Site Directed Mutagenesis, Expression and Enzymatic Studies of the 60 kDa Human HIV-TAT 1 Interactive Protein, TIP60

Tip60 is a 60 kDa nuclear protein which exists in three isoforms, belongs to the MYST/HAT family of proteins and was discovered after its interaction with the Human HIV-1 Tat. As a nuclear protein, Tip60 can act as a coactivator or repressor. To understand the HAT action of Tip60, two possible catalytic models exist; the ping-pong and the ternary complex formation models. In correlation with the exploration of HAT catalytic action, mutations of a Cys to Ala and a Glu to Gln on Esa1 (yeast homolog of Tip60 and MYST/HAT prototype), was reported to show wild type-like and decreased acetylating properties, respectively. In this work, Tip60 HAT action was explored. In Tip60, the Cys in the active site is important for acetylation of the H4(120) substrate and the Glu showed semi loss in acetylating the H4(1-20) peptide substrate. These data highlight a unique mechanism of Tip60 catalysis. INDEX WORDS: Tip60, Site-directed mutagenesis, HATs, MYST family HATs, Histones, Chromatin, Chromatin modification, Post-translational modification, Tip60 catalysis, HAT assay, His-tagged Protein Expression, Histone acetylation and HAT inhibitors SITE DIRECTED MUTAGENESIS, EXPRESSION, AND STEADY-STATE KINETIC STUDIES OF THE HUMAN HIV-TAT 1 INTERACTIVE PROTEIN, TIP60