Massively parallel determination and modeling of endonuclease substrate specificity.
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.
Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA sthyme@gmail.com.
Summer B
SB
Thyme
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Yifan
Y
Song
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
T J
TJ
Brunette
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Mindy D
MD
Szeto
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Lara
L
Kusak
Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.
Philip
P
Bradley
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue, Seattle, WA 98109, USA dabaker@u.washington.edu.
David
D
Baker
D001483:Base Sequence; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry; D053837:DNA Cleavage; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D008958:Models, Molecular; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D004247:DNA; D004706:Endodeoxyribonucleases