The biochemical status of the DNA synthesome can distinguish between permanent and temporary cell growth arrest.
We previously identified and characterized the human leukemia (HL-60) cell DNA synthetic machinery as a multiprotein form of DNA polymerase, which was designated the DNA synthesome. This multiprotein replication complex contains DNA polymerases alpha and delta, primase, replication factor C, replication protein A, helicase, poly(ADPribose) polymerase, proliferating cell nuclear antigen, DNA ligase I, and topoisomerases I and II. Recently, the HeLa cell-derived DNA synthesome was identified as a discrete high molecular weight protein band in native polyacrylamide gels. Here, we report our findings regarding the change in the organizational status of the DNA synthesome when HL-60 cells undergo either terminal differentiation or temporary G1 growth arrest. We observed that the HL-60 cell DNA synthesome also migrates as a discrete high molecular weight protein band in nondenaturing polyacrylamide gels. This high molecular weight protein band was present in nuclei derived from both actively cycling cells and aphidicolin-arrested cells but was absent in TPA-induced terminally differentiated cells. We also found that DNA polymerase delta, replication factor C, and proliferating cell nuclear antigen are absent in cells that are induced to differentiate in response to 12-O-tetradecanoyl phorbol-13-acetate treatment but are present in actively cycling cells. The level of replication protein A in differentiated cells was similar to that of cycling cells, whereas the level of annexin I, a cytoskeleton protein, is higher in differentiated cells than it is in actively cycling cells. We conclude that the DNA synthesome remains integrated and inactive in temporarily growth-arrested cells but is disassembled in differentiated cells. Furthermore, we conclude that disassembly of the organized replication complex is a specific cellular event in the process of permanent cell cycle exit and that the process leading to disassembly may be regulated, in part, at the level of gene transcription.
Lin
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S
Toxicology Programs, University of Maryland School of Medicine, Baltimore 21201, USA.
Hickey
R
R
Malkas
L
L
D016590:Aphidicolin / Q000494:pharmacology; D015152:Blotting, Northern; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects / Q000502:physiology*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D018922:HL-60 Cells; D018398:Homeodomain Proteins; D006801:Humans; D015778:Minor Histocompatibility Antigens; D009097:Multienzyme Complexes / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger / Q000032:analysis; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000099272:Replisomes
D016590:Aphidicolin; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D004791:Enzyme Inhibitors; D018398:Homeodomain Proteins; D015778:Minor Histocompatibility Antigens; D009097:Multienzyme Complexes; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D013755:Tetradecanoylphorbol Acetate; D000099272:Replisomes; C096816:BCL2-related protein A1; C096063:MATA1 protein, S cerevisiae