Identification and detailed characterization of a novel origin of DNA replication in the transcriptional promoter of the human DBF4 gene

Abstract

Origins of DNA replication (oris) are the genomic sites where DNA synthesis starts to duplicate a cell's genome. Although prokaryotic and yeast oris have been characterized in detail, the nature of mammalian oris is still ill-defined. In fact, of ∼30,000 oris estimated to exist in mammalian genomes, only the one localized at the human lamin B2 locus has been mapped with nucleotide resolution. This thesis describes the identification of a novel human on located around the transcriptional promoter of the DBF4 gene. Using a modified version of the replication initiation point (RIP) mapping technique, precise leading strand initiation sites have been determined at this new ori. Additionally, chromatin immunoprecipitation (ChIP) assays have been employed to study the interaction of replication and transcription proteins within the ori region. The data presented here demonstrate that, at the DBF4 ori, DNA replication can initiate from multiple potential start-sites which are distributed within two replication initiation zones. These zones are separated by ∼400 bp and are localized near binding sites for the origin recognition complex (ORC) and the Sp1 transcription factor. Strikingly, initiation of DNA synthesis from the two zones occurs sequentially and not simultaneously: replication starts first from initiation zone I and proceeds in the direction of DBF4 transcription, followed by activation of zone II and DNA synthesis from the complementary strand. This replication pattern, termed asymmetric bidirectional replication (ABR), is reminiscent of initiation at oriC in Escherichia coli but greatly differs from what is observed at the lamin B2 ori, where a single ORC-binding site and unique replication start-points have been reported. Overall, the data suggest, for the first time, that eukaryotic replication initiation can take place through either the traditional origin of bidirectional replication (OBR) model, such as is the case in budding yeast and at the human lamin B2 ori, or through the asymmetric ABR model described here for the DBF4 ori.