Positive and negative cooperativity of modularly assembled zinc fingers

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Abstract

One simple and widespread method to create engineered zinc fingers targeting the desired DNA sequences is to modularly assemble multiple finger modules pre-selected to recognize each DNA triplet. However, it has become known that a sufficient DNA binding affinity is not always obtained. In order to create successful zinc finger proteins, it is important to understand the context-dependent contribution of each finger module to the DNA binding ability of the assembled zinc finger proteins. Here, we have created finger-deletion mutants of zinc finger proteins and examined the DNA bindings of these zinc fingers to clarify the contributions of each finger module. Our results indicate that not only a positive cooperativity but also a context-dependent reduction in the DNA binding activity can be induced by assembling zinc finger modules.

Section snippets

Materials and methods

Chemicals. The modification enzymes and restriction enzymes were purchased from New England Biolabs, except for AgeI that was obtained from Nippon Gene. The Taq DNA polymerase was acquired from Nippon Gene. The synthesized oligonucleotides were supplied from Invitrogen. All other chemicals were of commercial reagent grade.

Construction of expression vectors and reporter vectors. The multi-cloning sites containing the XmaI, EcoRI, and BamHI restriction sites were introduced between the SacI and

Creation of 6-zinc finger proteins targeting a specific E-box

As the targets of the artificial zinc finger proteins, we selected the E-box-containing sequences on the mouse Period1 (mPer1) promoter involved in the regulation of the circadian clock. There are at least five E-box sequences (E1–E5) on the mPer1 promoter [19], but the contribution of each E-box is unclear. It is required to create site-specific zinc finger proteins that can recognize a specific E-box for future genomic promoter analyses. In order to target a specific E-box, we paid attention

Acknowledgments

This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan and Takeda Science Foundation to M.I. A.N. and T.M. are research fellows of the Japan Society for the Promotion of Science.

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