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 GDP or GTP bound state within the cytoplasm or the nucleus respectively, and by generating a gradient of RanGTP around the chromosomes. The spatial localization of RanGTP in the nucleus is achieved through chromatin bound RCC1 (regulator of chromosomal condensation) protein. RCC1 recruits Ran to the chromosomes and promotes the exchange of RanGDP for RanGTP, thereby creating a high concentration of RanGTP around chromosomes. We currently lack a molecular understanding of how RCC1 binds to the nucleosome and how RCC1 recruits Ran to the nucleosome, despite the critical importance of these interactions for basic cellular processes. Our overall goal is therefore to develop atomic models which describe how RCC1 and Ran bind to the nucleosome core particle.
Our specific aims are: 1. Define how RCC1 binds to nucleosomes through biochemical methods. We will challenge structural models for how RCC1 interacts with the nucleosome through pulldown, biolayer interferometry and fluorescence resonance energy transfer experiments. 2. Determine the structure of the RCC1/nucleosome complex. We will use single crystals of the RCC1/nucleosome complex we have grown to determine the structure of the complex. These crystallographic studies will be complemented with small angle X-ray and neutron scattering experiments to provide a solution structure of the complex. 3. Determine how chromatin-bound RCC1 binds to and activates Ran. We will test models for the Ran/RCC1/nucleosome complex by analyzing the effects of directed mutations on binding of Ran to the RCC1/nucleosome complex and on Ran's nucleotide exchange activity in the presence of RCC1 and the nucleosome.

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 regulate the equal distribution of chromosomes during cell division by creating a GPS or genome-positioning system for a eukaryotic cell.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM088236-04
Application #
8311671
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter C
Project Start
2009-08-01
Project End
2013-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
4
Fiscal Year
2012
Total Cost
$298,803
Indirect Cost
$92,982
Name
Pennsylvania State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
003403953
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