Identification of Primary Initiation Sites for DNA Replication in the Hamster Dihydrofolate Reductase Gene Initiation Zone

Abstract

Mammalian replication origins appear paradoxical. While some studies conclude that initiation occurs bidirectionally from specific loci, others conclude that initiation occurs at many sites distributed throughout large DNA regions. To clarify this issue, the relative number of early replication bubbles was determined at 26 sites in a 110-kb locus containing the dihydrofolate reductase (DHFR)-encoding gene in CHO cells; 19 sites were located within an 11-kb sequence containing ori-␤. The ratio of ϳ0.8-kb nascent DNA strands to nonreplicated DNA at each site was quantified by competitive PCR. Nascent DNA was defined either as DNA that was labeled by incorporation of bromodeoxyuridine in vivo or as RNA-primed DNA that was resistant to -exonuclease. Two primary initiation sites were identified within the 12-kb region, where two-dimensional gel electrophoresis previously detected a high frequency of replication bubbles. A sharp peak of nascent DNA occurred at the ori-␤ origin of bidirectional replication where initiation events were 12 times more frequent than at distal sequences. A second peak occurred 5 kb downstream at a previously unrecognized origin (ori-␤). Thus, the DHFR gene initiation zone contains at least three primary initiation sites (ori-␤, ori-␤, and ori-␥), suggesting that initiation zones in mammals, like those in fission yeast, consist of multiple replication origins.At least 22 replication origins have now been mapped in the chromosomes of flies, frogs, and mammals (references 14, 45, and 61 and references cited below) by using several different strategies (reviewed in reference 13). While all the data are consistent with bidirectional replication involving classical replication bubbles and forks, a complex and sometimes contradictory view of replication origins has emerged. While some studies conclude that most initiation events occur at specific sites analogous to those found in single-cell organisms such as yeast, Tetrahymena, and Physarum, other studies conclude that most initiation events are distributed throughout large DNA regions with no preference for one site over another. This paradox is best illustrated by studies on the mammalian rRNA and dihydrofolate reductase (DHFR) gene regions, where several different methods have been applied to the same genomic loci.When nascent DNA is labeled with nucleotide precursors during its biosynthesis, the data suggest that most initiation events occur at specific DNA sequences referred to as origins of bidirectional replication (OBRs). In the rRNA gene region, the earliest labeled DNA fragments have been identified (8, 21) and the growth and relative abundance of nascent DNA chains have been determined (21,29,60). These studies reveal two primary initiation sites: a 1-to 6-kb locus upstream of the rRNA gene promoter that is Ͼ10-fold more active than distal sites and a ϳ3-kb locus with weaker activity downstream of the 3Ј end of the gene. The major site appears to be conserved among species (37). In the DHFR gene region in Chinese hamster ovary (CHO) ce...

Overreplication of short DNA regions during S phase in human cells

“…The reliability of this approach for ORI identification is supported by the fact that several mammalian ORIs that were initially identified by a variety of non-PCR approaches have been validated later by Q-PCR. Examples include the preferential initiation sites within the disperse initiation region downstream from the hamster DHFR gene identified by two-dimensional electrophoresis (Pelizon et al 1996;Kobayashi et al 1998), the human GM-CSF Ori1 and Ori2 identified by subtractive hybridization for origin capture (Todorovic et al 2005), and several human ORIs recently identified by high-throughput array hybridization (Lucas et al 2007). Conversely, ORIs initially identified by Q-PCR have been shown to bind ORC and MCM proteins as in the case of the human Lamin B2 (Abdurashidova et al 2003), c-Myc , and FMR1 (Gray et al 2007).…”

Isolation of aDrosophilaamplification origin developmentally activated by transcription

“…To determine the site at which DNA synthesis initiates during amplification at DAFC-62D, nascent strand analysis was performed as described (16,17) on replicative intermediates isolated from stage 10B or 13 egg chambers. We observed a 1 kb region that was highly enriched in the 0.5-1 kb (Fig.…”

DNA Methylation at Mammalian Replication Origins

“…Nascent Strand Length-To isolate nascent DNA of different lengths, CHO K1 cells were cultured and synchronized at their G 1 /S transition by a double aphidicolin block as described previously (8). The fraction of cells at their G 1 /S boundary was determined by fluorescence-activated cell sorting analysis to be Ͼ90%.…”

“…For example, a 200-kb 1 region at the human ␤-globin gene (4, 5) and a 500-kb region at the mouse IgH gene (6) are both replicated from a single initiation locus. Moreover, what previously had been viewed as random initiation events distributed throughout "initiation zones" in Schizosaccharomyces pombe and hamster cells more likely reflects the presence of several strongly preferred initiation sites (7,8).Nevertheless, the precise size and composition of these initiation loci, as well as the parameters that define them remain the subject of intense investigation.The fact that specific sites for initiation of DNA replication can be developmentally acquired makes it clear that initiation sites in the metazoa are determined at least in part by epigenetic parameters. These parameters include nuclear structure, chromatin structure, and events that occur during G 1 phase of the cell division cycle (reviewed in Ref.…”

“…For example, a 200-kb 1 region at the human ␤-globin gene (4,5) and a 500-kb region at the mouse IgH gene (6) are both replicated from a single initiation locus. Moreover, what previously had been viewed as random initiation events distributed throughout "initiation zones" in Schizosaccharomyces pombe and hamster cells more likely reflects the presence of several strongly preferred initiation sites (7,8).…”