SINGLE-STRANDED NUCLEIC ACID BINDING PROTEINS
Principal Investigator: Kenneth Williams
Abstract: The single-stranded DNA binding proteins encoded by gene 32 of bacteriophage T4 (gp32) and ssb of E. coli (SSB) have served as prototypes for a class of proteins required in stoichiometric amounts in DNA replication, repair and recombination. In general, binding of these proteins imposes a conformation onto the ssDNA that is both resistant to nucleases and optimum to then serve as a substrate for other enzymes, such as DNA polymerases, involved in nucleic acid metabolism. Our research increasingly suggests that gp32 and SSB share many features in common with functionally unrelated eukaryotic nucleic acid binding proteins. Hence the function of the acidic COOH-terminus of these proteins appears analogous to the corresponding region in high mobility group proteins; as in the case of gp32 and SSB, hydrophobic interactions involving aromatic amino acid residues have also been implicated in the binding of heterogeneous nuclear RNA binding proteins. Finally, the zinc binding domain of gp32 appears to represent a nucleic acid binding motif shared by eukaryotic transcription factors as well as nucleic acid binding proteins encoded by murine leukemia and acquired immune deficiency viruses. To continue this line of research, we propose to more clearly define the role of the zinc ion in gp32 and to use an in vitro mutagenesis/1H-NMR/physicochemical approach to identify amino acids in gp32 and SSB that are directly involved in ssDNA binding. High priority will be given towards crystallizing gp32. Two new conditional lethal mutations in SSB will be characterized and limited proteolysis as well as photocross-linking experiments will be undertaken to determine how the tetrameric structure of SSB relates to ssDNA binding. A novel approach using a thermolabile SSB mutant and peptide competition experiments is planned to identify amino acids essential for SSB tetramer formation. This research will culminate in the synthesis and characterization of peptides corresponding to presumed functional domains in SSB and gp32 as well as to other related nucleic acid binding proteins. The structures of these synthetic peptides will then be varied in such a way as to test our ideas concerning the relationship of structure and function in nucleic acid binding proteins. These proposed studies are basic to our understanding of those physiological processes such as DNA replication, transcription and translation that require a single-stranded nucleic acid template.
Funding Period: 1988-03-01 - 1994-02-28
more information: NIH RePORT
- Insulin controls subcellular localization and multisite phosphorylation of the phosphatidic acid phosphatase, lipin 1Thurl E Harris
Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908 0735, USA
J Biol Chem 282:277-86. 2007..The results suggest that insulin and epinephrine control lipin primarily by changing localization rather than intrinsic PAP activity...