Journal
BIOCHEMISTRY
Volume 42, Issue 7, Pages 2137-2148Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi026806o
Keywords
-
Categories
Funding
- NCI NIH HHS [CA86258] Funding Source: Medline
- NIAID NIH HHS [AI41944] Funding Source: Medline
- NIDDK NIH HHS [DK61803] Funding Source: Medline
Ask authors/readers for more resources
In previous studies, we have developed a technology for the rapid construction of novel DNA-binding proteins with the potential to recognize any unique site in a given genome. This technology relies on the modular assembly of modified zinc finger DNA-binding domains, each of which recognizes a three bp subsite of DNA. A complete set of 64 domains would provide comprehensive recognition of any desired DNA sequence, and new proteins could be assembled by any laboratory in a matter of hours. However, a critical parameter for this approach is the extent to which each domain functions as an independent, modular unit, without influence or dependence on its neighboring domains. We therefore examined the detailed binding behavior of several modularly assembled polydactyl zinc finger proteins. We first demonstrated that 80 modularly assembled 3-finger proteins can recognize their DNA target with very high specificity using a multitarget ELISA-based specificity assay. A more detailed analysis of DNA binding specificity for eight 3-finger proteins and two 6-finger proteins was performed using a target site selection assay. Results showed that the specificity of these proteins was as good or better than that of zinc finger proteins constructed using methods that allow for interdependency. In some cases, near perfect specificity was achieved. Complications due to target site overlap were found to be restricted to only one particular amino acid interaction (involving an aspartate in position 2 of the a-helix) that occurs in a minority of cases. As this is the first report of target site selection for designed, well characterized 6-finger proteins, unique insights are discussed concerning the relationship of protein length and specificity. These results have important implications for the design of proteins that can recognize extended DNA sequences, as well as provide insights into the general rules of recognition for naturally occurring zinc finger proteins.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available