The long-term objective of the research is to determine the structural basis underlying the suggested biological effects mediated by mammalian HMG-I proteins. HMG-I is the principal member of an isoform group (the HMGI family) of the nuclear proteins and is the first mammalian nonhistone protein demonstrated to specifically bind, both in vitro and in vivo, to A T-rich regions of DNA. Recent reports indicate that all types of cancerous cells contain exceptionally high concentrations of HMGI proteins which have been proposed to be biochemical markers for the transformed state. In vitro, members of the HMGI family have been demonstrated to act as gene transcription stimulatory factors and also to specifically bind to important A.T-rich DNA regulatory regions, including gene promoters, enhancers and the DNA replication origins of several organisms. A peptide """"""""binding domain"""""""" (BD) common to all known HMGI proteins has been identified that mediates binding of these proteins to the narrow minor groove of DNA and is called """"""""A T-hook"""""""" because of its novel predicted secondary structure. In certain ways the BD peptide resembles the antitumor/antiviral drugs distamycin, netropsin and the dye Hoechst 33258, ligands which effectively compete with HMGI proteins for DNA binding both in vitro and in vivo. Individual HMGI proteins have three separate BD peptide motifs. Both in vitro and in vivo, HMGI proteins are preferred substrates for the cell division regulating enzyme cdc2 kinase which specifically phosphorylates the """"""""hook"""""""" region of the BD peptides. In vitro such phosphorylation has been demonstrated to significantly weaken the DNA binding affinity of both synthetic BD peptides and intact HMGI proteins.
Specific Aims of Project: (1) To determine the 3D solution structure of both the unphosphorylated and cdc2 kinase phosphorylated synthetic BD peptides employing 2D NMR techniques; (2) Determine the effects of specific deletions, duplications or mutations in one or more of the individual BD peptides present in wild type (wt) HMG-I proteins on the ability of such mutant proteins to strongly and specifically interact with A T-rich DNA. (3) Produce recombinant hybrid BD-peptide/""""""""tag-peptide"""""""" fusion proteins and determine whether these hybrid proteins can specifically """"""""target"""""""" and bind to stretches of A T-rich sequence in vitro and in vivo. The results of these studies will not only help establish the structural basis for understanding the biological effects of an important group of proteins, but will also contribute new detailed information about general molecular mechanisms involved in specific DNA-protein interactions. And, perhaps as importantly, the experiments may establish a mechanism for """"""""targeting"""""""" specific peptides or proteins A T-rich sequences in living cells.
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