Analysis of mechanism of elongation of ubiquitin chains

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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.