BIOCHEMICAL ANALYSIS OF NUCLEAR STRUCTURE

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The DNA in all eukaryotic cells is located in the nucleus. The outer boundary of the nucleus is defined by a complex structure called the nuclear envelope. The envelope is a semi-permeable barrier composed of two membrane bilayers linked to each other by nuclear pores. The envelope regulates traffic of molecules into and out of the nucleus and acts as a structural platform for the organization of DNA. Although the envelope has been implicated as playing a role in chromatin function, relatively little is known about how this occurs. This is largely due to the fact that the overall complexity of the nucleus has made it impossible to study a subset of interactions by using intact nuclei. To circumvent this problem we have been developing a simple model system using purified components to identify the structural components which mediate binding of chromatin to the nuclear envelope and to identify the proteins which regulate these interactions. the system consists of membrane vesicles, isolated from Xenopus eggs, which will bind to chromatin and fuse to form an intact nuclear envelope. Using this system we have shown that the interaction between the vesicles and chromatin is mediated by a membrane receptor and a chromatin bound protein. Further, we have shown that the interaction between these two proteins is regulated by a kinase/phosphatase system which phosphorylates the receptor. In this grant we propose to use this simple model system to investigate in detail how membrane-chromatin interactions occur, how they are regulated, and whether changes in these associations effect nuclear functions such as chromosome decondensation and DNA replication. Specifically, we propose to: 1) isolate the receptor and chromatin binding protein which mediate chromatin- membrane association; 2) isolate the kinase and phosphatase which regulate this association; 3) combine these purified components together to reconstruct a model system for studying chromatin-membrane dynamics; 4) determine whether changes in the dynamics of this system under "real" conditions perturbs nuclear function such as chromosome condensation and DNA replication. The results from these studies will provide valuable information about envelope function as well as extend our knowledge about how higher order DNA structure contributes to DNA function.