This project will study a human DNA replication factor, replication protein complex A (RPA), which has been purified from a human cancer cell line and is essential for DNA replication and repair in humans, other animals and yeasts. RPA is a complex of three subunits of 70 kD, 34 kD and 13 kD, and is the first human cellular DNA replication factor which enters the replication initiation complex after the """"""""origin binding protein"""""""" (as yet unidentified in animal cells). RPA p34 is phosphorylated at the G1-S transition of the cell cycle on multiple sites, and cdc2 kinase (a positive factor required at G1-S) has been implicated in part of this phosphorylation. In the last year we discovered that the tumor suppressor protein p53 interacts with and inhibits the function of RPA (binding of single stranded DNA) suggesting a mechanism by which p53 inhibits the onset of S phase. We shall determine by mutagenesis of p53 if its ability to inhibit RPA is important for its growth suppression activity. The normal regulation of this interaction, by cell cycle stage specific phosphorylation, will be studied. Also, the potential effect of RPA on p53, specifically its transactivation and growth suppression functions will be analyzed. The interaction of RPA with p53 and other transcriptional transactivators could recruit RPA to origins of replication, accounting for the interplay between transcription and replication observed in multiple systems. We shall explore whether the ability of RPA to interact with transcriptional factors is important for the normal functions of the protein, turning to yeast for these series of experiments (where they can be easily done without interference from wild type endogenous RPA). The three subunits of RPA (p7O, p34, and pl3) have to associate with each other to form the functional, essential DNA replication factor. Therefore, anti-proliferative drugs could potentially be targeted to interfere with these inter-subunit interactions.
The second aim of this project uses two novel systems (one in yeast, and the other in mouse cells) developed by us to study the association between the RPA subunits. Using these systems we shall delineate parts of the subunits that are essential for their association. The results will therefore (a) demonstrate the feasibility of designing small molecules to interfere with RPA holocomplex formation, and (b) in the long run may directly contribute to the designing of such molecules.
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