A binding site for Pur alpha and Pur beta is structurally unstable and is required for replication in vivo from the rat aldolase B origin

The rat aldolase B promoter acts as a replication origin in vivo, as well as an autonomously replicating sequence (ARS). Here, we examined roles of a polypurine stretch (site PPu) in this origin, which is indispensable to the ARS activity. Purification of site PPu-binding protein revealed that site PPu binds Puralpha and Purbeta, i.e., single-stranded DNA-binding proteins whose roles in replication have been implicated, but less clear. Biochemical analyses showed that site PPu even in a longer DNA fragment is unstable in terms of double-helix, implying that Puralpha/beta may stabilize single-stranded state. Deletion of site PPu from the origin DNA, which was ectopically positioned in the mouse chromosome, significantly reduced replicator activity. Chromatin immunoprecipitation experiments showed that deletion of site PPu abolishes binding of the Puralpha/beta proteins to the origin. These observations suggest functional roles of site PPu and Puralpha/beta proteins in replication initiation.     

Replication of ColE2 and ColE3 plasmids: The regions sufficient for autonomous replication

Summary We have localized the regions sufficient for autonomous replication on the genomes of the colicin E2 (ColE2) and colicin E3 (ColE3) plasmids and analyzed the replication functions carried by these regions. A 1.3 kb segment of each plasmid is sufficient for autonomous replication. Plasmids carrying this segment retain the replication properties of the original plasmid. The 1.3 kb segment consists of three functional portions. Firstly, a 0.9 kb region which specifies at least one trans-acting factor required for replication of each plasmid. Secondly, a 0.4 kb region located adjacent to one end of the 0.9 kb region, which is required for expression of the trans-acting factor(s) and probably contains the promoter. The region across the border of these two portions of ColE2 is involved in copy number control of the plasmid. The third portion is a 50 bp region adjacent to the other end of the 0.9 kb region, which contains a cis-acting site (origin) where replication initiates in the presence of the trans-acting factor(s). The action of the trans-acting factor(s) on the origin is plasmid specific. The 50 bp regions functioning as the origins of replication of ColE2 and ColE3 are the smallest among those in prokaryotic replicons so far identified and analyzed.     

Identification of a cis-element that determines autonomous DNA replication in eukaryotic cells

A 36-bp human consensus sequence (CCTMDAWKSGBYTSMAAWTWBCMYTTRSCAAATTCC) is capable of supporting autonomous replication of a plasmid after transfection into eukaryotic cells. After transfection and in vitro DNA replication, replicated plasmid DNA containing a mixture of oligonucleotides of this consensus was found to reiterate the consensus. Initiation of DNA replication in vitro occurs within the consensus. One version, A3/4, in pYACneo, could be maintained under selection in HeLa cells, unrearranged and replicating continuously for >170 cell doublings. Stability of plasmid without selection was high (> or =0.9/cell/generation). Homologs of the consensus are found consistently at mammalian chromosomal sites of initiation and within CpG islands. Versions of the consensus function as origins of DNA replication in normal and malignant human cells, immortalized monkey and mouse cells, and normal cow, chicken, and fruit fly cells. Random mutagenesis studies suggest an internal 20-bp consensus sequence of the 36 bp may be sufficient to act as a core origin element. This cis-element consensus sequence is an opportunity for focused analyses of core origin elements and the regulation of initiation of DNA replication.     

In vitro deletion analysis of ARS elements spanning the replication origin in the 5'' non-transcribed spacer of Tetrahymena thermophila ribosomal DNA.

Two adjacent but non-overlapping restriction fragments that encompass the replication origin of the macronuclear copy of rDNA from Tetrahymena thermophila allow autonomous replication of plasmids in the yeast Saccharomyces cerevisiae; i.e. they function as autonomously replicating segments (ARS). Deletions generated in vitro into these fragments yield an 82 bp segment from each as the smallest sequence specifying ARS function. These 82 bp segments are at the 5' end of a 220 bp region of homology between the two original ARS restriction fragments. A 39 bp region of almost complete sequence identity between the two 82 bp fragments is suggested to be a core sequence element necessary for ARS function. This 39 bp sequence contains a region identical or nearly identical to the 11 bp yeast ARS consensus sequence (T/ATTTATPuTTTA/T) which is suggested to be essential for ARS function. Detailed comparisons of the 82 bp segments and of the 39 bp core with other ARS sequences reveal no extensive homologies aside from the consensus.     

