Replication Protein-A (RPA) is a heterotrimeric single-stranded DNA binding protein (SSB) that is essential for the replication and maintenance of the genome in all eukaryotic organisms. Although each of the three subunits is essential for activity only the largest, RPA1, binds ssDNA in moderate or low salt. X-ray crystallography indicates that the ssDNA binding domain of RPA1 consists of a pair of homologous ssDNA binding domains (SBD's) that together bind eight nucleotides of ssDNA. Current evidence suggests that two additional regions may directly participate in binding ssDNA in a """"""""higher order"""""""" mode, similar to the mechanism of ssDNA binding in prokaryotic cells. To test this possibility and study the role of individual subunits in ssDNA binding, amino acid residues that contact ssDNA will be identified by photo-crosslinking and peptide sequencing; residues cross-linked to DNA will be mutated, and the mutant proteins expressed, to confirm their involvement in ssDNA binding. Mutations that give rise to biochemical defects in RPA will be correlated with the phenotypes of the same mutants expressed in vivo. To identify potential regulatory domains in RPA, conditional lethal mutations in regions of RPA that do not bind ssDNA will be isolated. The effect of these mutations on DNA replication, repair, and recombination will be examined. These mutants also will be used to search for interacting factors, using suppressor and synthetic lethal genetic screens. These genetically interacting factors are expected to reveal the biological function of these domains.
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