Increased origin activity in transformed versus normal cells: identification of novel protein players involved in DNA replication and cellular transformation.
Using libraries of replication origins generated previously, we identified three clones that supported the autonomous replication of their respective plasmids in transformed, but not in normal cells. Assessment of their in vivo replication activity by in situ chromosomal DNA replication assays revealed that the chromosomal loci corresponding to these clones coincided with chromosomal replication origins in all cell lines, which were more active by 2-3-fold in the transformed by comparison to the normal cells. Evaluation of pre-replication complex (pre-RC) protein abundance at these origins in transformed and normal cells by chromatin immunoprecipitation assays, using anti-ORC2, -cdc6 and -cdt1 antibodies, showed that they were bound by these pre-RC proteins in all cell lines, but a 2-3-fold higher abundance was observed in the transformed by comparison to the normal cells. Electrophoretic mobility shift assays (EMSAs) performed on the most efficiently replicating clone, using nuclear extracts from the transformed and normal cells, revealed the presence of a DNA replication complex in transformed cells, which was barely detectable in normal cells. Subsequent supershift EMSAs suggested the presence of transformation-specific complexes. Mass spectrometric analysis of these complexes revealed potential new protein players involved in DNA replication that appear to correlate with cellular transformation.
Di Paola
Domenic
D
Goodman Cancer Center, McGill University, Montreal, Quebec, H3G 1Y6, Canada.
Rampakakis
Emmanouil
E
Chan
Man Kid
MK
Arvanitis
Dina N
DN
Zannis-Hadjopoulos
Maria
M
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D056915:DNA Copy Number Variations; D004261:DNA Replication; D056426:Genetic Loci; D015894:Genome, Human; D006801:Humans; D013058:Mass Spectrometry; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D018741:Replication Origin
C103706:CDC6 protein, human; C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; D051738:Origin Recognition Complex; D004247:DNA