Figure 3.

Site-specific initation of DNA replication at the human lamin B2 origin in vivo and in vitro. (A) Map of the human lamin B2 origin and the primer sets used. Three primer sets were generated for quantification of nascent DNA strands by real-time PCR (see Materials and Methods). The primer set LB amplifies the lamin B2 replication initiation site (cf. 5,7). The two adjacent primer sets LBC1 and LBC2 amplify sequences 4 kb upstream and 3 kb downstream of the origin, respectively. (B) Initiation of DNA replication in vivo. Abundance of nascent DNA strands was quantified by real-time PCR using the LBC1, LB and LBC2 primer sets (see Materials and Methods). Nascent strands were prepared from HeLa cells arrested with mimosine in late G₁ phase (grey bars) and from cells that were released for 3 h from a mimosine block into early S phase in vivo (black bars). Means ± SD from two independent experiments are shown. (C) Initiation of DNA replication in vitro. Abundance of nascent strands from nuclei of mimosine-arrested cells incubated in a cytosolic extract of proliferating cells was analysed as detailed for (B).