The Chinese hamster dihydrofolate reductase replication origin beta is active at multiple ectopic chromosomal locations and requires specific DNA sequence elements for activity

Abstract

To identify cis-acting genetic elements essential for mammalian chromosomal DNA replication, a 5.8-kb fragment from the Chinese hamster dihydrofolate reductase (DHFR) locus containing the origin beta (ori-beta) initiation region was stably transfected into random ectopic chromosomal locations in a hamster cell line lacking the endogenous DHFR locus. Initiation at ectopic ori-beta in uncloned pools of transfected cells was measured using a competitive PCR-based nascent strand abundance assay and shown to mimic that at the endogenous ori-beta region in Chinese hamster ovary K1 cells. Initiation activity of three ectopic ori-beta deletion mutants was reduced, while the activity of another deletion mutant was enhanced. The results suggest that a 5.8-kb fragment of the DHFR ori-beta region is sufficient to direct initiation and that specific DNA sequences in the ori-beta region are required for efficient initiation activity.

FIG. 1

(A) Features of the endogenous DHFR ori-β IR. Preferred start sites of DNA replication—the ori-β, ori-β′, and ori-γ sites; a 55-kb initiation zone (shaded area) between the DHFR- and 2BE2121-coding sequences; and matrix-attached regions (MARs, stippled boxes)—are indicated (5, 14, 42, 43, 44, 52, 55, 57, 71, 85, 86, 89). The 5.8-kb fragment of the DHFR ori-β region (pMCD), extending from the BamHI to the KpnI recognition sequences, is indicated. Arrows denote the positions of the primers used in the competitive PCR assay for origin function and correspond to primer pairs used in previous DHFR ori-β mapping studies (71). (B) Strategy for quantitating initiation occurring in exogenous DHFR ori-β fragments in ectopic chromosomal locations. A 5.8-kb wild-type or mutated DHFR ori-β fragment was coelectroporated with a neomycin resistance gene into the DR12 cell line, a CHO derivative containing a 150-kb deletion encompassing the entire DHFR locus (47). After selection with G418, total DNA was isolated, heat denatured, and size fractionated on a 5 to 30% linear neutral sucrose gradient. The fraction containing single-stranded DNA with a length of 1 to 2 kb, representing nascent DNA, was isolated, and the abundance of ori-β target sequences contained in the fraction was quantitated by competitive PCR.

FIG. 2

Initiation of DNA replication at the endogenous DHFR ori-β site in asynchronous CHOK1 cells. (A) PCR amplifications were performed with each of the four primer pairs and with size-fractionated nascent DNA template from asynchronous CHOK1 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. Amplification reactions with pp2 used a 1:10 dilution of the nascent DNA. (B) PCRs were performed and analyzed as in panel A, except that the template was a 1:10 dilution of sheared, denatured total genomic CHOK1 DNA 1 to 2 kb in length. (C) Amplification products generated with either nascent DNA from asynchronous CHOK1 cells (black bars) or sheared total genomic DNA from asynchronous CHOK1 cells (white bars) were quantitatively evaluated for five independent experiments with each type of template. 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 example in Table 2), and the average of five experiments is shown. Bars indicate the standard error of the mean (SEM).

FIG. 3

Initiation of DNA replication in the exogenous 5.8-kb ori-β fragment in DR12 cells. (A) The integrated exogenous DHFR ori-β fragment in 5 ng of DNA from uncloned pools of DR12 cells and the endogenous ori-β in 5 ng of CHOK1 DNA were amplified by PCR. The ratio of the amplification products in DR12 relative to those of endogenous ori-β in CHOK1 is indicated, and this ratio suggests that the copy number of exogenous ori-β fragments present in the pool of transfectants mimics that of the endogenous locus in CHOK1. (B) The structure of the integrated DHFR ori-β fragments in DR12 pools was determined by PCR amplification of either pMCD plasmid DNA (lanes P) or large single-stranded genomic DNA isolated from uncloned drug-resistant pools of pMCD-transfected DR12 cells (lanes G). Amplification reactions were performed with a panel of six primer sets spanning the 5.8-kb DHFR ori-β fragment and the flanking vector sequences (bottom). The resulting overlapping PCR amplification products, labeled 1 through 6, were analyzed by PAGE and ethidium bromide staining. M indicates a DNA size marker. The sizes of the resulting PCR amplification products are indicated. (C) PCR amplifications were performed with each of the four primer pairs and size-fractionated nascent DNA from asynchronous pMCD-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. Amplification reactions for pp2 and pp6 used a 1:10 dilution of the nascent DNA. (D) Amplification products generated with either nascent DNA from a pool of asynchronous pMCD-transfected DR12 cells (black bars) or sheared total genomic DNA from asynchronous pMCD-transfected DR12 cells (white bars) were quantitatively evaluated for seven independent transfection experiments. 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 example in Table 2), and the average of the seven experiments is shown. Bars indicate the SEM.

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.