The majority of human replication protein A remains complexed throughout the cell cycle.
Replication Protein A (RPA), the replicative single-strand DNA binding protein 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 during different stages of the cell cycle with varying results. In this study, various HeLa cell fractions were subjected to separation by either immunoprecipitation or velocity sedimentation. These separations were evaluated by immunoblotting for specific RPA subunits to determine whether the RPA in these fractions retains its heterotrimeric association. Immunoprecipitation of either the large (RPA70) or middle-sized (RPA32) subunit of RPA followed by immunoblotting for the other subunits demonstrate that RPA remains complexed throughout the G(1), S and G(2) phases of the cell cycle. Immunoprecipitation and sedimentation separations of both the nucleosolic and chromatin-bound RPA populations from both cycling and nocodazole-blocked cells showed that the majority of RPA remains complexed under all conditions examined. Consistent with previous reports, hypotonic extracts from 293 cells were shown 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 complexed as a heterotrimer throughout the cell cycle.
Loo
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Department of Microbiology, School of Medicine and Biomedical Sciences, SUNY Buffalo, 138 Farber Hall, Buffalo, NY 14214-3000, USA.
Melendy
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D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D002457:Cell Extracts; D002499:Centrifugation, Density Gradient; D002843:Chromatin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D016193:G1 Phase / Q000187:drug effects; D016195:G2 Phase / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis / Q000187:drug effects; D015739:Nocodazole / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects; D051716:Replication Protein A; D016196:S Phase / Q000187:drug effects
D002457:Cell Extracts; D002843:Chromatin; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D015739:Nocodazole