Major DNA replication initiation sites in the c-myc locus in human cells

DNA replication initiation sites and initiation frequencies over 12.5 kb of the human c-myc locus, including 4.6 kb of new 5' sequence, were determine d based on short nascent DNA abundance measured by competitive polymerase c hain reaction using 21 primer sets. In previous measurements, no comparativ e quantitation of nascent strand abundance was performed, and distinction o f major from minor initiation sites was not feasible. Two major initiation sites were identified in this study. One predominant site has been located at similar to 0.5 kb upstream of exon 1 of the c-myc gene, and a second new major site is located in exon 2. The site in exon 2 has not been previousl y identified. In addition, there are other sites that may act as less frequ ently used initiation sites, some of wh ich may correspond to sites in prev ious reports. Furthermore, a comparison of the abundance of DNA replication intermediates over this same region of the c-myc locus between HeLa and no rmal skin fibroblast (NSF) cells indicated that the relative distribution w as very similar, but that nascent strand abundance in HeLa cells was approx imately twice that in NSF relative to the abundance at the lamin B2 origin. This increased activity at initiation sites in the c-myc locus may mainly be influenced by regulators at higher levels in transformed cells like HeLa . (C) 2000 Wiley-Liss, Inc.