Characterizing Replication Intermediates in the Amplified CHO Dihydrofolate Reductase Domain by Two Novel Gel Electrophoretic Techniques

Abstract

Using neutral/neutral and neutral/alkaline two-dimensional (2-D) gel techniques, we previously obtained evidence that initiation can occur at any of a large number of sites distributed throughout a broad initiation zone in the dihydrofolate reductase (DHFR) domain of Chinese hamster ovary (CHO) cells. However, other techniques have suggested a much more circumscribed mode of initiation in this locus. This dichotomy has raised the issue whether the patterns of replicating DNA on 2-D gels have been misinterpreted and, in some cases, may represent such noncanonical replication intermediates as broken bubbles or microbubbles. In an accompanying study (R. F. Kalejta and J. L. Hamlin, Mol. Cell. Biol. 16:4915-4922, 1996), we have shown that broken bubbles migrate to unique positions in three different gel systems and therefore are not likely to be confused with classic replication intermediates. Here, we have applied a broken bubble assay developed from that study to an analysis of the amplified DHFR locus in CHO cells. This assay gives information about the number and positions of initiation sites within a fragment. In addition, we have analyzed the DHFR locus by a novel stop-and-go-alkaline gel technique that measures the size of nascent strands at all positions along each arc in a neutral/neutral 2-D gel. Results of these analyses support the view that the 2-D gel patterns previously assigned to classic, intact replication bubbles and single-forked structures indeed correspond to these entities. Furthermore, potential nascent-strand start sites appear to be distributed at very frequent intervals along the template in the intergenic region in the DHFR domain.Because of the great complexity of higher eukaryotic genomes, the identification of origins of replication has proven to be extremely challenging. In the absence of a reliable genetic assay for identifying autonomously replicating sequence elements, several origin mapping strategies have been devised to localize start sites in a region of interest, with the assumption that the responsible genetic element (replicator) will lie close by. The Chinese hamster dihydrofolate reductase (DHFR) replicon (Fig. 1A) has been analyzed by almost every available replicon mapping method, and all methods suggest that an origin lies somewhere in the 55-kb spacer region between the DHFR and 2BE2121 genes (reviewed in references 8, 13, and 25). However, different methods of analysis have not provided an entirely coherent picture of initiation in this locus. For example, results from high-resolution intrinsic labelling in the early S period (1, 9, 23, 32), leading-and lagging-strand polarities (10, 24, 26), and nascent-strand size and/or abundance measurements (44) are all compatible with two preferred sites or zones of initiation (termed ori-␤ and ori-␥ [32]) that are separated by about 22 kb and that lie in the 55-kb spacer region between the DHFR and 2BE2121 genes (Fig. 1A) (1, 32). In contrast, both neutral/neutral (5) and neutral/alkaline (40) two-dimensional (2-D) gel replicon...

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

“…In two previous studies, novel variations of the neutralneutral and neutral-alkaline 2-D gel methods were developed to determine whether these methods could fail to detect a major initiation site near ori-␤ (22,23). Instead, the results supported our previous proposal that nascent-strand start sites are chosen from many potential sites scattered throughout the 55-kb intergenic region, with a concentration of sites in the central 30-to 35-kb encompassing the ori-␤ and ori-␥ regions (10,11,13,39).…”

“…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-␤ failed to do so (22,23), lending support to a model in which potential initiation sites are scattered throughout the intergenic region, with ori-␤ and ori-␥ being only somewhat preferred.…”

Mammalian Mitochondrial DNA Replicates Bidirectionally from an Initiation Zone

“…Furthermore, our previous suggestion that the Y-like species resulted from bubbles broken during processing (14) is undermined by other observations. First, Hamlin and co-workers have shown that broken replication bubbles form arcs distinct from a simple Y arc (23). Second, intact bubble arcs were seen with other enzymes such as HincII and ClaI (e.g.…”

“…Our original interpretation depended on the assumption that molecules derived from O H -containing fragments coincident with a simple Y arc represented broken bubbles (14). The assumption was tenuous, given that it had been shown earlier that broken bubbles form arcs distinct from a simple Y arc (23).…”

Functionally distinct, sequence-specific replicator and origin elements are required for Drosophila chorion gene amplification

“…However, different techniques yield somewhat contradictory data regarding the exact sites of initiation, and origin activity appears to involve initiations throughout the region, with some degree of preference for initiation at three sites, called ori-␤, ori-␤Ј, and ori-␥ (Burhans et al 1990;Vaughn et al 1990;Kalejta et al 1996;Kobayashi et al 1998;Wang et al 1998). Attempts to identify DHFR replicators have so far yielded conflicting results.…”