NMR Studies On Solution Structure Of The Human JARID1B-ARID Domain And Its Interaction With DNA

With the development of computer science, electronic technology, high field magnet and pulse sequences, the Nuclear Magnet Resonance (NMR) technology has gained more and more applications in organic chemistry, biochemistry, physical chemistry, inorganic chemistry, medicine and petroleum industry. It has become one of the most important methods in structure determination and dynamics analysis of biomolecules and synthetic compounds. As to the good precision and resolution that it could match the X-ray crystallography. Moreover, NMR methods process lot of other advantages, such as the simplicity in sample preparation, the proximity to in vivo environments of the bio-NMR sample and so on.The JARID1B/PLU-1, a 1,544 amino-acid multi-domain nuclear protein, belongs to the subfamily of JARID1. As a member of JmjC demethylase family, JARID1B/PLU-1 is considered to be a key component in the course of demethylation in H3K4me3 mode. Significantly, JARID1B/PLU-1 is a candidate protein, with strong transcriptional repression activity for regulating gene expression in breast cancer cells. It contains an ARID/BRIGHT DNA binding domain (110 aa.) which can binds the specific DNA motif for regulating the demethylation.There are four parts of the thesis as the following:Chapter one:we give a review on proceedings of the NMR technique and its application.Chapter two:we cloned, expressed, purified the JARID1B-ARID and we utilized NMR spectroscopy to assign backbone amino acids in order to study the solution structure. The predicted secondary structure elements of human JARID1B-ARID is comprised of 6 a-helix, which is consistent with other ARID domain.Chapter three:we mapped the DNA-binding sites of the human JARID1B ARID domain. Perturbations to1H-15N correlation spectra reveal that the flexible loop L1 of ARID is the main DNA-binding interface. EMSA and intrinsic fluorescence experiments demonstrate that mutations on loop L1 strongly reduce the DNA-binding activity of JARID1B ARID. Furthermore, transfection of mutant forms lead to a distinct loss of intrinsic H3K4 demethylase activity, implying that the flexible loop L1 makes a major contribution to sustaining the DNA-binding ability of J ARID1B ARID domain.Chapter four:Backbone dynamics studies were used to probe the molecular motion of the human JARID1B-ARID. Our results are indicated of an overall fairly rigid structure of J ARID1B-ARID and some conformational flexibility at different time scales around the binding site including loopl and part of loop2 region.