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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
2R01GM088236-05A1
Application #
8777837
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
University Park
State
PA
Country
United States
Zip Code
16802
McGinty, Robert K; Henrici, Ryan C; Tan, Song (2014) Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome. Nature 514:591-6
Tan, Song; Nagai, Kiyoshi (2013) Protein-nucleic interactions: 'I have a cunning planýýý'. Curr Opin Struct Biol 23:90-2
Makde, Ravindra D; Tan, Song (2013) Strategies for crystallizing a chromatin protein in complex with the nucleosome core particle. Anal Biochem 442:138-45
Tan, Song (2012) Deciphering how the chromatin factor RCC1 recognizes the nucleosome: the importance of individuals in the scientific discovery process. Biochem Soc Trans 40:351-6
Tan, Song; Davey, Curt A (2011) Nucleosome structural studies. Curr Opin Struct Biol 21:128-36
England, Joseph R; Huang, Jiehuan; Jennings, Matthew J et al. (2010) RCC1 uses a conformationally diverse loop region to interact with the nucleosome: a model for the RCC1-nucleosome complex. J Mol Biol 398:518-29