Yeast Dam1p has a role at the kinetochore in assembly of the mitotic spindle
Yeast Dam1p has a role at the kinetochore in assembly of the mitotic spindle
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DOI：
10.1073/pnas.241417098
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发表时间：
2001-11-20
影响因子：
11.1
通讯作者：
Winey, M
中科院分区：
文献类型：
--
作者：
Jones, MH;He, XW;Winey, M
在有丝分裂期间，复制的染色体通过基于微管的有丝分裂纺锤体分离到子细胞。染色体通过称为着丝粒的特殊 DNA/蛋白质结构附着在有丝分裂纺锤体上，但附着机制尚不清楚。我们在这里表明，酵母微管结合蛋白 Dam1p 在物理和功能上与着丝粒相关，表明在着丝粒附着到纺锤体中发挥作用。在染色体扩散的免疫荧光分析中，带有表位标记的 Dam1p 版本与完整动粒组件 Ndc10p/Cbf2p 共定位。此外，染色质免疫沉淀实验表明，Dam1p 优先与着丝粒 DNA 关联，并且这种关联依赖于 Ndc10p/Cbf2p。我们还证明了 DAM1 和编码动粒蛋白的 CTF19 或 SLK19 基因之间的遗传相互作用。尽管 dam1-1 突变引起的缺陷导致纺锤体检查点监视系统的激活以及随后姐妹染色单体凝聚力的持续存在，但中期停滞纺锤体异常伸长，导致几乎完全的染色体错误分离。执行点实验表明 Dam1p 在中期纺锤体的形成和后期纺锤体的伸长中发挥作用。最后，我们观察到 dam1-1 等位基因编码的蛋白质在不允许的温度下变得离域，与随后突变表型的发生相关。我们的研究与 Dam1p 在染色体分离之前通过着丝粒将姐妹染色单体附着到有丝分裂纺锤体中的作用一致。
During mitosis, replicated chromosomes are separated to daughter cells by the microtubule-based mitotic spindle. Chromosomes attach to the mitotic spindle through specialized DNA/protein structures called kinetochores, but the mechanism of attachment is not well understood. We show here that the yeast microtubule-binding protein, Dam1p, associates physically and functionally with kinetochores, suggesting a role in kinetochore attachment to the spindle. An epitope-tagged version of Dam1p colocalizes with the integral kinetochore component Ndc10p/Cbf2p in immunofluorescence analysis of chromosome spreads. in addition, Dam1p is associated preferentially with centromeric DNA as shown by chromatin immunoprecipitation experiments, and this association depends on Ndc10p/Cbf2p. We also demonstrate genetic interactions between DAM1 and CTF19 or SLK19 genes encoding kinetochore proteins. Although the defect caused by the dam1-1 mutation leads to activation of the spindle checkpoint surveillance system and consequent persistence of sister chromatid cohesion, the metaphase arrest spindle abnormally elongates, resulting in virtually complete chromosome missegregation. Execution point experiments indicate that Dam1p has a role in formation of a metaphase spindle and in anaphase spindle elongation. Finally, we have observed that the protein encoded by the dam1-1 allele becomes delocalized at the nonpermissive temperature, correlating with the subsequent onset of the mutant phenotype. Our studies are consistent with a role for Dam1p in attachment of sister chromatids through the kinetochore to the mitotic spindle before chromosome segregation.