Replication initiation sites are distributed widely in the amplified CHO dihydrofolate reductase domain

Abstract

In previous studies, we utilized a neutral/neutral twodimensional (2-D) gel replicon mapping method to analyze the pattern of DNA synthesis in the amplified dihydrofolate reductase (DHFR) domain of CHOC 400 cells. Replication forks appeared to initiate at any of a large number of sites scattered throughout the 55 kb region lying between the DHFR and 2BE2121 genes, and subsequently to move outward through the two genes. In the present study, we have analyzed this locus in detail by a complementary, neutral/alkaline 2-D gel technique that determines the direction in which replication forks move through a region of interest. In the early S period, forks are observed to travel in both directions through the intergenic region, but only outward through the DHFR gene. Surprisingly, however, replication forks also move in both directions through the 2BE2121 gene. Furthermore, in early S phase, small numbers of replication bubbles can be detected in the 2BE2121 gene on neutral/neutral 2-D gels. In contrast, replication bubbles have never been detected in the DHFR gene. Thus, replication initiates not only in the intergenic region, but also at a lower frequency in the 2BE2121 gene. We further show that only a small fraction of DHFR amplicons sustains an active initiation event, with the rest being replicated passively by forks from distant amplicons. These findings are discussed in light of other experimental approaches that suggest the presence of a much more narrowly circumscribed initiation zone within the intergenic region.

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

“…2C). However, by 100 min, when initiation is maximal in this locus (15,18), a typical composite pattern is detected in this fragment (Fig. 2D).…”

“…1A) (1, 32). In contrast, both neutral/neutral (5) and neutral/alkaline (40) two-dimensional (2-D) gel replicon mapping methods suggest that initiation can occur at any of a large number of sites distributed throughout the 55-kb intergenic region (15,16,18,45).Since it is relatively difficult to accurately compare initiation frequencies at different genomic sites by 2-D gel techniques (5, 36), it is possible that the frequencies of initiation at ori-␤ and ori-␥ have been greatly underestimated (10). Furthermore, 2-D gels are technically challenging, and interpretation is based on a comparison of the patterns obtained with those of well-defined replicons.…”

“…1A) (1, 32). In contrast, both neutral/neutral (5) and neutral/alkaline (40) two-dimensional (2-D) gel replicon mapping methods suggest that initiation can occur at any of a large number of sites distributed throughout the 55-kb intergenic region (15,16,18,45).…”

DNA replication origins in the Schizosaccharomyces pombe genome

“…In several cases initiation has been shown to occur with relatively low efficiency at many different sites in intergenic regions of the genome (47,48). Recent work suggests that human ORC binds preferentially to AT-rich sequences but otherwise has little sequence specificity (49).…”

Mammalian Mitochondrial DNA Replicates Bidirectionally from an Initiation Zone

“…However, this zone does not encompass the entire genome, much of which is free of origins that give rise to initiation arcs on N/N 2D-AGE. The fragments that define a contiguous origin-free zone covering the rest of the mitochondrial genome are as follows: in DISCUSSION N/N 2D-AGE has been employed widely since 1987 and is the pre-eminent method for analyzing replication intermediates, including origins of replication (1,30,31); to date, it has proved to be totally reliable. In the present study, extensive analysis of mitochondrial DNA of three mammals revealed a set of overlapping fragments associated with initiation arcs (Figs.…”