The yeast silent information regulator Sir4p anchors and partitions plasmids.
Circular plasmids containing telomeric TG1-3 arrays or the HMR E silencer segregate efficiently between dividing cells of the yeast Saccharomyces cerevisiae. Subtelomeric X repeats augment the TG1-3 partitioning activity by a process that requires the SIR2, SIR3, and SIR4 genes, which are also required for silencer-based partitioning. Here we show that targeting Sir4p to DNA directly via fusion to the bacterial repressor LexA confers efficient mitotic segregation to otherwise unstable plasmids. The Sir4p partitioning activity resides within a 300-amino-acid region (residues 950 to 1262) which precedes the coiled-coil dimerization motif at the extreme carboxy end of the protein. Using a topology-based assay, we demonstrate that the partitioning domain also retards the axial rotation of LexA operators in vivo. The anchoring and partitioning properties of LexA-Sir4p chimeras persist despite the loss of the endogenous SIR genes, indicating that these functions are intrinsic to Sir4p and not to a complex of Sir factors. In contrast, inactivation of the Sir4p-interacting protein Rap1p reduces partitioning by a LexA-Sir4p fusion. The data are consistent with a model in which the partitioning and anchoring domain of Sir4p (PAD4 domain) attaches to a nuclear component that divides symmetrically between cells at mitosis; DNA linked to Sir4p by LexA serves as a reporter of protein movement in these experiments. We infer that the segregation behavior of telomere- and silencer-based plasmids is, in part, a consequence of these Sir4p-mediated interactions. The assays presented herein illustrate two novel approaches to monitor the intracellular dynamics of nuclear proteins.
Ansari
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Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway 08854, USA.
Gartenberg
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D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D008954:Models, Biological; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D004271:DNA, Fungal; D005656:Fungal Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D000099160:LexA Repressor Protein; C061573:SIR4 protein, S cerevisiae