Differentially Active Origins of DNA Replication in Tumor versus Normal Cells

Abstract

Previously, a degenerate 36 bp human consensus sequence was identified as a determinant of autonomous replication in eukaryotic cells. Random mutagenesis analyses further identified an internal 20 bp of the 36 bp consensus sequence as sufficient for acting as a core origin element. Here, we have located six versions of the 20 bp consensus sequence (20mer) on human chromosome 19q13 over a region spanning f211 kb and tested them for ectopic and in situ replication activity by transient episomal replication assays and nascent DNA strand abundance analyses, respectively. The six versions of the 20mer alone were capable of supporting autonomous replication of their respective plasmids, unlike random genomic sequence of the same length. Furthermore, comparative analyses of the endogenous replication activity of these 20mers at their respective chromosomal sites, in five tumor/ transformed and two normal cell lines, done by in situ chromosomal DNA replication assays, involving preparation of nascent DNA by the L exonuclease method and quantification by real-time PCR, showed that these sites coincided with chromosomal origins of DNA replication in all cell lines. Moreover, a 2-to 3-fold higher origin activity in the tumor/ transformed cells by comparison to the normal cells was observed, suggesting a higher activation of these origins in tumor/transformed cell lines. (Cancer Res 2006; 66(10): 5094-103)

A novel role for fanconi anemia (FA) pathway effector protein FANCD2 in cell cycle progression of untransformed primary human cells

“…Indeed, increased activity of DNA replication origins in tumor cells or SV-40 transformed cells have been reported, suggesting that transformation may induce greater frequency of initiation of origins at certain loci. [39][40][41][42] The increased origin activity in tumor/transformed cells compared to normal cells may be influenced by many parameters including concentration and activity of initiation factors, chromatin structure and nuclear organization. 41 Potentially, FA proteins might be involved in these processes to regulate initiation of DNA replication.…”

Molecular basis for the actions of Hsp90 inhibitors and cancer therapy

“…Moreover, the origin activity in the tumor-transformed cells is two-to threefold higher than that of normal cells. 30 In chromosomal DNA replication, a plausible supermolecular assembly, the origin recognition complex, and the replisomes are thought to function in DNA replication initiation and the elongation of the DNA strands, respectively. The cell cycle starts from a Go quiescent state.…”

Metazoan origins of DNA replication: Regulation through dynamic chromatin structure

“…A twofold increase in origin activity across the same 12.5 kb region of the human c-myc locus was also found in the isogenic cell lines WI38 and their transformed counterpart WI38 (SV40) [Tao et al, 2001], ruling out the possibility that cell type effects were the cause of the differential origin usage found in HeLa and NSF cells. It was thus concluded that the increased origin activities were the result of cellular transformation.More recent studies have found two-to threefold higher origin activities in transformed/tumor cells compared to normal cells, suggesting higher activation of these origins located across this $211 kb region on human chromosome 19q13 [Di Paola et al, 2006]. Again, use of the isogenic cell lines of WI38 and WI38(SV40) showed that the origins are at least twice as active in the transformed cell line (WI38(SV40)) compared to that of its normal counterpart (WI38), ruling out the possibility that cell type effects were responsible for increased frequency of initiation.…”

“…The distribution of this 20mer consensus over 1 Mb of human chromosomal DNA is similar, quantitatively and qualitatively to the distribution of the ARS consensus sequence (ACS) on S. cerevisiae chromosomes . Finally, six versions of the 20mer which were analyzed ectopically and endogenously were found to act as origins of DNA replication in plasmids as well as in situ, at their chromosomal loci, suggesting that the 20-bp consensus sequence can be used to predict chromosomal regions that contain replication origins [Di Paola et al, 2006]. …”

“…The increased origin activity across the $211 kb region in tumor/transformed cells compared to normal cells may be influenced by one or many parameters regulating DNA replication, such as the concentration and conformation of initiator proteins [McNairn and Gilbert, 2005;Lau et al, 2007;Blow and Gillespie, 2008], specific DNA sequences within the initiation sites having differential affinities for ORC [Bell, 2002], gene transcription [DePamphilis, 1993], chromatin structure [Melendy and Li, 2001;Aladjem, 2007], nuclear organization [Taddei et al, 2004;Ottaviani et al, 2008], and nucleotide pool levels [Anglana et al, 2003]. Previous work suggests that there are at least two types of malignant changes in regulation of DNA replication, the unexpected increase in origin activity at some loci [Tao et al, 1997[Tao et al, , 2000[Tao et al, , 2001Di Paola et al, 2006] and activation of silent origins [Martin and Oppenheim, 1977;Oppenheim and Martin, 1978]. Thus, it appears that there are at least three subsets of origins; those that are normal and remain unchanged, those with increased activity in immortalized or malignant cells, and those that are activated solely in tumor cells.…”