The general long term goal is the determination of the topological structure of DNA in chromosomes and the protein DNA interactions that organize this structure. The research has a fundamental relationship to developing an understanding of gene regulation mediated by protein-DNA interactions and will therefore provide a basis for understanding abnormalities at this level that occur in many diseases.
The specific aims are focused on delineating the way that histone like proteins (HLPs) of bacteria and mammals wrap DNA in transient coils and simultaneously change the DNA helical repeat. The experiments investigate the hypothesis that certain HLPs, and especially the HU protein, mediate DNA structural transitions at many sites in the chromosome (such as looping or wrapping) while overwinding the DNA helical repeat to maintain levels of DNA torsional tension required for gene expression. Newly developed methods for obtaining covalent cyclization of small DNA molecules, while they are wrapped on a protein core of HLPs, provides an approach for determining precisely the DNA helical repeat of bound DNA and for determining parameters of DNA bending and wrapping. Recently discovered eukaryotic proteins that seem to wrap DNA in similar, transient, tight coils will be purified and characterized with respect to their possible analogous effects on DNA structure. Finally, a recently developed technique for introducing purified, labelled proteins into living bacterial cells will be exploited to define the distribution of certain HLPs in chromosomes of growing cells and to delineate the sites of transcriptionally active genes in the nucleoid. The latter experiments will test a postulated dynamic structure of the chromosome in living cells.
| Wagner, J P; Quill, D M; Pettijohn, D E (1995) Increased DNA-bending activity and higher affinity DNA binding of high mobility group protein HMG-1 prepared without acids. J Biol Chem 270:7394-8 |
