Massively parallel determination and modeling of endonuclease substrate specificity.

Publication Type:

Journal Article

Source:

Nucleic acids research, Volume 42, Issue 22, p.13839-52 (2014)

Keywords:

Base Sequence, Computer Simulation, DNA, DNA Cleavage, Endodeoxyribonucleases, High-Throughput Nucleotide Sequencing, Models, Molecular, Sequence Analysis, DNA, Substrate Specificity

Abstract:

We describe the identification and characterization of novel homing endonucleases using genome database mining to identify putative target sites, followed by high throughput activity screening in a bacterial selection system. We characterized the substrate specificity and kinetics of these endonucleases by monitoring DNA cleavage events with deep sequencing. The endonuclease specificities revealed by these experiments can be partially recapitulated using 3D structure-based computational models. Analysis of these models together with genome sequence data provide insights into how alternative endonuclease specificities were generated during natural evolution.