Abstract
Numerous studies have demonstrated that DNA replication initiates within the 30 kBnontranscribed spacer (NTS) region of the human ribosomal RNA gene (rDNA). Using a series ofclosely spaced primer pairs to measure nascent leading strand abundance in mid and late S phasecells isolated by centrifugal elutriation, we find evidence for one highly preferred initiation site andtwo less utilized sites within a 6 kb region of the NTS. The initiation sites co-localize withsignificant DNA unwinding elements (DUEs), matrix attachment regions (MARs), and ARS-likesequences. An intrinsic DNA bending site was localized by circular permutation analysis to withinseveral hundred base pairs of one initiation site. While DUE and MAR elements occur elsewherethroughout the 43 kb rDNA sequence, the close association of DUE and MAR elements occursonly near replication initiation sites, a juxtaposition also seen in other well-studied mammalianreplication initiation sites. The utilization of rDNA initiation sites close to DUE and MAR elementsin mid and late S phase, but not in very early S phase as previously shown, suggests that in rRNAgenes, contributions from these sequence-associated properties may be more significant to initiationsites associated with transcriptionally inactive genes than to initiation sites associated with activegenes.
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