Localization of a bidirectional DNA replication origin in the native locus and in episomally amplified murine adenosine deaminase loci.

Abstract

Gene amplification is frequently mediated by the initial production of acentric, autonomously replicating extrachromosomal elements. The 4,000 extrachromosomal copies of the mouse adenosine deaminase (ADA) amplicon in B-1/50 cells initiate their replication remarkably synchronously in early S phase and at approximately the same time as the single-copy chromosomal locus from which they were derived. The abundance ofADA sequences and favorable replication timing characteristics in this system led us to determine whether DNA replication initiates in ADA episomes within a preferred region and whether this region is the same as that used at the corresponding chromosomal locus prior to amplification. This study reports the detection and localization of a discrete set of DNA fragments in the ADA amplicon which label soon after release of synchronized B-1/50 cells into S phase. A switch in template strand complementarity of Okazaki fragments, indicative of the initiation of bidirectional DNA replication, was found to lie within the same region. This putative replication origin is located approximately 28.5 kbp upstream of the 5' end of the ADA gene. The same region initiated DNA replication in the single-copy ADA locus of the parental cells. These analyses provide the first evidence that the replication of episomal intermediates involved in gene amplification initiates within a preferred region and that the same region is used to initiate DNA synthesis within the native locus.Amplification and consequent overexpression of oncogenes and growth factor receptor genes occurs in many human and rodent cancer cells in vivo. Amplification of a large number of target genes has also been shown to underlie the drug resistance of many mammalian cell lines propagated in vitro (see references 4, 5, 9, 43, and 44 for recent reviews). It is reasonable to infer that oncogene amplification contributes to tumor cell growth or survival since recent studies reveal that reducing the expression of oncogenes by antisense strategies (51) or by elimination of amplified oncogenes (49) reduces tumorigenicity.In many cases, the initial molecular intermediates of gene amplification are genetically unstable (see reference 26 for an early example and reference 50 for a review) and consist of chromosome fragments lacking centromeres (see reference 52 for a recent study and reference 53 for a review). These fragments often appear as heterogeneously sized, paired chromatin bodies called double minute chromosomes (DMs) (3,10,17,39). DMs can be formed by chromosome breakage (40, 52), resulting in the generation and subsequent oligomerization of submicroscopic circular precursors referred to as episomes or amplisomes (7,13,37,38,48,52), or by the generation of microscopically visible fragments in a single step (34,40,52 amplification suggests either that such structures are preferred intermediates or that they offer more substantial survival advantages than chromosomal amplicons in vivo (50).The common occurrence of DMs and their submicroscopic precursor...

High-Resolution Mapping of the Origin of DNA Replication in the Hamster Dihydrofolate Reductase Gene Domain by Competitive PCR

“…While it is difficult to reconcile these results, it should be pointed out that the vast majority of studies analyzing newly synthesized DNA emanating from replication start sites (by analysis either of its distribution along the chromosome or of its polarity) have led to the identification of precise sites for initiation of DNA replication. Among the identified origins are those located within the ribosomal protein S14 gene in Chinese hamster cells (39), within the Syrian hamster CAD gene (28), and near the mouse adenosine deaminase gene (8,43), the Chinese hamster rhodopsin gene (19), the human ␤-globin gene (29), the c-myc gene (40), and the human lamin B2 gene (20). In contrast, most of the studies analyzing the structure of replication intermediates by the 2D-gel technique led to the conclusion that origins are dispersed in wide genomic areas or, at best, confined to still large preferred initiation zones (11,32,34,42).…”

Replication Initiation Patterns in the β-Globin Loci of Totipotent and Differentiated Murine Cells: Evidence for Multiple Initiation Regions

“…Nevertheless, several lines of evidence suggest that defined cis-acting elements are required for DNA replication in mammalian cells. First, replication initiates from identical regions in wild-type chromosomes and in rearranged loci resulting from naturally occurring processes, such as gene amplification (8,22,27). Second, an 8-kb deletion of the single biochemically defined IR in the human ␤-globin locus (Lepore deletion) prevents initiation within this locus (28), suggesting that the deleted sequence contained information essential for initiation.…”

Site-Specific Initiation of DNA Replication in Xenopus Egg Extract Requires Nuclear Structure

“…From 80 to 95% of DNA synthesis occurs bidirectionally from the OBR. This conclusion is based on the fraction of replication forks traveling in the same direction as determined by two-dimensional (2D) neutral/alkaline gels (64,83), the ratio of Okazaki fragments that hybridize to the two strands of a unique DNA probe (6,15,20,54,88), and the ratio of long leading nascent DNA strands from forward arms of replication forks that hybridize to the two strands of a unique DNA probe (16,46,54,57). In addition, quantitative analysis of specific DNA sequences within long nascent DNA strands reveals that most of them originate bidirectionally from a small chromosomal locus (39) and that this locus can reside within an initiation zone (97).…”

“…First, the same origins that are utilized in cells containing single copies of a unique genetic locus are also utilized in cells containing hundreds of copies of the same locus (20,26,54,91). Second, some origins have been found to retain their activity when translocated to other chromosomal sites (46,80), and origin activity near the human ␤-globin gene is eliminated by an 8-kb deletion (57).…”

“…If replication forks are initiated bidirectionally at or close to the same site in each cell, then Okazaki fragments prepared from these cells will hybridize preferentially to the DNA strand that serves as template for discontinuous DNA synthesis. Thus, an OBR can be identified by the transition from continuous to discontinuous DNA synthesis that will occur on each DNA strand (15,20,88). Therefore, to locate the OBR used in CHOC 400 cells, G 1 /S nuclei were incubated briefly with [␣-32 P]dATP, and Okazaki fragments were then isolated and hybridized to individual strands of the DHFR DNA probes.…”