DNA Replication Initiates at Different Sites in Early and Late S Phase within Human Ribosomal RNA Genes

Abstract

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