Generation of single-chain LAGLIDADG homing endonucleases from native homodimeric precursor proteins

Abstract

Homing endonucleases (HEs) cut long DNA target sites with high specificity to initiate and target the lateral transfer of mobile introns or inteins. This high site specificity of HEs makes them attractive reagents for gene targeting to promote DNA modification or repair. We have generated several hundred catalytically active, monomerized versions of the well-characterized homodimeric I-CreI and I-MsoI LAGLIDADG family homing endonuclease (LHE) proteins. Representative monomerized I-CreI and I-MsoI proteins (collectively termed mCreIs or mMsoIs) were characterized in detail by using a combination of biochemical, biophysical and structural approaches. We also demonstrated that both mCreI and mMsoI proteins can promote cleavage-dependent recombination in human cells. The use of single chain LHEs should simplify gene modification and targeting by requiring the expression of a single small protein in cells, rather than the coordinate expression of two separate protein coding genes as is required when using engineered heterodimeric zinc finger or homing endonuclease proteins.

The Structural Basis of Asymmetry in DNA Binding and Cleavage as Exhibited by the I-SmaMI LAGLIDADG Meganuclease

“…In turn, the observed conformational changes are likely a prerequisite for cleavage to proceed. This concept is in agreement with the published literature for these proteins: several previous studies have consistently demonstrated that their specificity of cleavage (which required both the formation of a tightly bound complex and then the precise ordering and arrangement of both active sites around the two scissile phosphates) is considerably higher than their specificity of binding (which is largely dependent only on the establishment of an initial series of contacts, often largely localized to one protein domain) [21,37,38]. …”

Homing endonucleases from mobile group I introns: discovery to genome engineering

“…Because wild-type I-CreI is a natural homodimeric enzyme, both efforts rely upon the ‘monomerization’ of the I-CreI protein to create a single-chain reagent in which the two subunits of the enzyme are linked by a peptide tether and then expressed in cis as a monomeric scaffold [110-112]. Armed with this construct, redesign efforts can then be conducted on individual protein domains (targeting corresponding half-sites of the desired genomic target) with the resulting constructs combined into a single polypeptide which is further optimized for optimal in vivo performance.…”

Engineered Meganucleases for Genome Engineering Purposes

“…Computational studies have given rise to not only speci fi city re-engineering (Ashworth et al 2006 ) , but have also addressed the dimerization issue via targeting protein-protein interactions of the subunits (Fajardo-Sanchez et al 2008 ) as well as through mimicking nature with single-chain constructs (Grizot et al 2009, Li et al 2009 ) . Exhaustive analysis of the I-AniI scaffold Thyme et al 2009 ) has led to better computational methods that can exploit binding energy for designs (Thyme et al 2009 ) .…”