Abstract
The controlled fragmentation of chromosomes by DNA double-strand breaks (DSBs) initiates meiotic recombination, which is essential for meiotic chromosome segregation in most eukaryotes. This chapter describes a straightforward microarray-based approach to measure the genome-wide distribution of meiotic DSBs by detecting the single-stranded DNA (ssDNA) that transiently accumulates at DSB sites during recombination. The protocol outlined here has been optimized to detect meiotic DSBs in Saccharomyces cerevisiae. However, because ssDNA is a universal intermediate of homologous recombination, this method can ostensibly be adapted to discover and analyze programmed or damage-induced DSB hotspots in other organisms whose genome sequence is available.
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References
Keeney, S., Giroux, C.N., and Kleckner, N. (1997) Meiosis-specific DNA double-strand breaks are catalyzed by spo11, a member of a widely conserved protein family. Cell 88, 375–384.
Bishop, D.K., and Zickler, D. (2004) Early decision; meiotic crossover interference prior to stable strand exchange and synapsis. Cell 117, 9–15.
Petes, T.D. (2001) Meiotic recombination hot spots and cold spots. Nat Rev Genet 2, 360–369.
Baudat, F., and Nicolas, A. (1997) Clustering of meiotic double-strand breaks on yeast chromosome III. Proc Natl Acad Sci USA 94, 5213–5218.
Game, J.C. (1992) Pulsed-field gel analysis of the pattern of DNA double-strand breaks in the Saccharomyces genome during meiosis. Dev Genet 13, 485–497.
Zenvirth, D., Arbel, T., Sherman, A., Goldway, M., Klein, S., and Simchen, G. (1992) Multiple sites for double-strand breaks in whole meiotic chromosomes of Saccharomyces cerevisiae. EMBO J 11, 3441–3447.
Gerton, J.L., DeRisi, J., Shroff, R., Lichten, M., Brown, P.O., and Petes, T.D. (2000) Inaugural article: global mapping of meiotic recombination hotspots and coldspots in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci USA 97, 11383–11390.
Pan, J., Sasaki, M., Kniewel, R., Murakami, H., Blitzblau, H.G., Tischfield, S.E., Zhu, X., Neale, M.J., Jasin, M., Socci, N.D., Hochwagen, A., and Keeney, S. (2011) A hierarchical combination of factors shapes the genome-wide topography of yeast meiotic recombination initiation. Cell 144, 719–731.
Blitzblau, H.G., Bell, G.W., Rodriguez, J., Bell, S.P., and Hochwagen, A. (2007) Mapping of meiotic single-stranded DNA reveals double-strand-break hotspots near centromeres and telomeres. Curr Biol 17, 2003–2012.
Buhler, C., Borde, V., and Lichten, M. (2007) Mapping meiotic single-strand DNA reveals a new landscape of DNA double strand breaks in Saccharomyces cerevisiae. PLoS Biol 5, 2797–2808.
Huberman, J.A., Spotila, L.D., Nawotka, K.A., el-Assouli, S.M., and Davis, L.R. (1987) The in vivo replication origin of the yeast 2 microns plasmid. Cell 51, 473–481.
Feng, W., Collingwood, D., Boeck, M.E., Fox, L.A., Alvino, G.M., Fangman, W.L., Raghuraman, M.K., and Brewer, B.J. (2006) Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication. Nat Cell Biol 8, 148–155.
Smyth, G.K. (2005) Limma: linear models for microarray data. In Bioinformatics and computational biology solutions using R and bioconductor, R.C. Gentleman, V.J. Carey, S. Dudoit, R. Irizarry, W. Huber, eds. (New York, NY: Springer), pp. 397–420.
Yang, Y.H., Dudoit, S., Luu, P., and Speed, T.P. (2001) Normalization of cDNA microarray data. In SPIE BiOS 2001. San Jose, CA.
Bishop, D.K., Park, D., Xu, L., and Kleckner, N. (1992) DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression. Cell 69, 439–456.
Yabuki, N., Terashima, H., and Kitada, K. (2002) Mapping of early firing origins on a replication profile of budding yeast. Genes Cells 7, 781–789.
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We would also like to thank Gerben Vader and Milan de Vries for technical discussions and critical reading of this protocol.
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Blitzblau, H.G., Hochwagen, A. (2011). Genome-Wide Detection of Meiotic DNA Double-Strand Break Hotspots Using Single-Stranded DNA. In: Tsubouchi, H. (eds) DNA Recombination. Methods in Molecular Biology, vol 745. Humana Press. https://doi.org/10.1007/978-1-61779-129-1_4
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DOI: https://doi.org/10.1007/978-1-61779-129-1_4
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