Targeting integration of the Saccharomyces Ty5 retrotransposon.
Many retrotransposons and retroviruses display integration site specificity. Increasingly, this specificity is found to result from recognition by the retroelement of specific chromatin states or DNA-bound protein complexes. A well-studied example of such a targeted retroelement is the Saccharomyces Ty5 retrotransposon, which integrates into heterochromatin at the telomeres and silent mating loci. Targeting is mediated by an interaction between Ty5 integrase (IN) and the heterochromatin protein silent information regulator 4 (Sir4). A small motif of IN, called the targeting domain, is responsible for this interaction. Ty5 integration can be directed to DNA sites outside of heterochromatin by tethering Sir4 to ectopic locations using fusion proteins between Sir4 and a DNA-binding domain. Alternatively, the targeting domain of Ty5 can be swapped with peptides that recognize other protein partners, thereby generating Ty5 elements with new target specificities. The mechanism of Ty5 target site choice suggests that integration specificity of other retrotransposons and retroviruses can be altered by engineering integrases to recognize DNA-bound protein partners. Retroelements can also be used to probe chromatin dynamics and the distribution of protein complexes on chromosomes. Here, we describe the basic assay by which Ty5 integration is monitored to sites of tethered Sir4.
Brady
Troy L
TL
Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA.
Schmidt
Clarice L
CL
Voytas
Daniel F
DF
D001665:Binding Sites / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D019426:Integrases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D018626:Retroelements / Q000235:genetics*; D012440:Saccharomyces / Q000235:genetics* / Q000378:metabolism; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D004271:DNA, Fungal; D018626:Retroelements; C061573:SIR4 protein, S cerevisiae; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D019426:Integrases