Massively parallel determination and modeling of endonuclease substrate specificity

Academic Article

Abstract

  • © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. 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.
  • Authors

    Digital Object Identifier (doi)

    Author List

  • Thyme SB; Song Y; Brunette TJ; Szeto MD; Kusak L; Bradley P; Baker D
  • Start Page

  • 13839
  • End Page

  • 13852
  • Volume

  • 42
  • Issue

  • 22