Species specificity of human RPA in simian virus 40 DNA replication lies in T-antigen-dependent RNA primer synthesis

Replication protein A (RPA) is a three-subunit protein complex with multipl e functions in DNA replication. Previous study indicated that human RPA (h- RPA) could not be replaced by Schizosaccharomyces pombe RPA (sp-RPA) in sim ian virus 40 (SV40) replication, suggesting that h-RPA may have a specific function in SV40 DNA replication. To understand the specificity of h-RPA in replication, we prepared heterologous RPAs containing the mixture of human and S. pombe subunits and compared these preparations for various enzymati c activities. Heterologous RPAs containing two human subunits supported SV4 0 DNA replication, whereas those containing only one human subunit poorly s upported DNA replication, suggesting that RPA complex requires at least two human subunits to support its function in SV40 DNA replication. All hetero logous RPAs effectively supported single-stranded (ss)DNA binding activity and an elongation of a primed DNA template catalyzed by DNA polymerase (pol ) alpha and delta. A strong correlation between SV40 DNA replication activi ty and large tumor antigen (T-ag)-dependent RNA primer synthesis by pol alp ha -primase complex was observed among the heterologous RPAs, Furthermore, T-ag showed a strong interaction with 70- and 34-kDa subunits from human, b ut poorly interacted with their S. pombe counterparts, indicating that the specificity of h-RPA is due to its role in RNA primer synthesis. In the SV4 0 replication reaction, the addition of increasing amounts of sp-RPA in the presence of fixed amount of h-RPA significantly reduced overall DNA synthe sis, but increased the size of lagging strand, supporting a specific role f or h-RPA in RNA primer synthesis. Together, these results suggest that the specificity of h-RPA in SV40 replication lies in T-ag-dependent RNA primer synthesis.