Start sites for bidirectional in vitro DNA replication inside ..

A critical point of DNA replication is the selection of the sites where DNA synthesis starts.

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DNA replication in prokaryotes and lower eukaryotes usually initiates from a discrete cis-acting DNA element termed the origin of DNA replication (ori),. There are lines of evidence that replication in the mammalian cells also starts from a defined location called an origin of bidirectional replication (OBR). However, there is equally compelling evidence that replication initiates from a broad initiation zone. It is now generally accepted that several potential ori sites exist within a small or large initiation zone, although certain sites within the zone may be preferentially used. However, there is no experimental data that can reconcile the OBR and diffused/delocalized ori models. Even in the case of known mammalian OBRs, the exact mode of replication is still to be demonstrated, since the resolution of all known OBRs is several hundreds to thousands of nucleotides range, except the one in the human lamin-B2 (LMNB2) locus.

Within each position are one or two start sites

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 ~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 ~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’.

We have shown that a 1,2 kb DNA fragment comprising the start site of DNA replication along with the OBP and the CpG island associated with the TIMM13 promoter behaves as an origin of DNA replication when integrated at ectopic positions of the human genome.