Metazoan replication origins often contain multiple potential initiation sites, and the selection of which of the potential sites are used appears to be dependent upon multiple factors, including the state of differentiation, cell metabolism, and local transcriptional activity. Numerous studies have shown that a replication origin exists within the non-transcribed spacer region of the human ribosomal RNA gene. We here analyze nascent leading strand DNA from S phase human lymphoid cells, and find that while the majority of rDNA replicates in mid- and late S phase and preferentially initiates replication 6 kbp from the transcription start site, in very early S phase the preferred initiation site is much closer to the transcription start site and may involve rDNA promoter sequences. This early site is coincident with a minimum GC skew value, diagnostic for replication origins in bacteria and yeast. These results suggest that replication timing can influence initiation site selection. The timing and nucleolar localization of rDNA further suggest that this site likely participates in the small number of perinucleolar initiation foci observed in very early S phase cells that represent the beginning of cellular DNA replication.

Numerous studies have demonstrated that DNA replication initiates within the 30 kB non-transcribed spacer (NTS) region of the human ribosomal RNA gene (rDNA). Using a series of closely spaced primer pairs to measure nascent leading strand abundance in mid and late S phase cells isolated by centrifugal elutriation, we find evidence for one highly preferred initiation site and two less utilized sites within a 6 kb region of the NTS. The initiation sites colocalize with significant DNA unwinding elements (DUEs), matrix attachment regions (MARs), and ARS-like sequences. An intrinsic DNA bending site was localized by circular permutation analysis to within several hundred base pairs of one initiation site. While DUE and MAR elements occur elsewhere throughout the 43 kb rDNA sequence, the close association of DUE and MAR elements occurs only near replication initiation sites, a juxtaposition also seen in other well-studied mammalian replication initiation sites. The utilization of rDNA initiation sites close to DUE and MAR elements in mid and late S phase, but not in very early S phase as previously shown, suggests that in rRNA genes, contributions from these sequence-associated properties may be more significant to initiation sites associated with transcriptionally inactive genes, than to initiation sites associated with transcriptionally active genes.