Abstract
Single-stranded DNA (ssDNA) is a DNA repair, replication, and recombination intermediate and a stimulus for checkpoint kinase-dependent cell cycle arrest. Current assays to detect ssDNA generated in vivo are indirect, laborious, and generally require the use of radioactivity. Here, we describe simple, quantitative approaches to measure ssDNA generated in yeast, at single- and multi-copy chromosomal loci and in highly repetitive telomeric sequences. We describe a fluorescence in-gel assay to measure ssDNA in the telomeric TG repeats of telomere cap-defective budding yeast yku70∆ and cdc13-1 mutants. We also describe a rapid method to prepare DNA for Quantitative Amplification of ssDNA, used to measure ssDNA in single-copy and repetitive sub-telomeric loci. These complementary methods are useful to understand the important roles of ssDNA in yeast cells and could be readily extended to other cell types.
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References
Garvik B, Carson M, Hartwell L (1995) Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint. Mol Cell Biol 15:6128–6138
Lydall D, Nikolsky Y, Bishop DK, Weinert T (1996) A meiotic recombination checkpoint controlled by mitotic checkpoint genes. Nature 383:840–843
Lydall D, Weinert T (1995) Yeast checkpoint genes in DNA damage processing: implications for repair and arrest. Science 270:1488–1491
Buhler C, Borde V, Lichten M (2007) Mapping meiotic single-strand DNA reveals a new landscape of DNA double-strand breaks in Saccharomyces cerevisiae. PLoS Biol 5:e324
White CI, Haber JE (1990) Intermediates of recombination during mating type switching in Saccharomyces cerevisiae. EMBO J 9:663–673
Lee SE, Moore JK, Holmes A, Umezu K, Kolodner RD, Haber JE (1998) Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage. Cell 94:399–409
Mimitou EP, Symington LS (2008) Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 455:770–774
Mimitou EP, Symington LS (2010) Ku prevents Exo1 and Sgs1-dependent resection of DNA ends in the absence of a functional MRX complex or Sae2. EMBO J 29:3358–3369
Nicolette ML, Lee K, Guo Z, Rani M, Chow JM, Lee SE, Paull TT (2010) Mre11-Rad50-Xrs2 and Sae2 promote 5′ strand resection of DNA double-strand breaks. Nat Struct Mol Biol 17:1478–1485
Zhu Z, Chung WH, Shim EY, Lee SE, Ira G (2008) Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell 134:981–994
Chung WH, Zhu Z, Papusha A, Malkova A, Ira G (2010) Defective resection at DNA double-strand breaks leads to de novo telomere formation and enhances gene targeting. PLoS Genet 6:e1000948
Terentyev N, Johnson M, Neale A, Bishop-Bailey M, Goldman AS (2010) Evidence that MEK1 could positively promotes interhomologue double-strand break repair. Nucleic Acids Res 38:4349–4360
Bonetti D, Clerici M, Anbalagan S, Martina M, Lucchini G, Longhese MP (2010) Shelterin-like proteins and Yku inhibit nucleolytic processing of Saccharomyces cerevisiae telomeres. PLoS Genet 6:e1000966
Booth C, Griffith E, Brady G, Lydall D (2001) Quantitative amplification of single-stranded DNA (QAOS) demonstrates that cdc13-1 mutants generate ssDNA in a telomere to centromere direction. Nucleic Acids Res 29:4414–4422
Maringele L, Lydall D (2002) EXO1-dependent single-stranded DNA at telomeres activates subsets of DNA damage and spindle checkpoint pathways in budding yeast yku70∆ mutants. Genes Dev 16:1919–1933
Zubko MK, Guillard S, Lydall D (2004) Exo1 and Rad24 differentially regulate generation of ssDNA at telomeres of Saccharomyces cerevisiae cdc13-1 mutants. Genetics 168:103–115
Lazzaro F, Sapountzi V, Granata M, Pellicioli A, Vaze M, Haber JE, Plevani P, Lydall D, Muzi-Falconi M (2008) Histone methyltransferase Dot1 and Rad9 inhibit single-stranded DNA accumulation at DSBs and uncapped telomeres. EMBO J 27:1502–1512
Dewar JM, Lydall D (2010) Pif1- and Exo1-dependent nucleases coordinate checkpoint activation following telomere uncapping. EMBO J 29:4020–4034
Ngo HP, Lydall D (2010) Survival and growth of yeast without telomere capping by Cdc13 in the absence of Sgs1, Exo1, and Rad9. PLoS Genet 6:e1001072
Zubko MK, Maringele L, Foster SS, Lydall D (2006) Detecting repair intermediates in vivo: effects of DNA damage response genes on single-stranded DNA accumulation at uncapped telomeres in budding yeast. Methods Enzymol 409:285–300
Chandra A, Hughes TR, Nugent CI, Lundblad V (2001) Cdc13 both positively and negatively regulates telomere replication. Genes Dev 15:404–414
Bertuch AA, Lundblad V (2004) EXO1 contributes to telomere maintenance in both telomerase-proficient and telomerase-deficient Saccharomyces cerevisiae. Genetics 166:1651–1659
Dionne I, Wellinger RJ (1996) Cell cycle-regulated generation of single-stranded G-rich DNA in the absence of telomerase. Proc Natl Acad Sci USA 93:13902–13907
Vodenicharov MD, Wellinger RJ (2006) DNA degradation at unprotected telomeres in yeast is regulated by the CDK1 (Cdc28/Clb) cell-cycle kinase. Mol Cell 24:127–137
Vodenicharov MD, Laterreur N, Wellinger RJ (2010) Telomere capping in non-dividing yeast cells requires Yku and Rap1. EMBO J 17:3007–3019
Miyake Y, Nakamura M, Nabetani A, Shimamura S, Tamura M, Yonehara S, Saito M, Ishikawa F (2009) RPA-like mammalian Ctc1-Stn1-Ten1 complex binds to single-stranded DNA and protects telomeres independently of the Pot1 pathway. Mol Cell 36:193–206
Surovtseva YV, Churikov D, Boltz KA, Song X, Lamb JC, Warrington R, Leehy K, Heacock M, Price CM, Shippen DE (2009) Conserved telomere maintenance component 1 interacts with STN1 and maintains chromosome ends in higher eukaryotes. Mol Cell 36:207–218
Celli GB, de Lange T (2005) DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion. Nat Cell Biol 7:712–718
Abramoff MD, Magelhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophotonics Int 11:36–42
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Dewar, J.M., Lydall, D. (2012). Simple, Non-radioactive Measurement of Single-Stranded DNA at Telomeric, Sub-telomeric, and Genomic Loci in Budding Yeast. 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_24
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DOI: https://doi.org/10.1007/978-1-61779-998-3_24
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