Abstract
Site-specific recombinases have been harnessed for a variety of genetic manipulations involving the gain, loss, or rearrangement of genomic DNA in a variety of organisms. The enzymes have been further exploited in the model eukaryote Saccharomyces cerevisiae for mechanistic studies involving chromosomal context. In these cases, a chromosomal element of interest is converted into a DNA circle within living cells, thereby uncoupling the element from neighboring regulatory sequences, obligatory chromosomal events, and other context-dependent effects that could alter or mask intrinsic functions of the element. In this chapter, I discuss general considerations in using site-specific recombination to create DNA circles in yeast and the specific application of the R recombinase.
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References
Raghuraman MK, Brewer BJ, Fangman WL (1997) Cell cycle-dependent establishment of a late replication program. Science 276: 806–809.
Megee PC, Koshland D (1999) A functional assay for centromere-associated sister chromatid cohesion. Science 285: 254–257.
Holmes SG, Broach JR (1996) Silencers are required for inheritence of the repressed state in yeast. Genes Dev 10: 1021–1032.
Bi X, Broach JR (1997) DNA in transcriptionally silent chromatin assumes a distinct topology that is sensitive to cell cycle progression. Mol Cell Biol 17: 7077–7087.
Cheng T-H, Li Y-C, Gartenberg MR (1998) Persistence of an alternate chromatin structure at silenced loci in the absence of silencers. Proc Natl Acad Sci USA 95: 5521–5526.
Cheng T-H, Gartenberg MR (2000) Yeast heterochromatin is a dynamic structure that requires silencers continuously. Genes Dev 14: 452–463.
Kirchmaier AL, Rine J (2001) DNA replication-independent silencing in S. cerevisiae. Science 291: 646–650.
Li Y-C, Cheng T-H, Gartenberg MR (2001) Establishment of transcriptional silencing in the absence of DNA replication. Science 291: 650–653.
Gartenberg MR, Neumann FN, Laroche T, Blaszczyk M, Gasser SM (2004) Sir-mediated repression can occur independently of chromosomal and subnuclear contexts. Cell 119: 955–967.
Wu CS, Chen YF, Gartenberg MR (2011) Targeted sister chromatid cohesion by Sir2. PLoS Genet 7: e1002000.
Ansari A, Cheng T-H, Gartenberg MR (1999) Isolation of selected chromatin fragments from yeast by site-specific recombination in vivo. Methods 17: 104–111.
Boeger H, Griesenbeck J, Strattan JS, Kornberg RD (2003) Nucleosomes unfold completely at a transcriptionally active promoter. Mol Cell 11: 1587–1598.
Griesenbeck J, Boeger H, Strattan JS, Kornberg RD (2004) Purification of defined chromosomal domains. Methods Enzymol 375: 170–178.
Branda CS, Dymecki SM (2004) Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice. Dev Cell 6: 7–28.
Bischof J, Basler K (2008) Recombinases and their use in gene activation, gene inactivation, and transgenesis. Methods Mol Biol 420: 175–195.
Logie C, Stewart AF (1995) Ligand-regulated site-specific recombination. Proc Natl Acad Sci USA 92: 5940–5944.
Cheng T-H, Chang C-R, Joy P, Yablok S, Gartenberg MR (2000) Controlling gene expression in yeast by inducible site-specific recombination. Nucleic Acids Res 28: E108.
Verzijlbergen KF, Menendez-Benito V, van Welsem T, van Deventer SJ, Lindstrom DL, et al. (2010) Recombination-induced tag exchange to track old and new proteins. Proc Natl Acad Sci USA 107: 64–68.
Tsalik EL, Gartenberg MR (1998) Curing Saccharomyces cerevisiae of the 2 Micron plasmid by targeted DNA damage. Yeast 14: 847–852.
Ghosh SK, Hajra S, Paek A, Jayaram M (2006) Mechanisms for chromosome and plasmid segregation. Annu Rev Biochem 75: 211–241.
Matsuzaki H, Nakajima R, Nishiyama J, Araki H, Oshima Y (1990) Chromosome engineering in Saccharomyces cerevisiae by using a site-specific recombination system of a yeast plasmid. J Bact 172: 610–618.
Gartenberg MR, Wang JC (1993) Identification of barriers to rotation of DNA segments in yeast from the topology of DNA rings excised by an inducible site-specific recombinase. Proc Natl Acad Sci USA 90: 10514–10518.
Goldstein AL, McCusker JH (1999) Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15: 1541–1553.
Güldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH (1996) New efficient gene disruption cassette for repeated use in budding yeast. Nucl Acids Res 24: 2519–2524.
Boeke JD, LaCroute F, Fink GR (1984) A positive selection for mutants lacking orotidine-5’-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet 197: 345–346.
Reid RJ, Lisby M, Rothstein R (2002) Cloning-free genome alterations in Saccharomyces cerevisiae using adaptamer-mediated PCR. Methods Enzymol 350: 258–277.
Ansari A, Gartenberg MR (1999) Persistence of an alternate chromatin structure at silenced loci in vitro. Proc Natl Acad Sci USA 96: 343–348.
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, et al., editors (2010) Current Protocols in Molecular Biology. New York: John Wiley & Sons.
Smith MC, Brown WR, McEwan AR, Rowley PA (2010) Site-specific recombination by phiC31 integrase and other large serine recombinases. Biochem Soc Trans 38: 388–394.
Weber SA, Gerton JL, Polancic JE, DeRisi JL, Koshland D, et al. (2004) The kinetochore is an enhancer of pericentric cohesin binding. PLoS Biol 2: E260.
Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, et al. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14: 115–132.
Rothstein R (1991) Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol 194: 281–301.
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Gartenberg, M.R. (2012). Generation of DNA Circles in Yeast by Inducible Site-Specific Recombination. In: Morse, R. (eds) Chromatin Remodeling. Methods in Molecular Biology, vol 833. Humana Press. https://doi.org/10.1007/978-1-61779-477-3_7
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