Lagging-Strand, Early-Labelling, and Two-Dimensional Gel Assays Suggest Multiple Potential Initiation Sites in the Chinese Hamster Dihydrofolate Reductase Origin

To analyze the replication pattern of a defined mammalian chromosomal locus, we have taken advantage of a methotrexate-resistant CHO cell line (CHOC 400) in which one allele of the early-replicating dihydrofolate reductase (DHFR) locus is amplified ca. 1,000 times (30). The high copy number of the 240-kb repeating unit (amplicon) has facilitated a number of studies designed to identify the replication start site(s) (reviewed in references 8 and 17). By radiolabelling the DNA synthesized at the beginning of the S phase, it was possible to show that replication initiates preferentially in the region lying between the convergently transcribed DHFR and 2BE2121 genes (20) (Fig. ​(Fig.1).1). In a more quantitative and higher-resolution in-gel renaturation approach, two somewhat preferred initiation regions (termed ori-β and ori-γ) were identified within the intergenic spacer (Fig. ​(Fig.1,1, ELF in-gel) (26). ori-β and ori-γ are ∼22 kb apart and lie on either side of a prominent matrix attachment region (MAR) (9). In recent years, several additional replicon-mapping techniques with potentially higher resolution and/or sensitivity have been applied to the DHFR locus either in CHOC 400 cells or in parental CHO cells. However, the results from these different approaches do not paint a unified picture. FIG. 1 The DHFR initiation locus. A 110-kb region encompassing the convergently transcribed DHFR and 2BE2121 genes and the intergenic region of the DHFR domain is shown. Positions of ori-β, ori-γ, and the MAR on the EcoRI map are indicated by ... One view was suggested by measurements of the template bias of lagging nascent strands, which should switch dramatically at a fixed origin of replication. When this technique was applied to the 11-kb region surrounding ori-β in synchronized CHOC 400 and CHO cells, a pronounced switch in template bias suggested that >80% of initiations occur within a 480-bp fragment centered in the ori-β region (Fig. ​(Fig.1,1, Lagging St) (5). Thus, ori-β has been designated an origin of bidirectional replication (5). The same result was reported for asynchronous CHOC 400 cells (5), which excluded the possibility of a second active origin in the region of ori-γ; this follows because Okazaki fragments in forks replicating through ori-β from either direction would not change templates at this site, resulting in a much lower switch in template bias than was actually observed. The results of PCR-based nascent-strand abundance assays (34, 38) also argue that ori-β is a highly preferred initiation locus (Fig. ​(Fig.1,1, PCR) (34). However, neither the lagging-strand nor nascent-strand abundance assay examined the region surrounding ori-γ. Thus, the presence of a preferred initiation site or zone in this region was not formally excluded. At the other end of the spectrum are results from two-dimensional (2-D) gel analyses of the DHFR locus. A neutral-neutral 2-D gel replicon-mapping method (3) suggests that initiation can occur at virtually any location within the 55-kb intergenic region, with relatively more initiations occurring in the central 30 to 35 kb encompassing ori-β and ori-γ (Fig. ​(Fig.1,1, N/N 2-D) (10, 11, 13, 39). In apparent agreement, a neutral-alkaline 2-D gel method (33) detects replication forks travelling in both directions within this central region in the early S phase (Fig. ​(Fig.1,1, N/A 2-D) (11, 13, 39). Two recent variations of the 2-D gel methods that were designed specifically to detect a highly favored start site at ori-β failed to do so (22, 23), lending support to a model in which potential initiation sites are scattered throughout the intergenic region, with ori-β and ori-γ being only somewhat preferred. The results of other approaches could accommodate either view. In an early-labelling fragment hybridization (ELFH) assay, DNA labelled either in vitro (4, 16) or in vivo (4) in the first 5 to 30 min of the S phase was used to probe immobilized clones from the intergenic region to determine which restriction fragments are the first to be synthesized at the beginning of the S phase. In two studies (4, 16), the resulting hybridization patterns identified a peak of early labelling that encompassed ori-β. However, because of the absence of clones containing and surrounding ori-γ, neither study examined the possibility of a second preferred initiation locus in this region (Fig. ​(Fig.1,1, ELFH) (16). Indeed, in an in vivo variation of this approach in which psoralen cross-links were used to confine labelling to the region immediately surrounding the origins, a very sharp peak was detected over ori-β and a much broader peak was