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Development of MS Compatible Methods for Characterizing Protein-DNA Interactions

Novel protein interactions are generally discovered using immunoprecipitation.  During this process, the chromatin is removed, along with the rest of the cell debris.  No knowledge of specific protein-DNA interactions can be obtained from this method and the integrity of any complex that would bind to DNA under physiological conditions is compromised.  The development of a robust toolbox for the characterization of protein-DNA interactions would be a significant step in our understanding of a broad range of biological process, from transcriptional regulation and chromatin regulation to the role that specific SNP’s play in disease states.

In the Laboratory of Proteomics and Analytical Technologies (LPAT), scientists are developing tools to identify unknown proteins that bind to a target DNA sequence, either through the incorporation of a tag through homologous recombination or through hybridization of the target to a tagged DNA probe. To study protein complex formation of transcription factors and chromatin remodeling in context of the chromatin structure we use antibodies against the protein of interest to perform immunoprecipitation of the protein complex as it associates with the chromatin.  Within the mixture DNA probes of various lengths and with different tags are added to simulate the chromatin environment of the protein in vivo.  In addition differently modified (methylation) probes will be used to reflect altered epigenetic status of the binding sequence of interest. We are also exploring different methods of data analysis with the goal of developing bioinformatics system that will help prioritize the potential interacting proteins for further study and validation.

MS Compatible Methods for Protein-DNA Interactions

Figure 1. General aim is to isolate DNA-binding protein complexes in the context of their DNA_binding site to provide a true representation of their structure in vivo.

Page last updated April 27, 2011 @ 4:32 pm