SITE SPECIFIC INCORPORATION OF PHOTOREACTIVE NON NATURAL AMINOACID IN THE ATPASE DOMAIN OF ISW2

Author

Samarth Bilash Upadhya, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2016

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

davie, judy

Second Advisor

Bartholomew, Blaine

Abstract

ATP dependent Chromatin Remodelers hydrolyze ATP in order to move DNA around nucleosomes. ISWI families of remodelers act throughout genes in promoter region, gene body and terminator to mobilize & space nucleosome to regulate gene expression. ISW2 is a four subunit protein complex which acts on an end positioned nucleosome and converts it into a centrally positioned nucleosome. Among the four subunits, the largest is the Itc1, the catalytic subunit is Isw2 and two other small subunits are Dpb4 and Dls1. The process of translocation starts from the evolutionary conserved core ATPase domain of catalytic subunit Isw2 which hydrolyses ATP and cause DNA movement around the histone octamers. Tracking the movement of ATPase domain along the DNA during nucleosomal movement would allow us to decipher the steps and kinetics of the enzyme translocating on DNA. To accomplish this goal, we expanded the genetic code to replace non conserved amino acid with photoreactive non-natural amino acid into the ATPase domain of ISW2 protein in a site specific manner. We used non sense suppression strategy to accomplish this. A total of nine different mutant proteins were purified. Through gel shift assays (binding, cross linking & remodeling) and ATPase assay, all nine mutants were characterized. Five out nine mutants lying between the residues 445 to 451 in the Isw2 protein showed cross linking with 100 base pair DNA. All these mutants lie in the protrusion 2 within lobe 2. This result suggests that the amino acids between residues 445 to 451 in an Isw2 protein are close to the DNA binding pocket of ATPase domain. It was found that the mutant K446X had the maximum binding affinity and cross linking ability with DNA. It was also shown that the binding and cross linking ability of the mutant ISW2 protein was enhanced in the presence of H4 peptide and gamma thio ATP. With successful optimization of this technique, we were able to produce native, active modified protein in large scale which can be used in in vitro experiments to study the steps and kinetics of enzyme translocation on DNA. This technique can also be used in in vivo studies for enzyme’s localization to different genic regions through ChIP.