ＣｏｌＥ７を用いたアロステリック制御機構を持った人工ヌクレアーゼの開発

2014 Fiscal Year Annual Research Report

• Project/Area Number: 14F04807
• Research Institution: University of Tsukuba
• Principal Investigator: 永田 恭介 筑波大学, 学長 (40180492)
• Co-Investigator(Kenkyū-buntansha): NEMETH Eszter 筑波大学, 医学医療系, 外国人特別研究員
• Project Period (FY): 2014-04-25 – 2017-03-31
• Keywords: Artificial nuclease / DNA repair / Zinc finger nuclease / Chromatin

Outline of Annual Research Achievements

Artificial nucleases are designed to induce double or single strand break to　the desired target side of chromosomal DNA, inducing the DNA repair mechanisms of cells. Thus gene correction or the insertion of a template sequence becomes feasible. However, current nucleases are not safe enough for gene therapy.
My project includes the design and examine novel zinc finger nucleases (ZFN) that are controlled on the molecular level. This control is assured by the cooperation of the N- and C-termini, upon binding to the DNA substrate. The most successful model so far is N4-ZF-C105. ZF is the zinc finger providing the sequence specific DNA-binding, C105 is the inactive nuclease domain. Based on computational simulations, the latter can be activated by the N4 residues, upon binding to the substrate.
In the first 5 months of my JSPS term I completed two points of my research plan. 1) As a continuation of my preceding work, I performed the in vitro analysis of double strand break by the designed ZFN. According to these studies, the enzyme cleaves DNA in a sequence specific manner as expected. However, the recognition site is ambiguous which is related to the sequence restrictions of the zinc finger motif itself. 2) After cloning of the desired recombinant protein gene, I expressed and purified the truncated control protein ZF-C105. The comparison of this protein with N4-ZF-C105 reveals the role of the N4 residues on the N-terminus. According to my recent studies, the nuclease activity of the truncated mutant is lower.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

I completed two important goals set in my research plan. I characterized the reaction between the purified enzyme and purified DNA molecule in vitro. The results gave the proof of concept regarding the computational modelling. The results of ZF-C105 protein are promising for the molecular control via the N4 N-terminus. This control is important for the safety during the applications. If the protein binds to DNA in a nonspecific manner, or the protein is injured and its sequence is not complete, the reaction is not possible. On this way the side reactions meaning the main danger of the artificial nucleases are diminished. The results achieved so far are consistent with my research plan and gave a positive base for my further research.
During the in vivo application of zinc finger nucleases, the substrate is the chromosome consisting of DNA and DNA-binding proteins. In order to improve such applications, it is required to understand the molecular mechanisms of chromatin remodelling. The laboratory of Prof. Kyosuke Nagata is specialized in this topic and my stay here is an excellent opportunity to conduct research related to the chromatin of mammalian cells. The linker histone H1 has a key role in this phenomenon, stabilizing the structure of the chromatosome. However, the chaperon-mediated association and dissociation of this protein from chromatin is not yet fully understood. Therefore, I became interested in the biochemistry of this phenomenon. So far, I performed the expression and purification of the histone H1.1 protein.

Strategy for Future Research Activity

The plan of future work is a continuation of my present research related to controlled artificial nucleases and chromatin remodeling. I plan to reveal the ambiguity of the DNA recognition of N4-ZF-C105 finishing the in vitro studies, and start to prepare a manuscript from the experimental results. In the active collaboration with Dr. Bela Gyurcsik in University of Szeged, Hungary, we will complement the results with structural studies, such as CD-spectroscopy. The next goal is to examine the designed proteins in mammalian cell lines after transfection with the nuclease expressing plasmid and appropriate DNA template. The detection of in vivo DNA cleavage is possible with a reporter plasmid and/or bacterial DNA selection experiments. In later stages, I plan to design and examine further controlled artificial nucleases.
Furthermore, I plan to study the dissociation of histone H1 from the chromatin, in biochemical assays under different conditions. First, I will reconstitute the chromatosome itself from a short DNA, core histone proteins and histone H1. Next, the study of different phenomena related to the molecular composition of the chromatosome are feasible.
My results are expected extend a new platform of artificial nucleases, that have a built in control mechanism, providing enhanced safety during applications. Furthermore, they will contribute to the understanding of the mechanism how artificial and endogenous enzymes can access the DNA embedded in chromatin.