Gfi-1 is a transcriptional repressor crucial for precise regulation of cell proliferation and differentiation in hematopoiesis. Recently, it has also been demonstrated to be capable of restricting the proliferation of hematopoietic stem cells (HSCs), a process which appears to be vital for the long term competency of HSCs. These two seemingly opposite outcomes of regulation are likely to arise from interactions with a variety of different cellular partners. Such protein-protein interactions can directly affect the genes Gfi-1 recognizes through its DNA binding zinc-finger domain. Interestingly, biochemical foot-printing studies by methylation suggested a novel minor groove DNA-binding model by these canonical C2H2 zinc fingers. The focus of the work herein is to determine the solution structure of Gfi-1 zinc fingers 3-5 (Gfizf35) bound to a 16-mer consensus DNA to provide insights into the mechanism of specific DNA recognition by Gfi-1. The structure of Gfizf35 bound with its consensus DNA has been solved by using multidimensional NMR, including NOE-based distance restraints, torsion angle restraints, and residual dipolar couplings from both the protein and the DNA. Unlike the proposed minor groove model, the solution structure reveals that Gfi-1 zinc fingers wrap around the target DNA by binding the major groove. Protein-DNA interactions are mediated by residues located on the entire outer-surface of the α-helices, the second β-strands and the linkers between them. The last zinc finger recognizes the AATC core sequence of the consensus site by forming specific hydrogen bonds between the side chains of Asn72, Gln69 and the two invariant adenosine bases. Similarly, the fourth zinc finger also exhibits specific interaction between Gln41 and a less conserved but still important adenosine base. The structure furnishes insights into the mechanism of specific DNA recognition by Gfi-1 and provides a rationale for understanding the disease-caused by N72S mutation, mutagenesis and methylation data.
|Commitee:||Foster, Mark, Gopalan, Venkat, Zhong, Dongping|
|School:||The Ohio State University|
|School Location:||United States -- Ohio|
|Source:||DAI-B 79/09(E), Dissertation Abstracts International|
|Keywords:||Cataract, Crystallin, Dna, Gamma s-crystallin, Gfi-1, Opj, Origins, Proteins, Recognition|
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