioli A, Liberi G, Plevani P, Muzi-Falconi M, Newlon CS, Foiani M (2001) The DNA replication checkpoint response stabilizes stalled replication forks. Nature 412:557–561
Lisby M, Mortensen UH, Rothstein R (2003) Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre. Nat Cell Biol 5:572–577
Beranek DT, Weis CC, Swenson DH (1980) A comprehensive quantitative analysis of methylated and ethylated DNA using high pressure liquid chromatography. Carcinogenesis 1:595–606
Friedland W, Jacob P, Paretzke HG, Merzagora M, Ottolenghi A (1999) Simulation of DNA fragment distributions after irradiation with photons. Radiat Environ Biophys 38:39–47
Fronza G, Campomenosi P, Iannone R, Abbondandolo A (1992) The 4-nitroquinoline 1-oxide mutational spectrum in single stranded DNA is characterized by guanine to pyrimidine transversions. Nucleic Acids Res 20:1283–1287
Wiltrout ME, Walker GC (2011) The DNA polymerase activity of Saccharomyces cerevisiae Rev1 is biologically significant. Genetics 187:21–35
Deng C, Brown JA, You D, Brown JM (2005) Multiple endonucleases function to repair covalent topoisomerase I complexes in Saccharomyces cerevisiae. Genetics 170:591–600
Eng WK, Faucette L, Johnson RK, Sternglanz R (1988) Evidence that DNA topoisomerase I is necessary for the cytotoxic effects of camptothecin. Mol Pharmacol 34:755–760
Moore CW, McKoy J, Dardalhon M, Davermann D, Martinez M, Averbeck D (2000) DNA damage-inducible and RAD52-independent repair of DNA double-strand breaks in Saccharomyces cerevisiae. Genetics 154:1085–1099
Nielsen I, Bentsen IB, Lisby M, Hansen S, Mundbjerg K, Andersen AH, Bjergbaek L (2009) A Flp-nick system to study repair of a single protein-bound nick in vivo. Nat Methods 6:753–757
McConnell Smith A, Takeuchi R, Pellenz S, Davis L, Maizels N, Monnat RJ Jr, Stoddard BL (2009) Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease. Proc Natl Acad Sci U S A 106:5099–5104
Jensen RE, Herskowitz I (1984) Directionality and regulation of cassette substitution in yeast. Cold Spring Harbor Symp Quant Biol 49:97–104
Gietz D, St Jean A, Woods RA, Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425
Acknowledgment
We thank Dr. Neta Dean, Stony Brook University, for sharing the yEmRFP construct. This work was supported by Fundação para a Ciência e a Tecnologia (SS), The Danish Agency for Science, Technology and Innovation (ML, NEB), the Villum Kann Rasmussen Foundation (ML), and the European Research Council (ML).
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Silva, S., Gallina, I., Eckert-Boulet, N., Lisby, M. (2012). Live Cell Microscopy of DNA Damage Response in Saccharomyces cerevisiae . In: Bjergbæk, L. (eds) DNA Repair Protocols. Methods in Molecular Biology, vol 920. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-998-3_30
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DOI: https://doi.org/10.1007/978-1-61779-998-3_30
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