PDF《protein》

Int.

J. Biol. Sci. 2007,

3 205 International Journal of Biological Sciences ISSN 1449-2288 www.biolsci.org

2007 3(4):205-211 ? Ivyspring International Publisher. All rights reserved Research Paper Structural stability and endonuclease activity of a PI-SceI GFP-fusion protein Alireza G. Senejani and J. Peter Gogarten Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT, 06269-3125 U.S.A. Correspondence to: J. Peter Gogarten, E-mail: [email protected], Phone: (860) 486-4061, Fax: (860) 486-4331 Received: 2006.11.16;

Accepted: 2007.02.15;

Published: 2007.02.16 Homing endonucleases are site-specific and rare cutting endonucleases often encoded by intron or intein containing genes. They lead to the rapid spread of the genetic element that hosts them by a process termed '

homing'

;

and ultimately the allele containing the element will be fixed in the population. PI-SceI, an endonuclease encoded as a protein insert or intein within the yeast V-ATPase catalytic subunit encoding gene (vma1), is among the best characterized homing endonucleases. The structures of the Sce VMA1 intein and of the intein bound to its target site are known. Extensive biochemical studies performed on the PI-SceI enzyme provide information useful to recognize critical amino acids involved in self-splicing and endonuclease functions of the protein. Here we describe an insertion of the Green Fluorescence Protein (GFP) into a loop which is located between the endonuclease and splicing domains of the Sce VMA1 intein. The GFP is functional and the additional GFP domain does not prevent intein excision and endonuclease activity. However, the endonuclease activity of the newly engineered protein was different from the wild-type protein in that it required the presence of Mn2+ and not Mg2+ metal cations for activity. Key words: Homing endonuclease, intein, GFP-fusion protein, PI-SceI, Sce VMA 1. Introduction Homing endonucleases are found as domains in inteins, or encoded by open reading frames in genetically mobile introns [1]. Similar to restriction enzymes, homing endonucleases cleave double stranded DNA, but recognize a larger DNA sequence of 14-40 nucleotides. Based on the type of conserved sequence motifs, homing endonucleases are classified into four major families: LAGLIDADG, GIY-X-YIG, H-N-H, and His-Cys [2]. Homing endonucleases of the '

LAGLIDADG'

type are the largest known family [3], and the ones most frequently found in inteins [4]. Homing endonucleases facilitate transfer of their host genetic elements into intronless, or inteinless sites via a process called '

homing'

. The homing process begins when the homing endonuclease makes a site-specific, double-strand break in the intronless, or inteinless allele [5, 6]. During the repair of the cleaved gene, which relies on the host'

s repair machinery, the intron/intein is copied to the previously intron/intein free homolog [1, 7]. Homing endonucleases and the process of homing have been more intensively studied in self splicing introns [1, 2, 7], and the process is assumed to be similar for inteins. PI-SceI homing endonuclease is encoded as the only active intein identified in Saccharomyces cerevisiae (Sce VMA1 intein). PI-SceI is the name used in the homing endonuclease field to denote the Sce VMA1 intein [1], which is also known as VMA1 Derived Endonuclease (VDE) [8]. The structure of the Sce VMA1 intein and of the intein bound to its target site are known, with the self-spicing and endonuclease catalytic domains clearly defined [9, 10]. In addition to these two major domains the Sce VMA1 intein contains a sub-domain, near the self-splicing domain, that is involved in DNA recognition [10]. The PI-SceI homing endonuclease recognizes a long DNA sequence of more than