Selection and mapping of replication origins from a 500-kb region of the human X chromosome and their relationship to gene expression

In higher eukaryotes the mechanism controlling initiation of DNA replicatio n remains largely unknown. New technologies are needed to shed light on how DNA replication initiates along the genome in specific regions, To identif y the human DNA sequence requirements for initiation of replication, we dev eloped a new method that allows selection of replication origins starting f rom large genomic regions of human DNA. We repeatedly isolated 15 new putat ive replication origins (PROs) from a human DNA region of 500 kb in which 1 7 genes have previously been characterized. Fine-mapping of these PROs show ed that DNA replication can initiate at many specific points along actively transcribed DNA in the cell lines used for our selection. In conclusion, i n this paper we describe a new method to identify PROs that suggests that t he availability of initiation sites is dependent on the transcriptional sta te of the DNA. (C) 1999 Academic Press.