Howard Hughes Medical Institute, 3520 San Martin Drive, Baltimore, MD 21218, USA.
Curr Biol. 2010 May 25;20(10):957-63. doi: 10.1016/j.cub.2010.04.018. Epub 2010 May 6.
Cohesin's complex distribution on chromosomes and its implication in numerous cellular processes makes it an excellent paradigm for studying the relationship between the in vivo concentration of a protein and its in vivo function. Here, we report a method to generate systematic quantized reductions (QR) in the in vivo concentration of any yeast protein. With QR, we generate strains with 13% and 30% of wild-type levels of the limiting subunit of cohesin, Mcd1p/Scc1p/Rad21p. Reducing cohesin levels reveals a preferential binding of cohesin to pericentric regions over cohesin-associated regions (CAR) on chromosome arms. Chromosome condensation, repetitive DNA stability, and DNA repair are compromised by decreasing cohesin levels to 30% of wild-type levels. In contrast, sister-chromatid cohesion and chromosome segregation are unaffected even when cohesin levels are reduced to 13% of wild-type levels. The requirement for different in vivo cohesin concentrations to achieve distinct cohesin functions provides an explanation for how cohesin mutations can specifically lead to adult disorders such as Cornelia de Lange Syndrome and Roberts Syndrome without compromising the cell divisions needed for development and maturation. Our successful application of QR to cohesin suggests that QR is a powerful tool to study other proteins/pathways with multiple functions.
黏连蛋白复合物在染色体上的复杂分布及其在众多细胞过程中的作用，使其成为研究蛋白质体内浓度与其体内功能之间关系的理想范例。在这里，我们报告了一种系统地产生任何酵母蛋白体内浓度定量减少（QR）的方法。通过 QR，我们生成了限制亚基黏连蛋白 Mcd1p/Scc1p/Rad21p 的体内浓度分别为野生型的 13%和 30%的菌株。降低黏连蛋白水平表明，黏连蛋白优先与着丝粒区域结合，而不是与染色体臂上的黏连蛋白相关区域（CAR）结合。当黏连蛋白水平降低到野生型的 30%时，染色体浓缩、重复 DNA 稳定性和 DNA 修复受到损害。相比之下，当黏连蛋白水平降低到野生型的 13%时，姐妹染色单体的黏合和染色体分离不受影响。需要不同的体内黏连蛋白浓度来实现不同的黏连蛋白功能，这为黏连蛋白突变如何特异性导致 Cornelia de Lange 综合征和 Roberts 综合征等成人疾病而不影响发育和成熟所需的细胞分裂提供了一个解释。我们成功地将 QR 应用于黏连蛋白，表明 QR 是研究具有多种功能的其他蛋白质/途径的有力工具。
