Asymmetric bidirectional replication at the human DBF4 origin

Abstract

Faithful replication of the entire genome once per cell cycle is essential for maintaining genetic integrity, and the origin of DNA replication is key in this regulation. Unlike that in unicellular organisms, the replication initiation mechanism in mammalian cells is not well understood. We have identified a strong origin of replication at the DBF4 promoter locus, which contains two initiation zones, two origin recognition complex (ORC) binding sites and two DNase I-hypersensitive regions within approximately 1.5 kb. Notably, similar to the Escherichia coli oriC, replication at the DBF4 locus starts from initiation zone I, which contains an ORC-binding site, and progresses in the direction of transcription toward initiation zone II, located approximately 0.4 kb downstream. Replication on the opposite strand from zone II, which contains another ORC-binding site, may be activated or facilitated by replication from zone I. We term this new mammalian replication mode 'asymmetric bidirectional replication'.

One-way PCR-based mapping of a replication initiation point (RIP)

“…Since this method is difficult to apply in mammalian cells, we and others used emetine, which inhibits new production of lagging strands without affecting leading strand synthesis 1,11 . Although concern was raised previously regarding drug treatments and the generation of potential artifactual results 2 , we did not observe any notable problemwhenwe determined DBF4 RIPs with cells maintained in the presence and absence of emetine 10 . However, we cannot completely rule out the potential effects of emetine on RIP-mapping results because we have not systematically compared RIP patterns of cells grown in the absence and presence of emetine.…”

“…RIPs from exponentially growing HeLa cells, the results were much more pronounced when nascent DNA fragments were isolated from synchronous HeLa cells at 1 h in S phase 10 . This was achieved by releasing HeLa cells synchronized at the G 1 /S border by a doublethymidine treatment into complete medium for 1 h as described previously 10 .…”

“…This was achieved by releasing HeLa cells synchronized at the G 1 /S border by a doublethymidine treatment into complete medium for 1 h as described previously 10 .…”

“…This procedure minimizes sample handling and has been previously employed by others to map oris in mammalian cells 13,14 . This simple method also appears to minimize DNA damage because origin enrichment was not found with nonreplicating control DNA 10 . If substantial damage occurred during nascent strand preparation, we would have observed the enrichment of origin sequence from the control DNA.…”

“…The steps that we could omit include cesium chloride gradient centrifugation, BND chromatography, treatment with T4 polynucleotide kinase to phosphorylate DNA molecules lacking an RNA-primer and λ-exonuclease treatments. Using this new approach, we successfully mapped RIPs at the human DBF4 locus, where we have recently identified a strong ori 10 . This new RIPmappingmethod combines one-way PCR-based primer extension with several previously reported techniques to effectively isolate nascent DNA fragments as described below (see Table 1 for comparison of different methods).…”