The Dihydrofolate Reductase Origin of Replication Does Not Contain Any Nonredundant Genetic Elements Required for Origin Activity

Abstract

The Chinese hamster dihydrofolate reductase (DHFR) origin of replication consists of a broad zone of potential initiation sites scattered throughout a 55-kb intergenic spacer, with at least three sites being preferred (ori-␤, ori-␤, and ori-␥). We previously showed that deletion of the most active site or region (ori-␤) has no demonstrable effect on initiation in the remainder of the intergenic spacer nor on the time of replication of the DHFR locus as a whole. In the present study, we have now deleted ori-␤, both ori-␤ and ori-␤, an 11-kb region just downstream from the DHFR gene, or the central ϳ40-kb core of the spacer. The latter two deletions together encompass >95% of the initiation sites that are normally used in this locus. Two-dimensional gel analysis shows that initiation still occurs in the early S phase in the remainder of the intergenic spacer in each of these deletion variants. Even removal of the 40-kb core fails to elicit a significant effect on the time of replication of the DHFR locus in the S period; indeed, in the truncated spacer that remains, the efficiency of initiation actually appears to increase relative to the corresponding region in the wild-type locus. Thus, if replicators control the positions of nascent strand start sites in this complex origin, either (i) there must be a very large number of redundant elements in the spacer, each of which regulates initiation only in its immediate environment, or (ii) they must lie outside the central core in which the vast majority of nascent strand starts occur.In the chromosomes of bacteria, bacterial plasmids, viruses, and lower eukaryotes such as Saccharomyces cerevisiae, replication initiates at genetically defined sites known as replicators, which serve as recognition signals for cognate sequencespecific DNA binding protein complexes termed initiators (34,42). In the chromosomes of metazoans, however, many replication origins consist of broad zones of closely spaced initiation sites, some of which are used with greater efficiency than others (for reviews, see references 14, 19, and 30).An example of the latter is the early-firing Chinese hamster dihydrofolate reductase (DHFR) origin, which resides in the 55-kb spacer between the convergently transcribed DHFR and 2BE2121 genes (Fig. 1A) (17,18,22,58). Neutral/neutral (7) and neutral/alkaline (49) two-dimensional (2-D) gel studies on several adjacent and overlapping restriction fragments have demonstrated that there are at least 20 nascent strand start sites (and probably many more) scattered throughout the 55-kb spacer (Fig. 1) (23). However, more-quantitative assays showed that the central 40-kb core, and particularly two subregions within it known as ori-␤ and ori-␥, are preferred ( Fig. 1) (23, 43, 44). The results of an in-gel renaturation analysis of early labeled restriction fragments are shown in Fig. 1A (44), as is the frequency of initiation measured in a quantitative early labeled fragment hybridization (ELFH) assay that used very small origin-containing nascent DNA as a probe (23...

Mammalian Mitochondrial DNA Replicates Bidirectionally from an Initiation Zone

“…O H is always preserved in partially deleted mtDNAs, whereas most, if not the entire, initiation zone of human mtDNA as defined in this report is redundant (33). Similarly, in the most extensively studied replication initiation zone, the human nuclear dihydrofolate reductase locus, all sequence elements are known to be dispensable (34). The most obvious candidate for an essential cis-element for mtDNA replication outside the initiation zone is the triple-stranded D-loop region, located in the major noncoding region (35,36).…”

“…Perhaps any DNA located downstream of the D-loop could function as an origin zone because of its proximity to the D-loop and membrane components (42). Indeed, this may be a general feature of initiation zones; as mentioned above, the signals essential for nuclear dihydrofolate reductase gene replication have recently been shown to lie outside the well characterized initiation zone (34).…”

DNA Replication Origins Fire Stochastically in Fission Yeast

“…Alternatively, cells could have a mechanism that evenly distributes origin firing across the genome; they would thus avoid the random gap problem, and replication would be efficient. Models for such mechanisms have been proposed; for instance, origins within specific clusters could be selected to fire, or active origins could suppress their neighbors by lateral inhibition (Mesner et al, 2003;Shechter and Gautier, 2005). However, little direct evidence exists to either support or refute these models.…”

The promoter of the Chinese hamster ovary dihydrofolate reductase gene regulates the activity of the local origin and helps define its boundaries

“…In previous studies, we used a homologous recombination strategy to specifically delete various subregions of the 55-kb intergenic spacer in an effort to identify any required cis-regulatory elements residing within the origin itself (Kalejta et al 1998;Mesner et al 2003). Surprisingly, the entire central 45 kb of the zone could be deleted without affecting the time at which the locus normally replicates in the S period.…”

“…The DHFR initiation zone in Chinese hamster ovary (CHO) cells was the first mammalian origin identified (Heintz and Hamlin 1982;Heintz et al 1983), and is presently the most thoroughly characterized of the several mammalian origins that have since been localized (Leu and Hamlin 1989;Vaughn et al 1990;Dijkwel and Hamlin 1995;Pelizon et al 1996;Kalejta et al 1998;Kobayashi et al 1998;Mesner et al 2003). This origin consists of a zone of >20 potential initiation sites distributed throughout the 55-kb spacer between the convergently transcribed 2BE2121 and DHFR genes (Fig.…”