ROLE OF PROTEIN-PROTEIN INTERACTIONS IN THE FUNCTION OF REPLICATION PROTEIN-A (RPA) - RPA MODULATES THE ACTIVITY OF DNA-POLYMERASE A BY MULTIPLE MECHANISMS

Replication Protein A (RPA) from human cells is a stable complex of 70 -, 32-, and 14-kDa subunits that is required for multiple processes in DNA metabolism. RPA binds with high affinity to single-stranded DNA a nd interacts with multiple proteins, including proteins required for t he initiation of SV40 DNA replication, DNA polymerase alpha and SV40 l arge T antigen, We have used a series of mutant derivatives of RPA to map the regions of RPA required for specific protein-protein interacti ons and have examined the roles of these interactions in DNA replicati on T antigen, DNA polymerase alpha and the activation domain of VP16 a ll have overlapping sites of interaction in the N-terminal half (resid ues 1-327) of the 70-kDa subunit of RPA. In addition, the interaction site for DNA polymerase alpha is composed of two functionally distinct regions, one (residues 1-similar to 170) which stimulates polymerase activity and a second (residues similar to 170-327) which increases po lymerase processivity. In the latter, both the direct protein-protein interaction and ssDNA-binding activities of RPA were needed for RPA to modulate polymerase processivity. We also found that SV40 T antigen i nhibited the ability of RPA to increase processivity of DNA polymerase alpha, suggesting that this activity of RPA may be important for elon gation but not during the initiation of DNA replication, DNA polymeras e alpha, but not T antigen also interacted with the 32- and/or 14-kDa subunits of RPA, but these interactions did not seem to effect polymer ase activity.