Dicentric chromosome stretching during anaphase reveals roles of Sir2/Ku in chromatin compaction in budding yeast.
We have used mitotic spindle forces to examine the role of Sir2 and Ku in chromatin compaction. Escherichia coli lac operator DNA was placed between two centromeres on a conditional dicentric chromosome in budding yeast cells and made visible by expression of a lac repressor-green fluorescent fusion protein. Centromeres on the same chromatid of a dicentric chromosome attach to opposite poles approximately 50% of the time, resulting in chromosome bridges during anaphase. In cells deleted for yKU70, yKU80, or SIR2, a 10-kb region of the dicentric chromosome stretched along the spindle axis to a length of 6 microm during anaphase. On spindle disassembly, stretched chromatin recoiled to the bud neck and was partitioned to mother and daughter cells after cytokinesis and cell separation. Chromatin immunoprecipitation revealed that Sir2 localizes to the lacO region in response to activation of the dicentric chromosome. These findings indicate that Ku and Sir proteins are required for proper chromatin compaction within regions of a chromosome experiencing tension or DNA damage. The association of Sir2 with the affected region suggests a direct role in this process, which may include the formation of heterochromatic DNA.
Thrower
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Department of Biology, CB3280 University of North Carolina, Chapel Hill, NC 27599-3280, USA.
Bloom
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D000705:Anaphase; D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins / Q000378:metabolism; D002503:Centromere / Q000235:genetics; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D019457:Chromosome Breakage; D020090:Chromosome Segregation; D015825:Chromosomes, Fungal / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D017930:Genes, Reporter; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism*; D000072200:Ku Autoantigen; D007763:Lac Operon / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009875:Operator Regions, Genetic / Q000235:genetics; D011233:Precipitin Tests; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D056564:Sirtuin 1; D056565:Sirtuin 2; D037761:Sirtuins; D008941:Spindle Apparatus / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D015534:Trans-Activators; C520769:YKU70 protein, S cerevisiae; C520770:YKU80 protein, S cerevisiae; C081647:high affinity DNA-binding factor, S cerevisiae; C069930:rad9 protein; C087778:SIR2 protein, S cerevisiae; C447939:SIRT1 protein, human; D056564:Sirtuin 1; D056565:Sirtuin 2; D037761:Sirtuins; D006655:Histone Deacetylases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen