Functional genomic mapping of an early-activated centromeric mammalian origin of DNA replication

Ors12, a mammalian autonomously replicating sequence (812 bp), was previous ly isolated by extrusion of African green monkey (CV-1 cells) nascent DNA f rom active replication bubbles. It contains a region of a-satellite extendi ng 168-bp from the 5'-end, and a nonrepetitive portion extending from nucle otide position 169 to nucleotide 812 that is present in less than nine copi es per haploid genome. Ors12 is capable of transient autonomous DNA replica tion in vivo and in vitro, associates with the nuclear matrix in a cell cyc le-dependent manner, and hybridizes at the centromeric region of six CV-1 c ell chromosomes as well as a marker chromosome. To demonstrate that DNA rep lication initiates at ors12 at a native chromosomal locus, a 14.2 kb Africa n green monkey genomic clone was isolated and sequence information was obta ined that allowed us to generate eight sets of PCR primers spanning a regio n of 8 kb containing ors12. One set of primers occurred inside ors12. These primers were used to amplify nascent DNA strands from asynchronously growi ng CV-1 and African green monkey kidney (AGMK) cells, using noncompetitive and competitive PCR-based mapping methodologies. Both assays showed that DN A replication in vivo initiates preferentially in a 2.3 kb region containin g ors12, as well as at a second site located 1.7 kb upstream of ors12. This study provides the first demonstration of genomic function for a centromer ic mammalian origin of DNA replication, originally isolated by nascent stra nd extrusion. (C) 1999 Wiley-Liss, Inc.