Analysis of an activatable promoter: sequences in the simian virus 40 late promoter required for T-antigen-mediated trans activation.

The late promoter of simian virus 40 (SV40) is activated in trans by the viral early gene product, T antigen. We inserted the wild-type late-promoter region, and deletion mutants of it, into chloramphenicol acetyltransferase transient expression vectors to identify promoter sequences which are active in the presence of T antigen. We defined two promoter activities. One activity was mediated by a promoter element within simian virus 40 nucleotides 200 to 270. The activity of this element was detectable only in the presence of an intact, functioning origin of replication and accounted for 25 to 35% of the wild-type late-promoter activity in the presence of T antigen. The other activity was mediated by an element located within a 33-base-pair sequence (simian virus nucleotides 168 to 200) which spans the junction of the 72-base-pair repeats. This element functioned in the absence of both the origin of replication and the T-antigen-binding sites and appeared to be responsible for trans-activated gene expression. When inserted into an essentially promoterless plasmid, the 33-base-pair element functioned in an orientation-dependent manner. Under wild-type conditions in the presence of T antigen, the activity of this element accounted for 65 to 75% of the late-promoter activity. The roles of the 33-base-pair element and T antigen in trans-activation are discussed.     

Structural properties of the beta origin of replication of plasmid R6K

The beta origin of replication of plasmid R6K, one of three active R6K origins of replication, requires most or all of a 1962-base pair (bp) sequence for activity. The nucleotide sequence of a portion of this functional beta origin was determined in an earlier study (Stalker, D., Kolter, R., and Helinski, D. (1982) J. Mol. Biol. 161, 33-43). In this work, the sequence of the remaining portion of this 1964-bp segment was obtained. In addition to its activity as an origin of replication, this sequence also contains sufficient information for autonomous replication in Escherichia coli. A 277-bp region containing seven 22-bp direct repeats is present at one end of the beta origin segment (Stalker, D., Kolter, R., and Helinski, D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1150-1154) while the other end contains a 140-bp sequence that includes a relaxation complex site. The 277-bp direct repeat region is required for activity of the beta origin. The start of the beta origin of replication as mapped by electron microscopy (Crosa, J. (1980) J. Biol. Chem. 255, 11075-11077) lies approximately 1000 bp away from the 277-bp region. The pi structural gene, which makes up most of the sequence between the direct repeats and the beta origin, is required in cis for beta origin activity. The pi protein also is required for beta origin activity but can be provided in trans. The nucleotide sequence just beyond the pi structural gene and within or near the start of beta origin of replication contains an open reading frame for a 151-amino acid protein. Deletions ranging from 94 bp to 1590 bp were obtained within the 1964-bp beta origin region. In every case, the deletion results in loss of origin activity even when the deleted sequence plus adjacent regions are provided in trans. These observations suggest a requirement for a specific secondary structure over an extensive region for beta origin activity.     

Domain B of ARS307 contains two functional elements and contributes to chromosomal replication origin function.

ARS307 is highly active as a replication origin in its native location on chromosome III of Saccharomyces cerevisiae. Its ability to confer autonomous replication activity on plasmids requires the presence of an 11-bp autonomously replicating sequence (ARS) consensus sequence (ACS), which is also required for chromosomal origin function, as well as approximately 100 bp of sequence flanking the ACS called domain B. To further define the sequences required for ARS function, a linker substitution mutagenesis of domain B was carried out. The mutations defined two sequences, B1 and B2, that contribute to ARS activity. Therefore, like ARS1, domain B of ARS307 is composed of functional subdomains. Constructs carrying mutations in the B1 element were used to replace the chromosomal copy of ARS307. These mutations caused a reduction in chromosomal origin activity, demonstrating that the B1 element is required for efficient chromosomal origin function.