Eucaryotes contain a wide variety of single-stranded (ss) nucleic acid binding proteins that are required in stoichiometric amounts for DNA replication, transcription, hetergeneous nuclear RNA (hnRNA) processing and translation. One of the best characterized ssDNA binding proteins that may play a role in DNA replication is the UP1 protein from calf thymus. Our major objectives are to better understand the function of UP1 and to examine its interaction with ssDNA at a molecular level. In addition, we would like to determine if UP1 shares certain structural features that are essential for binding single-stranded nucleic acids in common with other ssDNA and ssRNA binding proteins. UP1 has already been sequenced and its gene cloned, thus it is perhaps the only mammalian ssDNA bindign protein that is currently suitable for detailed structure/function analysis. Specifically, protein chemistry studies are planned to determine if several calf ssDNA binding proteins that are slightly larger than UP1 are, in fact, structurally related to UP1. Homologous ssDNA binding proteins from calf liver, mouse myeloma and HeLa cells will be sequenced so that highly conserved and therefore probably essential regions can be identified in UP1. Similar studies on proteins associated with hnRNA will determine if, as immunological studies suggest, these ssRNA binding proteins are structurally related to UP1. Physicochemical techniques such as fluorescence, chemical modification, photo-crosslinking, and NMR will be used to identify amino acids in UP1 that are involved in DNA binding and to determine how the presence of two 10,000 dalton globular domains in UP1 relates to its ability to bind ssDNA. High expression UP1 vectors will be constructed to help supply the large quantities of protein required by these studies and to allow future in vitro mutagenesis experiments. The function of UP1 will be examined by comparing the relative tissue distribution and ability of UP1 and UP1-like proteins to stimulate DNA polymerase alpha. Protein affinity chromatography will be used to identify those proteins that UP1 interacts with in vivo. These proposed studies are basic to our understanding of those physiological processes that require a single-stranded nucleic acid template.
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