Abstract
The fission yeast Schizosaccharomyces pombe is especially well suited for studying meiosis in molecular detail. Experiments with S. pombe strains that undergo a nearly synchronous meiosis—at variable temperatures—have elucidated the mechanisms of meiotic progression and the proteins that are involved. For example, studies focused on the initiation of meiotic recombination by programmed DNA double-strand breaks (DSBs) have proven exceptionally informative. In meiosis, some regions of DNA have more frequent DSBs than the surrounding regions. These DSB hotspots can be visualized by Southern blot hybridization of restriction fragments ranging from kilobases (kb) to megabases (Mb) in size. More recently, the benefits of genome-wide analysis to map the distribution and frequency of meiotic DSBs have been attained, with resolution down to the nucleotide level. Infrequent, non-hotspot DSBs previously not detectable have been observed, creating a better understanding of how recombination is regulated. Additional genome-wide analyses have shown proteins that bind specifically to DSB hotspots, providing insight into how the DSB initiation complex functions. We describe here detailed methods for achieving these results.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cervantes MD, Farah JA, Smith GR (2000) Meiotic DNA breaks associated with recombination in S. pombe. Mol Cell 5:883–888
Iino Y, Yamamoto M (1985) Mutants of Schizosaccharomyces pombe which sporulate in the haploid state. Mol Gen Genet 198:416–421
Guerra-Moreno A, Alves-Rodrigues I, Hidalgo E, Ayte J (2012) Chemical genetic induction of meiosis in Schizosaccharomyces pombe. Cell Cycle 11:1621–1625
Cipak L, Hyppa RW, Smith GR, Gregan J (2012) ATP analog-sensitive Pat1 protein kinase for synchronous fission yeast meiosis at physiological temperature. Cell Cycle 11:1626–1633
Milman N, Higuchi E, Smith GR (2009) Meiotic DNA double-strand break repair requires two nucleases, MRN and Ctp1, to produce a single size class of Rec12 (Spo11)-oligonucleotide complexes. Mol Cell Biol 29:5998–6005
Cromie GA, Hyppa RW, Cam HE, Farah JA, Grewal SHIS, Smith GR (2007) A discrete class of intergenic DNA dictates meiotic DNA break hotspots in fission yeast. PLoS Genet 3:e141
Keeney S, Giroux CN, 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
Fowler KR, Sasaki M, Milman N, Keeney S, Smith GR (2014) Evolutionarily diverse determinants of meiotic DNA break and recombination landscapes across the genome. Genome Res 24:1650–1664
Young JA, Schreckhise RW, Steiner WW, Smith GR (2002) Meiotic recombination remote from prominent DNA break sites in S. pombe. Mol. Cell 9:253–263
Ren B, Robert F, Wyrick JJ, Aparicio O, Jennings EG, Simon I, Zeitlinger J, Schreiber J, Hannett N, Kanin E et al (2000) Genome-wide location and function of DNA binding proteins. Science 290:2306–2309
Iyer VR, Horak CE, Scafe CS, Botstein D, Snyder M, Brown PO (2001) Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF. Nature 409:533–538
Strahl-Bolsinger S, Hecht A, Luo K, Grunstein M (1997) SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. Genes Dev 11:83–93
Schena M, Shalon D, Davis RW, Brown PO (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270:467–470
Fowler KR, GutiĂ©rrez-Velasco S, MartĂn-Castellanos C, Smith GR (2013) Protein determinants of meiotic DNA break hotspots. Mol Cell 49:983–996
Estreicher A, Lorenz A, Loidl J (2012) Mug20, a novel protein associated with linear elements in fission yeast meiosis. Curr Genet 58:119–127
Spirek M, Estreicher A, Csaszar E, Wells JL, McFarlane RJ, Watts FZ, Loidl J (2010) SUMOylation is required for normal development of linear elements and wild-type meiotic recombination in Schizosaccharomyces pombe. Chromosoma 119:59–72
Davis L, Rozalén AE, Moreno S, Smith GR, Martin-Castellanos C (2008) Rec25 and Rec27, novel components of meiotic linear elements, link cohesin to DNA breakage and recombination in fission yeast. Curr Biol 18:849–854
Buck MJ, Nobel AB, Lieb JD (2005) ChIPOTle: a user-friendly tool for the analysis of ChIP-chip data. Genome Biol 6:R97
Hyppa RW, Cromie GA, Smith GR (2008) Indistinguishable landscapes of meiotic DNA breaks in rad50 + and rad50S strains of fission yeast revealed by a novel rad50 + recombination intermediate. PLoS Genet 4:e1000267
Steiner WW, Schreckhise RW, Smith GR (2002) Meiotic DNA breaks at the S. pombe recombination hotspot M26. Mol Cell 9:847–855
Cromie GA, Rubio CA, Hyppa RW, Smith GR (2005) A natural meiotic DNA break site in Schizosaccharomyces pombe is a hotspot of gene conversion, highly associated with crossing over. Genetics 169:595–605
Li YF, Smith GR (1997) The Schizosaccharomyces pombe rec16 gene product regulates multiple meiotic events. Genetics 146:57–67
Cam HP, Sugiyama T, Chen ES, Chen X, FitzGerald PC, Grewal SI (2005) Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome. Nat Genet 37:809–819
Acknowledgments
We are grateful to Sue Amundsen and Joshua Cho for helpful comments on the manuscript. Technical assistance and advice for this protocol were generously given by Hugh Cam and Shiv Grewal (NIH); Gareth Cromie and Neta Milman (FHCRC); Jing Pan, Mariko Sasaki, and Scott Keeney (MSKCC); and Jeff Delrow and Cassie Sather (FHCRC Genomics Facility). Our laboratory is supported by research grants GM031693 and GM032194 from the National Institutes of Health of the USA.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Hyppa, R.W., Fowler, K.R., Smith, G.R. (2017). Quantitative Genome-Wide Measurements of Meiotic DNA Double-Strand Breaks and Protein Binding in S. pombe . In: Stuart, D. (eds) Meiosis. Methods in Molecular Biology, vol 1471. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6340-9_2
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6340-9_2
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6338-6
Online ISBN: 978-1-4939-6340-9
eBook Packages: Springer Protocols