CEN plasmid segregation is destabilized by tethered determinants of Ty 5 integration specificity: a role for double-strand breaks in CEN antagonism.
The yeast retrotransposon Ty 5 integrates preferentially into heterochromatin at the telomeres and HM loci. Target specificity is mediated by a six amino acid sequence motif (the targeting domain, TD) of integrase that interacts with Sir4p, a structural component of heterochromatin. When tethered to CEN plasmids as part of a Gal4p DNA binding domain (GBD) fusion protein, TD destabilizes plasmid segregation in a manner similar to that observed for CEN + HM or CEN +TEL antagonism. This instability is caused by the ability of TD to nucleate components of heterochromatin on the CEN plasmid, because CEN +TD antagonism is abrogated by sir2, sir3 and sir4 mutations and by TD mutations that prevent interaction with Sir4p. In strains that acquire resistance to CEN +TD antagonism, the CEN plasmid has either recombined with a 2 mu plasmid or sustained deletions in sequences required to bind GBD-TD. CEN +TD and CEN + HM antagonism is exacerbated by mutations in components of the Ku-mediated non-homologous end-joining pathway. These observations suggest that CEN antagonism is caused by DNA breaks that result from competition between CEN - and Sir-specific segregation pathways.
Fuerst
Peter G
PG
Department of Zoology and Genetics, Iowa State University, Ames, IA 50011, USA.
Voytas
Daniel F
DF
D020816:Amino Acid Motifs; D004249:DNA Damage; D004260:DNA Repair / Q000502:physiology*; D004271:DNA, Fungal / Q000378:metabolism*; D006570:Heterochromatin / Q000378:metabolism; D019426:Integrases / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D010957:Plasmids / Q000037:antagonists & inhibitors*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018626:Retroelements; D012441:Saccharomyces cerevisiae / Q000235:genetics; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000235:genetics / Q000378:metabolism*
D004271:DNA, Fungal; D006570:Heterochromatin; D011993:Recombinant Fusion Proteins; D018626:Retroelements; C061573:SIR4 protein, S cerevisiae; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D019426:Integrases