detected over ori-γ (1). However, the signal from the ori-β region was subsequently shown to arise, at least in part, from two closely spaced copies of the AluI-like repeated sequence family in this region (6, 25), and hybridization could be completely eliminated by inclusion of excess unlabelled CHO DNA in the hybridization solution (25). Thus, although the work described in reference 1 was subsequently cited as evidence for a highly favored initiation site in the region of ori-β (5, 8, 38), the specific labelling of ori-β itself (but not ori-γ, which contains no repeated elements) was indeterminate owing to cross-hybridization with other early-replicating repetitive sequences in the genome. Finally, determinations of the template bias of leading strands could accommodate either highly preferred sites at ori-β and ori-γ or a much broader zone with ori-β and ori-γ being only somewhat preferred, since the nearest probes lay 5 to 8 kb on either side of the two regions and since there was significant hybridization to both strands at all positions (Fig. ​(Fig.1,1, Leading St) (19). There are other inconsistencies among the data sets. As mentioned above, the lagging-strand assay was reported to detect a template switch at the ori-β locus even in asynchronous CHOC 400 cell populations (5). Even if one excludes the possible existence of an ori-γ, this data implies that the DHFR origin is extremely efficient, with initiation occurring in all of the amplicons almost simultaneously in the early S phase in every cell cycle. Thus, at a fork rate of 3 kb/min (21), it should take less than 1 h to replicate most of the 240-kb amplicons. However, intrinsic labelling studies showed that it takes at least 8 h to replicate all of the amplicons (20); furthermore, neutral-neutral and neutral-alkaline 2-D gel methods detected bubbles in the intergenic zone only in the first 2 h of the S phase but single forks persisted for more than 6 h (10, 11, 13, 18, 39). Thus, initiation must occur in only 10 to 15% of the amplicons in any one S phase, with the remainder being replicated passively by forks from distant amplicons. Thus, in an asynchronous culture, a strand switch at ori-β in 10 to 15% of amplicons should be largely obscured by readthrough of this locus in the remaining 80 to 85% that is passively replicated. Clearly, each replicon-mapping method measures different properties of replicating DNA and each has its own strengths and weaknesses. For example, the neutral-neutral 2-D gel technique can detect initiation events even at minor sites but cannot detect small differences in the relative number of bubbles from one position to the next along a template, owing primarily to differences in restriction fragment sizes along a region of interest (29). The leading- and lagging-strand assays have the advantage that hybridization biases to the two template strands can be reasonably accurately quantified within a given experiment; however, the bias to one strand or the other is never absolute (5, 19, 24, 36) and, unlike 2-D gel approaches, leading- and lagging-strand assays cannot distinguish between minor initiation sites distributed throughout the replicon under study and unavoidable background hybridization. It is also significant that the indicators in the lagging-strand and ELFH assays are fragments that were labelled in vitro by a 1.5-min pulse of radioactive deoxyribonucleotides (5) and therefore qualify as short-lived replication intermediates whereas 2-D gel methods measure the steady-state in vivo distribution of intermediates and therefore bias toward longer-lived species. Furthermore, the lagging-strand and ELFH assays were performed on cells just as they were entering the S phase (i.e., ∼4 min after release from aphidicolin [5, 16]), while most 2-D gel measurements were performed when initiation in the DHFR locus was maximal (either 20 to 30 min after release from aphidicolin [39] or 80 to 90 min after release from mimosine [10, 11, 13]). We assume that each assay is valid and accurately reports some aspect of the biological initiation reaction. Therefore, in the present study we attempted to fix several of the experimental variables that could complicate the interpretations of the data. We have applied the lagging-strand, ELFH, neutral-neutral 2-D gel, and/or neutral-alkaline 2-D gel assays to replication intermediates prepared from CHOC 400 cells under the same experimental conditions. Results from these assays appear to be consistent with one another and suggest that there is a broad zone of initiation in the intergenic region of the amplified DHFR domain, with the region encompassing ori-β and ori-γ being somewhat preferred. (This work fulfills part of the requirement for a Ph.D. in biochemistry from the University of Virginia for S.W.)