Structural analysis of the human neuroblastoma DNA replication complex: insights into faulty proliferation.
BACKGROUND
Whether faulty DNA synthesis contributes to neuroblastoma (NB) pathogenesis has received little attention. We have shown that NB DNA replication is orchestrated by a multiprotein DNA replication complex (DNA synthesome) which mediates error-prone DNA synthesis. We sought to define proteomic alterations of the NB DNA synthesome, which lead to lowered replication fidelity.


METHODS
DNA synthesomes from NB and neural stem (NS) cell culture were purified. Proteomic differences of the DNA synthesome between NB and NS cells were determined using 2-dimensional polyacrylamide gel electrophoresis and immunodetection.


RESULTS
DNA synthesome proteins from NB and NS cells were compared. Only replication protein A (RPA) showed distinct changes. RPA from NS cells resolved as a single spot (isoelectric point [pI] 5.75), whereas RPA in NB showed a main spot (pI 5.75) along with 3 additional isoforms. RPA binds to single-stranded DNA and participates in protein-protein interactions.


CONCLUSIONS
NB DNA synthesome showed 3 distinct isoforms of RPA when compared with NS cells. These findings are significant in that it is possible to link changes in the fidelity of DNA replication with a specific protein alteration of the NB DNA synthetic apparatus. The novel RPA forms may be a new signature of NB malignancy.
Sandoval
John A
JA
Department of Surgery, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Grosfeld
Jay L
JL
Hickey
Robert J
RJ
Malkas
Linda H
LH
D015153:Blotting, Western; D004261:DNA Replication / Q000502:physiology*; D004259:DNA-Directed DNA Polymerase / Q000032:analysis / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D009097:Multienzyme Complexes / Q000032:analysis / Q000737:chemistry*; D009447:Neuroblastoma / Q000235:genetics* / Q000503:physiopathology*; D020033:Protein Isoforms; D040901:Proteomics; D014407:Tumor Cells, Cultured; D000099272:Replisomes
D004259:DNA-Directed DNA Polymerase; D009097:Multienzyme Complexes; D020033:Protein Isoforms; D000099272:Replisomes