SWI/SNF-NUCLEOSOME INTERACTIONS AND DISASSEMBLY OF NUCLEOSOMES: NOVEL METHODOLOGIES FOR MAPPING PROTEIN-PROTEIN AND PROTEIN-DNA INTERACTION

Author

Mekonnen Lemma Dechassa, Southern Illinois University CarbondaleFollow

Date of Award

1-1-2008

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Bartholomew, Blaine

Abstract

AN ABSTRACT OF THE DISSERTATION OF MEKONNEN LEMMA DECHASSA, for the Doctor of Philosophy degree in MOLECULAR BIOLOGY, MICROBIOLOGY, AND BIOCHEMISTRY, presented on December 18, 2008, at Southern Illinois University Carbondale. TITLE: SWI/SNF-NUCLEOSOME INTERACTIONS AND DISASSEMBLY OF NUCLEOSOMES: NOVEL METHODOLOGIES FOR MAPPING PROTEIN-PROTEIN AND PROTEIN-DNA INTERACTION MAJOR PROFESSOR: Dr. Blaine Bartholomew The SWI/SNF complex disrupts and mobilizes chromatin in an ATP-dependent manner. A site-directed photoaffinity crosslinking approach in which photoreactive moieties attached at specific sites within histone octamer was developed and used to map the interactions of SWI/SNF with the histone octamer face of the nucleosome. We identified the subunits that contact the nucleosomal histone proteins. The catalytic Swi2/Snf2 and Snf5 subunit were found to interact with a large surface of nucleosomal histone proteins. Affinity proteolysis, using FeEDTA that is attached to DNA or the histone octamer as a probe, was applied to identify the domains and motifs of SWI/SNF subunits that interact with DNA and nucleosome. It was found that motifs in the N-terminal lobe of ATPase/helicase domain of Sw2/Snf2 are in close contact with both DNA and histone octamer. Crosslinking and peptide mapping by chemical proteolysis revealed that the region in the C-terminal lobe of ATPase/helicase domain of Swi2/Snf2 is bound to the internal nucleosome region that is two helical turns from the dyad axis. The impact on nucleosome remodeling by adjacent nucleosomes and the recruitment of SWI/SNF by transcriptional activator was examined using a high resolution histone-DNA contact mapping and a single molecule MAP-IT technique. The data shows that the presence of adjacent nucleosomes promotes nucleosome eviction and the recruitment of SWI/SNF by Gal4-VP16 to dinucleosomes restricted the nucleosome mobilization in one direction. Finally, based on the data from this study and previous reports, the mechanism how recruitment and neighboring nucleosomes alter the outcome of SWI/SNF remodeling is discussed.