Abstract
A protein roadblock forms when a protein binds DNA and hinders translocation of other DNA binding proteins. These roadblocks can have significant effects on gene expression and regulation as well as DNA binding. Experimental methods for studying the effects of such roadblocks often target endogenous sites or introduce non-variable specific sites into DNAs to create binding sites for artificially introduced protein roadblocks. In this work, we describe a method to create programmable roadblocks using dCas9, a cleavage deficient mutant of the CRISPR effector nuclease Cas9. The programmability allows us to custom design target sites in a synthetic gene intended for in vitro studies. These target sites can be coded with multivalency—in our case, internal restriction sites which can be used in validation studies to verify complete binding of the roadblock. We provide full protocols and sequences and demonstrate how to use the internal restriction sites to verify complete binding of the roadblock. We also provide example results of the effect of DNA roadblocks on the translocation of the restriction endonuclease NdeI, which searches for its cognate site using one dimensional diffusion along DNA.
Funder
Directorate for Biological Sciences
Publisher
Public Library of Science (PLoS)
Reference17 articles.
1. DNA replication roadblocks caused by Cascade interference complexes are alleviated by RecG DNA repair helicase;Tom Killelea;RNA biology,2019
2. Dynamic DNA binding licenses a repair factor to bypass roadblocks in search of DNA lesions;Maxwell W Brown;Nature communications,2016
3. Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase;Mari-Liis Visnapuu Ilya J Finkelstein;Nature,2010
4. The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair;Yannicka SN Mardenborough;Nucleic acids research,2019
5. Nuclease dead Cas9 is a programmable roadblock for DNA replication;Kelsey S Whinn;Scientific reports,2019
Cited by
1 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献