THE 32-KILODALTON AND 14-KILODALTON SUBUNITS OF REPLICATION PROTEIN-AARE RESPONSIBLE FOR SPECIES-SPECIFIC INTERACTIONS WITH SINGLE-STRANDED-DNA

Replication protein A (RPA) is a multisubunit single-stranded DNA-bind ing (ssDNA) protein that is required for cellular DNA metabolism. RPA homologues have been identified in all eukaryotes examined. All homolo gues are heterotrimeric complexes with subunits of similar to 70, simi lar to 32, and similar to 14 kDa. While RPA homologues are evolutionar ily conserved, they are not functionally equivalent. To gain a better understanding of the functional differences between RPA homologues, we analyzed the DNA-binding parameters of RPA from human cells and the b udding yeast Saccharomyces cerevisiae (hRPA and scRPA, respectively). Both yeast and human RPA bind ssDNA with high affinity and low coopera tivity. However, scRPA has a larger occluded binding site (45 nucleoti des versus 34 nucleotides) and a higher affinity for oligothymidine th an hRPA. Mutant forms of hRPA and scRPA containing the high-affinity D NA-binding domain from the 70-kDa subunit had nearly identical DNA bin ding properties. In contrast, subcomplexes of the 32- and 14-kDa subun its from both yeast and human RPA had weak ssDNA binding activity. How ever, the binding constants for the yeast and human subcomplexes were 3 and greater than 6 orders of magnitude lower than those for the RPA heterotrimer, respectively. We conclude that differences in the activi ty of the 32- and 14-kDa subunits of RPA are responsible for variation s in the ssDNA-binding properties of scRPA and hRPA. These data also i ndicate that hRPA and scRPA have different modes of binding to ssDNA, which may contribute to the functional disparities between the two pro teins.