A eukaryotic cell must be able to transport macromolecules directionally between its nucleus and cytoplasm, and to divide the cell through mitosis. These fundamental processes are controlled by localizing the small Ran guanosine triphosphatase (GTPase) protein in its nucleotide bound state within the cytoplasm or the nucleus, and by generating a gradient of RanGTP around the chromosomes. This spatial localization of RanGTP in the nucleus is achieved through chromatin bound RCC1 (regulator of chromosome condensation) protein. RCC1 recruits Ran to the nucleosome repeating unit of chromosomes and promotes the exchange of RanGDP for RanGTP, thereby creating a high concentration of RanGTP around chromosomes. The Ran/RCC1/nucleosome complex thus regulates fundamental processes critical for a eukaryotic cell to function properly. Our biochemical studies and crystal structure of the RCC1/nucleosome determined in the last funding period showed how RCC1 binds to the nucleosome. These studies also present new questions about the Ran/RCC1/nucleosome complex, including (a) what regions of Ran interact with RCC1/nucleosome in the Ran/RCC1/nucleosome complex, (b) the role of conformational changes in RCC1/nucleosome upon Ran binding and (c) possible species differences in how Ran and RCC1 interact with the nucleosome. Our goal is therefore to describe how RCC1 and Ran from different species interact with the nucleosome core particle in atomic detail. This proposal focuses on three specific aims: 1. Determine the structure of the Ran/RCC1/nucleosome complex. We will improve single crystals of the Ran/RCC1/nucleosome complex using pre- and post-crystallization strategies to determine the atomic structure of the Ran/RCC1/nucleosome complex. 2. Define how Ran interacts with the nucleosome in Ran/RCC1/nucleosome complexes. We propose biochemical studies to understand which Ran regions interact with the nucleosome in Ran/RCC1/nucleosome complexes from different species, and how these interactions affect Ran's nucleotide exchange activity. 3. Determine role of conformational changes in RCC1/nucleosome & Ran/RCC1/nucleosome complexes. We will use fluorescent probes installed on the nucleosome to study how Ran and RCC1 interact with the nucleosome, and the role of conformational changes in this binding.
When a cell divides, each daughter cell must receive an equal share of the chromosomes which carry the cell's genetic blueprint. Unequal or improper distribution of the chromosomes can result in genetic instabilities and cancer. Our studies are directed at visualizing the molecules which create a GPS or genome-positioning system for a eukaryotic cell and which regulate the equal distribution of chromosomes during cell division.
Showing the most recent 10 out of 19 publications
