The human cruciform-binding protein, CBP, is involved in DNA replication and associates in vivo with mammalian replication origins.
We previously identified and purified from human (HeLa) cells a 66-kDa cruciform-binding protein, CBP, with binding specificity for cruciform DNA regardless of its sequence. DNA cruciforms have been implicated in the regulation of initiation of DNA replication. CBP is a member of the 14-3-3 family of proteins, which are conserved regulatory molecules expressed in all eukaryotes. Here, the in vivo association of CBP/14-3-3 with mammalian origins of DNA replication was analyzed by studying its association with the monkey replication origins ors8 and ors12, as assayed by a chromatin immunoprecipitation assay and quantitative PCR analysis. The association of the 14-3-3beta, -epsilon, -gamma, and -zeta isoforms with these origins was found to be approximately 9-fold higher, compared with other portions of the genome, in logarithmically growing cells. In addition, the association of these isoforms with ors8 and ors12 was also analyzed as a function of the cell cycle. Higher binding of 14-3-3beta, -epsilon, -gamma, and -zeta isoforms with ors8 and ors12 was found at the G(1)/S border, by comparison with other stages of the cell cycle. The CBP/14-3-3 cruciform binding activity was also found to be maximal at the G(1)/S boundary. The involvement of 14-3-3 in mammalian DNA replication was analyzed by studying the effect of anti-14-3-3beta, -epsilon, -gamma, and -zeta antibodies in the in vitro replication of p186, a plasmid containing the minimal replication origin of ors8. Anti-14-3-3epsilon, -gamma, and -zeta antibodies alone or in combination inhibited p186 replication by approximately 50-80%, while anti-14-3-3beta antibodies had a lesser effect ( approximately 25-50%). All of the antibodies tested were also able to interfere with CBP binding to cruciform DNA. The results indicate that CBP/14-3-3 is an origin-binding protein, acting at the initiation step of DNA replication by binding to cruciform-containing molecules, and dissociates after origin firing.
Novac
Olivia
O
McGill Cancer Center and Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada.
Alvarez
David
D
Pearson
Christopher E
CE
Price
Gerald B
GB
Zannis-Hadjopoulos
Maria
M
D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011485:Protein Binding
D017931:DNA Primers; D004268:DNA-Binding Proteins; C115090:cruciform-specific DNA-binding protein, human