FIG. 4.

Initiation of DNA replication in wild-type and mutant exogenous ori-β DNA fragments. (A) Unusual DNA sequences in the 5.8-kb ori-β fragment and the location of the deletion mutants are indicated on the restriction map of the region. Positions of AluI repetitive elements (white boxes) and AT-rich sequences homologous to the S. pombe origin consensus motifs D, C, Z, and M (50) (small black boxes) are marked. AT-rich sequences homologous to a cell cycle-dependent DNase I genomic footprint in the human lamin B2 IR (1, 29, 39) (hatched box) and to the ORC-binding region in the Drosophila chorion ACE3 (6, 80) (checkered boxes) are noted. AT-rich sequences containing stably bent DNA (B) and binding sites for a zinc finger protein of unknown function, RIP60 (black arrowheads), are indicated (15, 16, 21, 45, 64). The position of a cell cycle-dependent nuclease-hypersensitive site (72) is indicated (striped box). (B) The integrated mutant DHFR ori-β fragments in 5 ng of DNA from uncloned pools of DR12 cells and the endogenous ori-β region in 5 ng of CHOK1 DNA were amplified by PCR and visualized by gel electrophoresis and ethidium bromide staining. The ratio of the mutant amplification products in DR12 relative to those of the endogenous ori-β region in CHOK1 is indicated and suggests that the copy number of the exogenous ori-β region present in the pool of transfectants mimics that of the endogenous locus in CHOK1. (C) PCR amplifications were performed with each of the four primer pairs and size-fractionated nascent DNA from asynchronous mutant-transfected DR12 cells in the presence of a precalibrated amount of the corresponding competitor DNA. Amplification products were analyzed by PAGE and ethidium bromide staining. Control lanes: C, competitor template only; G, nascent genomic DNA template only; −, no template. Numbers above each lane represent the volume in microliters of nascent DNA added to the PCR mixture; note that for pMCDΔTR, the nascent genomic DNA was used at a 1:10 dilution with pp2 and pp6. (D) The abundance of nascent DNA from pools of mutant-transfected DR12 cells from three independent transfection experiments was quantitatively evaluated. As a measure of initiation activity, the abundance of each target sequence in nascent genomic DNA was normalized to the abundance of pp3 target sequences in the corresponding experiment, which was set equal to 1 (see Table 4 for a typical experiment), and the average of three experiments with each mutant is shown. For comparison, the average initiation activity measured with nascent DNA from a pool of asynchronous wild-type pMCD-transfected DR12 cells (Fig. 3D) is also shown. Bars indicate the SEM.