THE MECHANISMS OF ISW1A, ISW1B AND ISW2 CHROMATIN REMODELERS

Author

Saurabh Kant Bhardwaj, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2015

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

KADYROV, FARID

Abstract

The focus of this study was to understand the nucleosome remodeling mechanism of three ISWI complexes in yeast using various biochemical approaches. The aspects investigated were, identification of the suitable substrate for ISW1a, role of accessory subunits and various domains/motifs of ISWI complexes in nucleosome remodeling, and detailed remodeling mechanism of nucleosomes by ISW1a. ISW1a and ISW1b share the same catalytic subunit, but in vivo they are localized at different locations and is also known to have different roles. This suggests that the accessory subunits play a very crucial role in determining the identity of each complex. In in-vitro studies, ISW1a showed preference for a dinucleosomal substrate for efficient binding and remodeling with no defect in ATP hydrolyzing ability with any sort of substrates. The footprinting analysis of ISW1a bound mononucleosomal substrate revealed the unique protection of nucleosome at dyad axis never reported before with any other remodeler. In case of a dinucleosomal substrate, ISW1a could selectively dock its ATPase domain to one of the nucleosome with the bias for the one with single flanking DNA. Deletion of the portion of HLB domain of Ioc3 subunit resulted in the loss of spacing ability of ISW1a. It also reduced the nucleosome remodeling and binding ability of ISW1a and the ability to orient on an asymmetric dinucleosomal substrate. Therefore, Ioc3 subunit of ISW1a is important for the remodeler to sense that the uneven linker DNA flanking the nucleosomes and spacing them equally and efficiently. ISW1b on the other hand exhibits very different remodeling properties and could remodel mono and dinucleosomes efficiently. It lacks the spacing activity moving nucleosomes in bi-directions and site directed mapping revealed that ISW1b could push the nucleosomes off the edge of the DNA. Deletion of the PHD domain of Ioc2 subunit results in the unidirectional movement with inability to push the DNA off the edge of the DNA. The C-terminus region of ISWI complexes consists of signature HAND-SANT-SLIDE domains. This module is important for the functioning of the ATPase domain for efficient remodeling of the nucleosome. ATPase domain could push the DNA out of the exit side but requires SLIDE domain to push the DNA in at the entry site. The lack of pushing in of the DNA at the entry site by SLIDE domain results in inability of the DNA to pushed further out by ATPase domain. Therefore, it is a concerted effort of the C-terminus of Isw2 and the ATPase domain to mobilize the nucleosomal DNA.