The majority of human replication protein A remains complexed throughout the cell cycle

Replication Protein A (RPA), the replicative single-strand DNA binding prot ein from eukaryotic cells, is a stable heterotrimeric complex consisting of three polypeptides, Cytological studies have investigated the subcellular distribution and association characteristics of the three RPA subunits duri ng different stages of the cell cycle with varying results, In this study, various HeLa cell fractions were subjected to separation by either immunopr ecipitation or velocity sedimentation. These separations were evaluated by immunoblotting for specific RPA subunits to determine whether the RPA in th ese fractions retains its heterotrimeric association. Immunoprecipitation o f either the large (RPA70) or middle-sized (RPA32) subunit of RPA followed by immunoblotting far the other subunits demonstrate that RPA remains compl exed throughout the G(1), S and G(2) phases of the cell cycle, Immunoprecip itation and sedimentation separations of both the nucleosolic and chromatin -bound RPA populations from both cycling and nocodazole-blocked cells showe d that the majority of RPA remains complexed under all conditions examined. Consistent with previous reports, hypotonic extracts from 293 cells were s hown to contain some RPA32 not complexed with RPA70. These results indicate that in some cell types, extracts may contain small amounts of RPA32 free of RPA70; however, in HeLa cells the majority of RPA clearly remains comple xed as a heterotrimer throughout the cell cycle.