Poster abstracts
Poster number 80 submitted by Stephanie Mack
The Effect of Metal Ions on Lariat Debranching Enzyme Cleavage Activity
Stephanie Mack (Chemistry, Carnegie Mellon University), Subha R. Das (Chemistry, Carnegie Mellon University)
Abstract:
Lariat debranching enzyme (Dbr1p) is a metallophosphoesterase that specifically cleaves the 2'-5' phosphate linkage in lariats introns formed during splicing. It is a crucial enzyme and deletion is lethal in most organisms. The homology of the Dbr1p conserved active site residues to those of the DNA repair enzyme MRE11 as well as early in vitro experiments on Saccharomyces cerevisiae Dbr1p suggested that the active site harbors two Mn2+ ions for proper function. However, recent crystal structures of Entamoeba histolytica Dbr1p suggest Fe2+ and Zn2+ or Mn2+ and Zn2+ as the active site metal ions needed for full activity. We have found, intriguingly, while S. cerevisiae Dbr1p is inactivated by EDTA, E. histolytica Dbr1p retains significant activity. Here we present our assays on the debranching activity of E. histolytica Dbr1p with different metal ions following EDTA treatment. We report the effect of ions of Group II metals as well as transition metals on the observed rate constant for cleavage of branched RNA substrates.
Keywords: Lariat debranching enzyme, Branched RNA, Metallophosphoesterase