title: The spatio-temporal organization of DNA replication sites is identical in primary, immortalized and transformed mammalian cells.
abstract: We investigated the organization of DNA replication sites in primary (young or presenescent), immortalized and transformed mammalian cells. Four different methods were used to visualize replication sites: in vivo pulse-labeling with 5-bromo-2'-deoxyuridine (BrdU), followed by either acid depurination, or incubation in nuclease cocktail to expose single-stranded BrdU-substituted DNA regions for immunolabeling; biotin-dUTP labeling of nascent DNA by run-on replication within intact nuclei and staining with fluorescent streptavidin; and, finally, immunolabeling of the replication fork proteins PCNA and RPA. All methods produced identical results, demonstrating no fundamental differences in the spatio-temporal organization of replication patterns between primary, immortal or transformed mammalian cells. In addition, we did not detect a spatial coincidence between the early firing replicons and nuclear lamin proteins, the retinoblastoma protein or the nucleolus in primary human and rodent cells. The retinoblastoma protein does not colocalize in vivo with members of the Mcm family of proteins (Mcm2, 3 and 7) at any point of the cell cycle and neither in the chromatin-bound nor in the soluble nucleoplasmic fraction. These results argue against a direct role for the retinoblastoma or nuclear lamin proteins in mammalian DNA synthesis under normal physiological conditions.
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D002455:Cell Division / Q000235:genetics*; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis* / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D005057:Eukaryotic Cells / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D034882:Lamins / Q000235:genetics; D064111:Minichromosome Maintenance Complex Component 2; D009687:Nuclear Proteins / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics; D051716:Replication Protein A; D012093:Replicon / Q000235:genetics; D016160:Retinoblastoma Protein / Q000235:genetics; D013997:Time Factors
chemical_list: D004268:DNA-Binding Proteins; D034882:Lamins; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; C497167:RPA1 protein, human; D051716:Replication Protein A; D016160:Retinoblastoma Protein; D004247:DNA; D064111:Minichromosome Maintenance Complex Component 2