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.
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.
Authors: Heqiao Dai; Robert J Hickey; Jianying Liu; Robert M Bigsby; Carita Lanner; Linda H Malkas Journal: Gynecol Oncol Date: 2013-07-12 Impact factor: 5.482