Analysis of mechanism of elongation of ubiquitin chains

编码泛素结合酶UbcP1/Ubc4和UbcP4/Ubc11的基因突变导致分裂酵母有丝分裂异常。有丝分裂周期蛋白CDc13在突变细胞中稳定并积累。此外，激活的APC/C(后期促进复合体/环体)降解CdC13需要两种泛素偶联酶。在ubcP4/ubc11突变细胞中，cdc13的泛素化完全降低，而在ubcP1/ubc4突变细胞中，cdc13的泛素链较短。这些结果表明，Cc13的多泛素化是一个多步骤的反应，UbcP4/Ubc11参与了Ccc13泛素化的启动，而UbcP1/Ubc4参与了Cc13上泛素链的延长。为了证明Cd13的泛素化是一个多步骤反应，有必要建立Cd13泛素化的体外重组体系。由于利用裂解酵母全细胞提取液构建重组体系的试验没有取得良好的效果，因此将各组分表达为重组蛋白并进行纯化，现尝试按各因子构建重组体系。此外，ubcP4/ubc11突变株还表现出间期延迟的细胞伸长表型。这种表型依赖于DNA损伤检查点。此外，作为DNA损伤检查点效应蛋白的Chk1在ubcP4/ubc11突变细胞中被激活。由于ubcP4/ubc11突变体对DNA损伤不敏感，因此认为涉及UbcP4/Ubc11的泛素途径是DNA损伤检查点的调节因子。最近，我确定了靶蛋白的候选者，即调节DNA损伤检查点。我正在研究这个候选蛋白质的生物学意义、稳定性的调节等。

The mutation of the gene coding ubiquitin-conjugating enzymes UbcP1/Ubc4 and UbcP4/Ubc11 causes mitotic abnormality fission yeast. Mitotic cyclin Cdc13 was stabilized and accumulated in mutant cells. Furthermore, both ubiquitin-conjugating enzyme was required for degradation of Cdc13 by activated APC/C (Anaphase Promoting Complex/Cyclosome). Ubiquitination of Cdc13 was totally decreasing in the ubcP4/ubc11 mutant cells, while ubiquitin chains of Cdc13 were short in the ubcP1/ubc4 mutant cells. These results suggest that polyubiquitination of Cdc13 is a multi-step reaction and UbcP4/Ubc11 is involved in initiation of ubiquitination of Ccc13 while UbcP1/Ubc4 is involved in elongation of ubiquitin chains on Cdc13. In order to prove that polyubiquitination of Cdc13 is a multi-step reaction as mentioned above, it is necessary to establish the in vitro reconstitution system of ubiquitination of Cdc13. Since the trial of the reconstitution system construction using fission yeast whole cell extract was not able to obtain a good result, each component was expressed as recombinant protein and purified, and I try to construct a reconstitution system by each factor now. In addition, ubcP4/ubc11 mutant strain shows a cell elongation phenotype characteristic of delay of an interphase. This phenotype was dependent on a DNA damage checkpoint. Moreover, Chk1 that is effector kinase of DNA damage checkpoint was activated in the ubcP4/ubc11 mutant cells. Because ubcP4/ubc11 mutant did not show hypersensitivity to DNA damage, it is suggested that the ubiquitin pathway involving UbcP4/Ubc11 functions as a regulator of a DNA damage checkpoint. Recently I identified the candidate of target protein that is regulator DNA damage checkpoint. I am studding the biological significance, regulation of stability etc, of this candidate protein.