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Massively parallel determination and modeling of endonuclease 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.

SUBMITTER: Thyme SB

PROVIDER: S-EPMC4267613 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Massively parallel determination and modeling of endonuclease substrate specificity.

Thyme Summer B SB Song Yifan Y Brunette T J TJ Szeto Mindy D MD Kusak Lara L Bradley Philip P Baker David D

Nucleic acids research 20141111 22

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. ...[more]

PMID: 25389263

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