Map position of the replication origin on the E. coli chromosome.

Summary Strains carrying a dnaA temperature sensitive (t.s.) mutation and a Mu-1 prophage inserted within different genes near the origin of replication have been constructed. For each strain, integratively suppressed Hfrs, named G and D, in which the ori region was replicated clockwise and counterclockwise respectively, were isolated. The strand preferences of Mu-1 specific Okazaki fragments were subsequently determined for each t.s. strain and its Hfr derivatives. Their comparison led us to establish the direction of replication of the Mu-1 marker from ori. The site ori was thus confined to the bglB-C-rbsK-P interval.     

Identification of a predominant replication origin in fission yeast.

We have identified five autonomously replicating sequences (ARSs) in a 100 kbp region of the Schizosaccharomyces pombe chromosome II. Analyses of replicative intermediates of the chromosome DNA by neutral/neutral two-dimensional gel electrophoresis demonstrated that at least three of these ARS loci operate as chromosomal replication origins. One of the loci,ori2004, was utilized in almost every cell cycle, while the others were used less frequently. The frequency of initiation from the respective chromosomal replication origin was found to be roughly proportional to the efficiency of autonomous replication of the corresponding ARS plasmid. Replication from ori2004 was initiated within a distinct region almost the same as that for replication of the ARS plasmid. These results showed that the ori2004 region of approximately 3 kbp contains all the cis elements essential for initiation of chromosome replication.     

A yeast origin of replication is activated late in S phase

The mechanism that causes large regions of eukaryotic chromosomes to remain unreplicated until late in S phase is not understood. We have found that 67 kb of telomere-adjacent DNA at the right end of chromosome V in S. cerevisiae is replicated late in S phase. An ARS element in this region, ARS501, was shown by two-dimensional gel analysis to be an active origin of replication. Kinetic analyses indicate that the rate of replication fork movement within this late region is similar to that in early replicating regions. Therefore, the delayed replication of the region is a consequence of late origin activation. The results also support the idea that the pattern of interspersed early and late replication along the chromosomes of higher eukaryotes is a consequence of the temporal regulation of origin activation.     

The regulation of replication origin activation

At the start of the cell-division programme, proteins must be assembled onto replication origins to establish competence for initiation of DNA synthesis. At the correct moment, other effectors must then coordinate appropriate firing of the various origins to control entry into and progress through S phase. These processes are key targets of cell-cycle control, and understanding their regulation will provide a deeper knowledge of the mechanisms controlling cell proliferation.     

The in vivo replication origin of the yeast 2 microns plasmid

We have used two-dimensional neutral/alkaline agarose gel electrophoresis to separate the nascent strands of replicating yeast 2 micron plasmid DNA molecules according to extent of replication, away from nonreplicating molecules and parental strands. Analysis of the lengths of nascent strands by sequential hybridization with short probes shows that replication proceeds bidirectionally from a single origin at map position 3700 +/- 100, coincident with the genetically mapped ARS element. The two recombinational isomers of 2 microns plasmid (forms A and B) replicate with equal efficiency. These results suggest that ARS elements may prove to be replication origins for chromosomal DNA.     

DnaA boxes in the P1 plasmid origin: the effect of their position on the directionality of replication and plasmid copy number.

The DnaA protein is essential for initiation of DNA replication in a wide variety of bacterial and plasmid replicons. The replication origin in these replicons invariably contains specific binding sites for the protein, called DnaA boxes. Plasmid P1 contains a set of DnaA boxes at each end of its origin but can function with either one of the sets. Here we report that the location of origin-opening, initiation site of replication forks and directionality of replication do not change whether the boxes are present at both or at one of the ends of the origin. Replication was bidirectional in all cases. These results imply that DnaA functions similarly from the two ends of the origin. However, origins with DnaA boxes proximal to the origin-opening location opened more efficiently and maintained plasmids at higher copy numbers. Origins with the distal set were inactive unless the adjacent P1 DNA sequences beyond the boxes were included. At either end, phasing of the boxes with respect to the remainder of the origin influenced the copy number. Thus, although the boxes can be at either end, their precise context is critical for efficient origin function.     

Histone acetylation regulates the time of replication origin firing

The temporal firing of replication origins throughout S phase in yeast depends on unknown determinants within the adjacent chromosomal environment. We demonstrate here that the state of histone acetylation of surrounding chromatin is an important regulator of temporal firing. Deletion of RPD3 histone deacetylase causes earlier origin firing and concurrent binding of the replication factor Cdc45p to origins. In addition, increased acetylation of histones in the vicinity of the late origin ARS1412 by recruitment of the histone acetyltransferase Gcn5p causes ARS1412 alone to fire earlier. These data indicate that histone acetylation is a direct determinant of the timing of origin firing.     

Architecture of the yeast origin recognition complex bound to origins of DNA replication.

In many organisms, the replication of DNA requires the binding of a protein called the initiator to DNA sites referred to as origins of replication. Analyses of multiple initiator proteins bound to their cognate origins have provided important insights into the mechanism by which DNA replication is initiated. To extend this level of analysis to the study of eukaryotic chromosomal replication, we have investigated the architecture of the Saccharomyces cerevisiae origin recognition complex (ORC) bound to yeast origins of replication. Determination of DNA residues important for ORC-origin association indicated that ORC interacts preferentially with one strand of the ARS1 origin of replication. DNA binding assays using ORC complexes lacking one of the six subunits demonstrated that the DNA binding domain of ORC requires the coordinate action of five of the six ORC subunits. Protein-DNA cross-linking studies suggested that recognition of origin sequences is mediated primarily by two different groups of ORC subunits that make sequence-specific contacts with two distinct regions of the DNA. Implications of these findings for ORC function and the mechanism of initiation of eukaryotic DNA replication are discussed.     

