Research Interest

DNA supercoiling is a fundamental property of the DNA double helix and plays many essential roles in the cell. In vivo, DNA is typically negatively supercoiled. Free energy constrained in negative superhelical tension greatly promotes a number of biologically important DNA transactions, such as DNA replication, recombination, and gene expression. The current view regarding activation of gene expression by DNA supercoiling is that negative supercoils stabilize the formation of the transcription “open” complexes and therefore activate transcription initiation. Transcription by a RNA polymerase, on the other hand, can stimulate negative DNA supercoiling in vitro and in E. coli topA strains. This phenomenon has been nicely explained by a “twin-supercoiled-domain” model in which positive DNA supercoils are generated in front of a translocating RNA polymerase and negative supercoils behind it. Removal of either supercoiled domain results in production of positively or hypernegatively supercoiled DNA. However, a detailed mechanism regarding transcription-coupled DNA supercoiling (TCDS) remains elusive and many aspects of this important process have yet to be explored. Our laboratory is using molecular biology techniques, biochemical methods, and biophysical assays to study the mechanisms of TCDS in vitro and in vivo. The following articles present our views regarding this important biological process:

Samul, R. and Leng, F. (2007) Transcription-induced Hypernegative Supercoiling of Plasmid DNA by T7 RNA Polymerase in E. coli Topoisomerase I Deficient Strains. Journal of Molecular Biology, 374, 925-935.

Leng F, Amado L, McMacken R. (2004) Coupling DNA Supercoiling to Transcription in Defined Protein Systems. Journal of Biological Chemistry 279, 47564-47571.

Leng, F. and McMacken, R. “Potent Stimulation of Transcription-coupled DNA Supercoiling by Sequence-Specific DNA-Binding Proteins” Proceedings of the National Academy of Sciences U S A, 2002, 99, 9139-9144.

The mammalian high mobility group protein A2 (HMGA2) is a transcriptional factor regulating mesenchymal cell development and differentiation. Disruption of the normal expression pattern is directly linked to oncogenesis. The expression level is correlated with the degree of malignancy and metastasis potential of the transformed cells. In addition, HMGA2 is a key player in fat cell proliferation and is a potential target for the treatment of obesity. HMGA2, an intrinsically “unstructured” DNA binding protein, mediates all of these biological effects through its interaction with AT-rich DNA in the promoter regions and through the disordered-to-ordered conformational transition upon binding to DNA. Our laboratory is currently studying HMGA2-DNA interactions using isothermal titration calorimetry and other biochemical and biophysical techniques. We have published a few articles in the field:

Cui, T., Wei, S., Brew, K., and Leng, F. (2005) Energetics of Binding the Mammalian High Mobility Group Protein HMGA2 to poly(dA-dT)₂ and poly(dA)poly(dT). Journal of Molecular Biology, 325, 629-645.

Cui, T., Joynt, S., Morillo, V., Baez, M., Hua, Z., Wang, X., and Leng, F. (2007) Large Scale Preparation of the Mammalian High Mobility Group Protein A2 for Biophysical Studies. Protein & Peptide Letters, 14, 87-91.

Cui, T. and Leng, F. (2007) Specific Recognition of AT-rich DNA Sequences by the Mammalian High Mobility Group Protein AT hook 2: a SELEX study. Biochemistry, 46, 13059-66.

Miao, Y., Cui, T., Leng, F., and W. D. Wilson (2008) Evaluation of inhibition of HMGA-DNA interaction by netropsin using surface plasmon resonance. Analytical Biochemistry, 374(1):7-15.