Isolation and characterization of a DNA synthesome from a neuroblastoma cell line.
BACKGROUND
Despite significant analysis of the chromosomal abnormalities associated with neuroblastoma (NB), the role that NB DNA replication may play in the accumulation of genetic damage is poorly understood. For that matter, the mechanisms involved in NB DNA synthesis have yet to be elucidated. In an effort to investigate this process in NB, we have isolated and purified a multiprotein DNA replication complex from human NB cells (IMR-32).


METHODS
Using a series of subcellular fractionations, ion-exchange chromatography, and gradient sedimentation steps, we have isolated a simian virus 40 replication competent multiprotein complex from IMR-32 NB cells, which has been designated the DNA synthesome. Enzymatic and immunodetection techniques were used to characterize the multiple components of the multiprotein DNA replication complex.


RESULTS
The NB DNA synthesome was found to remain intact and functional through all the steps of its purification. The proteins and enzymatic activities that were found to copurify with the NB DNA synthesome include: DNA polymerases alpha , delta , and epsilon , proliferating cell nuclear antigen, replication factor A, replication factor C, topoisomerases I and II, flap endonuclease 1, and DNA ligase I.


CONCLUSIONS
Although the cooperative integration of a DNA replication macromolecular complex (DNA synthesome) is not new, we extend the view of the DNA synthesome mediating DNA synthesis for human NB. The data reported here characterize the human NB DNA synthesome for the first time and provide the groundwork for investigating whether the NB DNA synthesome contributes to faulty DNA replication and tumor pathogenesis for this childhood malignancy.
Sandoval
John A
JA
Department of Surgery and Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Hickey
Robert J
RJ
Malkas
Linda H
LH
D002852:Chromatography, Ion Exchange; D004247:DNA / Q000096:biosynthesis*; D004256:DNA Polymerase I / Q000302:isolation & purification / Q000378:metabolism; D000074002:DNA Polymerase gamma; D004261:DNA Replication / Q000235:genetics*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007118:Immunoassay; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000302:isolation & purification; D009447:Neuroblastoma / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D014407:Tumor Cells, Cultured
D046912:Multiprotein Complexes; D004247:DNA; D004256:DNA Polymerase I; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II