期刊
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
卷 396, 期 2, 页码 323-328出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2010.04.091
关键词
ARID; JARID1B/PLU-1; EMSA; Protein-DNA interaction; NMR; Intrinsic fluorescence
资金
- Natural Science Foundation of China [30730026]
- Chinese Academy of Sciences (CAS) [KSCX2-YW-R-104]
- Shanghai Institute of Materia Medica, CAS [07G603N058]
JARID1B, a member of the JmjC demethylase family, has a crucial role in H3K4me3 demethylation. The ARID domain is a potential DNA-binding domain of JARID1B. Previous studies indicate that a GC-rich DNA motif is the specific target of the ARID domain. However, the details of the interaction between the ARID domain and duplex DNA require further study. Here, we utilized NMR spectroscopy to assign the backbone amino acids and mapped the DNA-binding sites of the human JARID1B ARID domain. Perturbations to H-1-N-15 correlation spectra revealed that the flexible loop L1 of ARID was the main DNA-binding interface. EMSA and intrinsic fluorescence experiments demonstrated that mutations on loop L1 strongly reduced the DNA-binding activity of JARID1B ARID. Furthermore, transfection of mutant forms resulted in a distinct loss of intrinsic H3K4 demethylase activity, implying that the flexible loop L1 made a major contribution to sustaining the DNA-binding ability of JARID1B ARID domain. (C) 2010 Elsevier Inc. All rights reserved.
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