ROLES OF ACCESSORY DOMAINS IN THE CATALYTIC SUBUNIT OF ISW2

Author

Yuan-chi Lin, Southern Illinois University CarbondaleFollow

Date of Award

8-1-2017

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Bartholomew, Blaine

Abstract

Regulation of chromatin structures in eukaryotes involves ATP-dependent chromatin remodeler complexes. ISW2 belongs to the ISWI family of chromatin remodelers in Saccharomyces cerevisiae and has been shown to be involved in transcription repression and DNA replication. The catalytic subunit of ISW2 consists of the ATPase domain and three domains at the C-terminus called HAND, SANT and SLIDE (HSS) domains. The individual roles of each HSS domains and how they coordinate with the ATPase domain during remodeling are not completely understood. Initial studies of SLIDE domain from our lab reveal its role in nucleosomal DNA propagation during remodeling process. In this study we focused on the SANT domain and found other mechanism of regulation that are independent of the H4 tail. We deleted SANT domain (ΔSANT) and also created mutations in three basic amino acid residues (mSANT) on the surface of SANT domain predicted to be interacting with nucleosomal DNA. The absence of SANT domain affected ISW2 activity neither through ATP hydrolysis nor DNA translocation, but in the mechanism of remodeling process. The effects of SANT domain on ISW2 remodeling were analyzed by site-directed mapping at specific histone octamer positions to track nucleosomal DNA movement near the nucleosome entry/exit and DNA translocation sites. Without SANT domain, ISW2 was unable to efficiently move DNA in the appropriate direction and the remodeling process is halted after making the first complete step of DNA movement. We provide evidence that SANT domain regulated ISW2 activity without affecting ATPase and DNA translocation activity and was dependent on conformation changes of histone octamers instead of histone H4 tail. Residues in the Isw2 ATPase domain acidic patch as identified in previous studies have been selected and mutated to examine the effects. The biochemical characteristics of ATPase domain mutations and structure comparison with solved ISWI ATPase domain indicate the acidic patch is unlikely to interact with DNA as predicted previously. In summary, the understandings of Isw2 ATPase domain and C-terminus domains support the assumption that there is no redundancy of their roles in nucleosome remodeling.