Elucidation of the nonspecific DNA-binding mechanism of Oct3/4 using NMR

2014 Fiscal Year Final Research Report

• Project/Area Number: 25840059
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Biophysics
• Research Institution: Suntory Foundation for Life Sciences
• Principal Investigator: TSUYOSHI Konuma 公益財団法人サントリー生命科学財団, その他部局等, 研究員 (10631682)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Keywords: 非特異的DNA結合

Outline of Final Research Achievements

In this study, we have investigated dynamics of POU homeodomain (POUHD) of Oct3/4 upon DNA binding using NMR. Firstly, we titrated DNA into POUHD to characterize the conformational changes and binding kinetics upon DNA binding. The quantitative analysis of the chemical shift changes yielded kinetic parameters for POUHD binding to DNA. Subsequently, to obtain structural information on the nonspecific bound states, CLEANEX-PM was applied to POUHD in the presence of excess amounts of POUHD over DNA to increase the population of the nonspecific bound states. As a result, the amide protons of the N-terminal loop are protected from solvent by the bound DNA in the nonspecific binding states, whereas those of the helix region remain to be exchangeable with water protons. Therefore, it is suggested that the N-terminal loop first interacts with nonspecific sites of DNA, and then the stable complex with DNA is formed by locating the helix region to its specific target site.

Free Research Field

構造生物学