COMMUNICATION Differential DNA Replication Origin Activities in Human Normal Skin Fibroblast and HeLa Cell Lines Liang Tao, Torsten Nielsen, Paula Friedlander Maria Zannis-Hadjopoulos and Gerald Price* McGill Cancer Centre, McGill University, Montreal, Quebec Canada, H3G 1Y6 A modi®cation of the extrusion method for the isolation of nascent DNA from mammalian cells and a PCR-based assay has been used in order to compare the in vivo activities of DNA replication origins in different cell lines. Conventional PCR was ®rstly applied to detect the chromosomal activities of several known (origins associated with c-myc, hsp70, b-globin, immunoglobulin m-chain enhancer) and putative DNA replication origins (autonomously replicating sequences obtained from enriched libraries of human origins of DNA replication from normal and transformed cells) in four human cell lines (HeLa, NSF, WI-38 and SK-MG-1). Then, in nascent DNA samples from normal skin ®broblast (NSF) and HeLa cells, abun- dance of DNA sequences in the regions of ®ve of these origins was deter- mined by competitive PCR. Our results suggest that autonomously replicating sequences NOA3, S14, S3 and F15 are associated with functional chromosomal origins of replication. Quantitative comparison of origin activities demonstrates that origins associated with c-myc and NOA3 are approximately twice as active in HeLa cells as in NSF cells. The described approach can facilitate the identi®cation of origins which may be differentially active in normal cells and transformed cells or in different cell types. # 1997 Academic Press Limited Keywords: DNA replication; origin; human; nascent DNA; PCR *Corresponding author Chromosomal DNA fragments are replicated in units, replicons, with an average size of 50 to 300 kb in animal cells during the S phase (Edenberg & Huberman, 1975; Huberman, 1995). There are estimated to be 10 4 to 10 6 replicons on chromosomes in an animal cell, with each of the replicons containing one functional origin (ori). This large number of origins are well-regulated spatially and temporally (Dif¯ey & Stillman, 1990; Coverley & Laskey, 1994). The activation of eukaryotic mammalian origins may be regulated at different levels. Firstly, speci®c sequences that act as replication origins in higher eukaryotic cells may have differential af®nities for the different protein components of the origin recognition com- plex (Dif¯ey & Stillman, 1990; Pearson et al., 1991; Dijkwel & Hamlin, 1996; Boulikas, 1996). Secondly, the concentration and conformation of initiator proteins may also affect the activation origins (Dif¯ey & Stillman, 1990; Huberman, 1995; Walter & Newport, 1997; Stillman, 1996; Chevalier & Blow, 1996). Thirdly, transcription factors may also play important roles in initiation of replication and replication timing for different cell types (DePamphilis, 1993b; Diller & Raghuraman, 1994; Ohba et al., 1996). Transcriptional activation of cer- tain gene loci in cells has been associated with initiation of replication beginning earlier in S phase than in cells in which the same loci are not tran- scribed (Dhar et al., 1988; Hatton et al., 1988). Finally, chromatin structure and nuclear organiz- ation, essential for spatial positioning and inter- action of origin sequences with replication proteins in order to initiate replication, also affect origin activation (Hozak et al., 1993; DePamphilis, 1993a; Lawlis et al., 1996; Newport & Yan, 1996). Cell transformation may also modify the regu- lation of origin activation, resulting in differential origin usage between normal and transformed cells. This is supported by previous studies, which have shown that the average size of replicons, as measured by DNA ®ber autoradiography, is decreased with malignant transformation (Martin Abbreviations used: NSF, normal skin ®broblast; BrdUrd, bromodeoxyuridine. J. Mol. Biol. (1997) 273, 509±518 0022±2836/97/430509±10 $25.00/0/mb971352 # 1997 Academic Press Limited