Study On The Correlation Between Function And Subcellular Localization Of Monopolin Complex And Its Functional Connection With Nucleolar Protein Dnt1 In Fission Yeast

The monopolin complex plays a role in inhibiting merotelic attachment by recruiting condensins at kinetochores,and it also regulates the rDNA recombination.Cells with defects in the former function are sensitive to TBZ and the latter leads to HU sensitivity.The nucleolar protein Dntl also takes part in regulating the accurate separation of sister chromatids and the recombination of rDNA.The genetic interaction between the monopolin and Dntl is still not well understood.In this research,we transformed components of monopolin complex,so that they can be recognized and cut by TEV protease,thus destructing the complex.On this background,we localize TEV protease at kinetochores and cut one of the monopolin components-Mde4 specially at kinetochores.It shows that the destruction of monopolin at the kinetochores leads to strong sensitivity to TBZ,and the proportion of lagging chromosomes has a significant increase in anaphase.However,this specific destruction of kinetochore Mde4 does not cause the sensitivity to HU.Therefore,monopolin complex locating at kinetochores only participates in inhibiting merotelic attachment,but not in regulating the rDNA recombination.We also localize TEV protease to the nucleolus.The destruction of another monopolin component--Pcs--at nucleolus,results in HU sensitivity.Surprisingly,this strain is also sensitive to TBZ and the proportion of lagging chromosomes is also increased in anaphase.Fluorescence observation of PcsI-GFP shows that the localization of Pcsl at kinetochores also disappears after the destruction nucleolus Pcsl.In addition,we want to find out the mechanisms underlying the lethalify of combination of dntl??pcsl? or dnt/?/mde4A.Introducing dntl A into the strain in which kinetochore Mde4 was cut,increases the TBZ sensitivity and the proportion of lagging chromosomes in anaphase cells,but it does not affect the strain's sensitivity to HU,and the cells can grow normally.When the dntl? is introduced to the strain in which the Pcs1 was cut at nucleolus,not only the TBZ sensitivity but also the HU sensitivity is greatly increased,and the cells cannot grow normally.These results show that the cell death caused by the double mutation--dnt1?/pcs1? or dnt1?/mde4?,is due to the defects in both the separation of sister chromatids and the recombination of rDNA.In this article,we mainly studied the relations between the monopolin complex's location and its functions,and explored the genetic interaction between monopolin complex and Dntl in mitosis.The accurate separation of sister chromatids and the recombination of rDNA are regulated by the monopolin complex and Dntl,so that cells will grow and proliferate normally.