Similarly, Orc2 started to be detected (3-fold enrichment) in early G1 and remained rather constantly bound to the origin. progression to the activation of DNA replication. == Introduction == Fundamental work initially carried out in S. cerevisiae and then extended to other organisms, has shown that origins of DNA replication are the sites at which the ordered assembly of the multi-protein pre-replicative complex (pre-RC) takes place. During the G1 phase of the cell cycle, the six-subunit origin recognition complex (ORC) is first recruited onto DNA; the CDC6 and Cdt1 proteins are then required together for loading the putative replicative helicase (Mcm2-7) onto chromatin. Initiation of DNA replication occurs through the function of Cdc7 and S-phase CDKs, which activate the pre-RC and promote further recruitment of proteins required for DNA synthesis[1],[2]. In S. cerevisiae, origin selection is essentially dictated by the binding of ORC to a specific, A/T rich, 11-bp DNA sequence. In higher eukaryotes, however, despite the impressive conservation of proteins participating in the pre-RC formation, primary DNA sequence recognition by ORC is loose, and origin selection requires further information beyond ORC-DNA interaction, possibly indicating that other proteins participate in ORC positioning either directly or through the induction of epigenetic modifications of chromatin[3]. The expression of a large number of factors that participate in the formation of the pre-RC, or that are required for the G1-S transition and the progression of the replicative fork, is transcriptionally controlled by the interaction of the E2F1-3 transcription factors with the retinoblastoma (Rb) protein. In early G1 cells, E2F-bound, hypo-phosphorylated Rb inhibits E2F activity through both direct and indirect mechanisms, which include masking the E2F activation domain or recruiting transcriptional repressors and chromatin modifiers[4],[5]. Upon phosphorylation by the G1 phase CDKs, repression by Rb is relieved and transcription of the E2F-target genes ensues. This is, therefore, an effective mechanism to couple cell cycle progression to the expression of genes that are required for DNA replication. Over the last few years, however, a few observations have indicated that E2F and Rb might also participate more directly in the regulation of DNA replication. Rb was found to affect the spatial organization of DNA replication in primary mammalian cells[6]and to prevent genomic re-replication in cells experiencing S phase DNA damage, G2/M arrest and M phase block[7],[8]. More specifically, in cells irradiated in early S phase, Rb was found associated with an early firing origin of DNA replication (the Lamin B2 origin) during the S-phase block, and (S)-(+)-Flurbiprofen then with (S)-(+)-Flurbiprofen additional origins in the order in which they fired[9]. Finally, and most notably, studies performed in D. melanogaster have shown that the E2F1 and Rb homologues form a complex with Drosophila ORC and associate with the chorion gene cluster origin of DNA replication, thereby limiting the physiological amplification of this cluster in ovarian follicle cells[10],[11]. Taken collectively, this information strongly suggests that E2F and Rb might be part of the complex of proteins that associate with origins of DNA replication also in vertebrates. Indeed, here we show that both Rb and E2F1 bind three human origins of DNA replication and that this association (S)-(+)-Flurbiprofen is strictly regulated during the cell cycle. We describe that, both in vitro and inside the cells, the largest ORC subunit (Orc1) directly interacts with the under-phosphorylated form of Rb, and that this interaction is mutually exclusive with the binding of Rb to E2F1. Consistent with these findings, down-regulation of Orc1 by RNA interference in human cells favors E2F1 recruitment onto origins and induces a marked G1 arrest. == Results == == Rb and E2F1 proteins are recruited to human origins of DNA replication == We investigated recruitment of E2F and Rb to three human origins of DNA replication that our laboratory has described in molecular detail over the last few years. One of these origins encompasses the 3 end of the lamin B2 gene and the promoter of the downstream TIMM13 gene in chromosome 19q Lamin B2 origin[12],[13]; the other two origins (GM-CSF Ori1 and Ori2) are located downstream (S)-(+)-Flurbiprofen of the GM-CSF gene in human chromosome 5q[14]. Using a high resolution chromatin immunoprecipitation (ChIP) procedure, we have recently mapped the regions of binding of several components of the pre-RC, including Orc1, Orc2, Rabbit Polyclonal to CDCA7 Mcm5 and CDC6, at these origins in close correspondence to the sites.