Thyme Summer B, Song Yifan, Brunette T J, Szeto Mindy D, Kusak Lara, Bradley Philip, Baker David
Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
Nucleic Acids Res. 2014 Dec 16;42(22):13839-52. doi: 10.1093/nar/gku1096. Epub 2014 Nov 11.
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.
我们描述了使用基因组数据库挖掘来识别假定靶位点，随后在细菌筛选系统中进行高通量活性筛选，从而鉴定和表征新型归巢内切酶的过程。我们通过深度测序监测DNA切割事件，对这些内切酶的底物特异性和动力学进行了表征。通过基于三维结构的计算模型，可以部分重现这些实验所揭示的内切酶特异性。对这些模型以及基因组序列数据的分析，为深入了解自然进化过程中如何产生替代内切酶特异性提供了思路。