A replication enhancer of viral origin increases the mitotic stability of yeast transformants]

It is shown in this paper that a DNA fragment of Hepatitis B virus possessing structural features of yeast replication enhancer increases the mitotic stability of yeast transformants containing hybrid plasmids of episomal and replicative types. The mitotic stability of transformants with plasmid of the replicative type and with the replication enhancer increases only in [cir+] cells. Comparison of primary sequences of HBV DNA of different subtypes revealed that only DNA has unique structural features of the yeast enhancer of replication.     

Supercoiling unwinds two-micrometer plasmid yeast DNA at the origin of replication

All studied origins of replication of DNA in Saccharomyces cerevisiae contain DNA unwinding elements. The introduction of unrestrained negative supercoiling leads to melting of the two DNA strands in DNA unwinding elements. To understand the mechanism of DNA replication it is important to know whether the most unstable region of DNA coincides with the origin of replication. Two-micrometer plasmid DNA from S. cerevisiae inserted in pBR322 was investigated by cleaving with snake venom phosphodiesterase. Its single-strand endonucleolytic activity allows cutting of negatively supercoiled DNA in the DNA unwinding elements. The sites of the venom phosphodiesterase hydrolysis were mapped by restriction enzymes. This study shows that the unwinding of the two-micrometers plasmid DNA of S. cerevisiae takes place only in the origin of replication as a result of unrestrained negative supercoiling.     

A yeast replication origin consists of multiple copies of a small conserved sequence

Autonomously replicating sequences (ARSs) of the yeast S. cerevisiae function as replication origins on plasmids and probably also on chromosomes. ARS function requires a copy of the ARS core consensus (5'-[A/T]TTTAT[A/G]TTT[A/T]-3') and additional sequences 3' to the T-rich strand of the consensus. Our analysis of an ARS from chromosome III, the C2G1 ARS, suggests that ARS function depends on the presence of an exact match to the core consensus and the presence of additional near matches in the 3' flanking region. We have demonstrated that ARS function can be mediated by multiple matches to the core consensus by constructing synthetic ARS elements from oligonucleotides containing copies of the consensus sequence. We find that two copies of the core consensus are sufficient for ARS activity and that an artificial ARS as efficient as a natural chromosomal ARS can be constructed from multiple core consensus elements in a specific orientation.     

The effect of DNA replication mutations on CAG tract stability in yeast.

CAG repeat tracts are unstable in yeast, leading to frequent contractions and infrequent expansions in repeat tract length. To compare CAG repeats to other simple repeats and palindromic sequences, we examined the effect of DNA replication mutations, including alleles of pol alpha, pol delta, pol epsilon, and PCNA (proliferating cell nuclear antigen), on tract stability. Among the polymerase mutations, the pol delta mutation (pol3-14) destabilizes tracts with either CAG or CTG as the lagging strand template. One pol alpha mutation, pol1-1, destabilizes the orientation with CAG as the lagging strand template, but it has little effect on the CTG orientation. In contrast, the pol1-17 mutation has no effect on either orientation. Similarly, mutations in the proofreading functions of pol delta and pol epsilon, as well as a temperature-sensitive pol epsilon mutation, pol2-18, have no effect on tract stability. Three PCNA mutations, pol30-52, pol30-79, and pol30-90, all have drastic effects on tract stability. Of the three, pol30-52 is unique in yielding small tract changes that are indicative of an impairment in mismatch repair. These results show that while CAG repeats are destabilized by many of the same mutations that destabilize other simple repeats, they also have some behaviors that are suggestive of their potential to form hairpin structures.     

Ribosomal DNA replication time coordinates completion of genome replication and anaphase in yeast

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Developmental differences in genome replication program and origin activation

To ensure error-free duplication of all (epi)genetic information once per cell cycle, DNA replication follows a cell type and developmental stage specific spatio-temporal program. Here, we analyze the spatio-temporal DNA replication progression in (un)differentiated mouse embryonic stem (mES) cells. Whereas telomeres replicate throughout S-phase, we observe mid S-phase replication of (peri)centromeric heterochromatin in mES cells, which switches to late S-phase replication upon differentiation. This replication timing reversal correlates with and depends on an increase in condensation and a decrease in acetylation of chromatin. We further find synchronous duplication of the Y chromosome, marking the end of S-phase, irrespectively of the pluripotency state. Using a combination of single-molecule and super-resolution microscopy, we measure molecular properties of the mES cell replicon, the number of replication foci active in parallel and their spatial clustering. We conclude that each replication nanofocus in mES cells corresponds to an individual replicon, with up to one quarter representing unidirectional forks. Furthermore, with molecular combing and genome-wide origin mapping analyses, we find that mES cells activate twice as many origins spaced at half the distance than somatic cells. Altogether, our results highlight fundamental developmental differences on progression of genome replication and origin activation in pluripotent cells.