Nucleoprotein complexes released from lymphoma nuclei that contain the abl oncogene and RNA and DNA polymerase and RNA primase activities.
We report on the discovery and isolation of DNA- and RNA-containing macromolecular nuclear complexes whose purified major DNA possessed electrophoretic mobilities of approximately 90 and approximately 25 kbp. The deoxyribonucleoprotein-ribonucleoprotein complexes contain RNA and DNA polymerase and primase activities and were isolated from nuclei of murine RAW117 large-cell lymphoma cells by restriction digestion with Msp-I, gentle extraction with solutions containing MgCl2, but without chelating agents, and low ionic strength gel electrophoresis. Two-dimensional (isoelectric focusing/M(r)) gel electrophoresis and silver staining of the proteins of the complexes after treatment with DNase I indicated the presence of approximately 30 protein components. In vitro DNA and RNA polymerase/primase assays showed that the DNP/RNP complexes had very high enzyme specific activities. Using the DNP/RNP complexes a discrete DNA polymerase alpha product of approximately 85 kbp was synthesized that was not synthesized in the presence of the DNA polymerase alpha inhibitor aphidicolin. RNA polymerase assays in the presence of excess alpha-amanitin indicated that the complexes possessed significant RNA polymerase I activity. Preparing the complexes at various times after the release of cells from a double thymidine block showed the complexes as well as the complex-associated enzyme activities to be cell-cycle dependent. The DNA and RNA polymerase-related activities were highest in late S phase, 7 and 9 h, respectively, after release from the double thymidine block. The complexes synthesized a specific in vitro DNA polymerase product using endogenous substrate and nucleotide precursors.(ABSTRACT TRUNCATED AT 250 WORDS)
Rosenberg-Nicolson
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Department of Tumor Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Nicolson
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GL
D000818:Animals; D002453:Cell Cycle; D019915:DNA Primase; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000302:isolation & purification / Q000378:metabolism*; D003852:Deoxyribonucleoproteins / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D016313:Genes, abl; D016403:Lymphoma, Large B-Cell, Diffuse / Q000201:enzymology* / Q000235:genetics*; D051379:Mice; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D012316:RNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000737:chemistry; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D003852:Deoxyribonucleoproteins; D009097:Multienzyme Complexes; D012261:Ribonucleoproteins; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D012321:DNA-Directed RNA Polymerases; D004259:DNA-Directed DNA Polymerase