Distribution of DNA replication origins between matrix-attached and loop DNA in mammalian cells

Using a previously developed procedure (Gencheva et al. [1996] J Biol Chem 271:2608-2614), we isolated a DNA fraction consisting of short fragments originating from the regions of initiation of DNA synthesis from exponentially growing Chinese hamster ovary cells. This fraction arbitrarily designated as "collective origin fraction" was labeled in vitro and used to probe the abundance of origin containing sequences in preparations of matrix-attached and loop DNA isolated by two different procedures from Chinese hamster ovary cells. Alternatively, an individual DNA replication origin sequence - a 478-bp long DNA fragment located at about 17-kb downstream of the dihydrofolate reductase gene - was used to probe the same matrix-attached and loop DNA fractions. The results with both the collective and individual DNA replication origins showed that there was random distribution of the origin sequences between DNA attached to the matrix and DNA from the loops.     

DNA replication and chromatin

The study of DNA replication in eukaryotic chromosomes has revealed a multitude of different regulatory levels. Nuclear and chromosomal location as well as chromatin structure may affect the activity of replication origins and their modulation during development.     

Initiation of adenovirus DNA replication.

In an attempt to study the mechanism of initiation of adenovirus DNA replication, an assay was developed to investigate the pattern of DNA synthesis in early replicative intermediates of adenovirus DNA. By using wild-type virus-infected cells, it was possible to place the origin of adenovirus type 2 DNA replication within the terminal 350 to 500 base pairs from either of the two molecular termini. In addition, a variety of parameters characteristic of adenovirus DNA replication were compared with those obtained in a soluble nuclear extract competent for viral DNA replication. It was observed that in vitro DNA replication, which is dependent on the exogenously added viral DNA-protein complex as its optimal template, occurs in a manner apparently indistinguishable from the situation in virus-infected cells. This includes the presence of proteinaceous material on the molecular termini of newly initiated viral DNA.     

Initiation of DNA replication at cloned origins of bacteriophage T7

Bacteriophage T7 DNA replication is initiated at a site 15% of the distance from the genetic left end of the chromosome. This primary origin contains two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by an A + T-rich region. When the primary origin region is deleted replication initiates at secondary origins. We have analyzed the ability of plasmids containing cloned fragments of T7 to replicate after infection of Escherichia coli with bacteriophage T7. All cloned T7 fragments that support plasmid replication contain a T7 promoter but a T7 promoter alone is not sufficient for replication. Replication of plasmids containing the primary origin is dependent on T7 DNA polymerase and gene 4 protein (helicase/primase) and a portion of the A + T-rich region. The other T7 fragments that support plasmid replication after T7 infection are promoter regions phi OR, phi 13 and phi 6.5 (secondary origins). When both the primary and secondary origins are present simultaneously on compatible plasmids, replication of each is temporally regulated. Such regulation may play a role during T7 DNA replication.     

Initiation of DNA replication at the human beta-globin 3' enhancer

The origin of DNA replication in the human β-globin gene contains an initiation region (IR) and two flanking auxiliary elements. Two replicator modules are located within the upstream auxiliary sequence and the IR core, but the functional sequences in the downstream auxiliary element are unknown. Here, we use a combination of benzoylated-naphthoylated DEAE (BND) cellulose purification and nascent strand abundance assays to show that replication initiation occurs at the β-globin 3′ enhancer on human chromosome 11 in the Hu11 hybrid murine erythroleukemia (MEL) cell line. To examine replicator function, 3′ enhancer fragments were inserted into an ectopic site in MEL cells via an optimized FRT/EGFP-FLP integration system. These experiments demonstrate that the 1.6 kb downstream auxiliary element is a third replicator module called bGRep-E in erythroid cells. The minimal 260 bp 3′ enhancer is required but not sufficient to initiate efficient replication, suggesting cooperation with adjacent sequences. The minimal 3′ enhancer also cooperates with elements in an expressing HS3β/γ-globin construct to initiate replication. These data indicate that the β-globin replicator has multiple initiation sites in three closely spaced replicator modules. We conclude that a mammalian enhancer can cooperate with adjacent sequences to create an efficient replicator module.     

Cell cycle regulation of chromatin at an origin of DNA replication

Selection and licensing of mammalian DNA replication origins may be regulated by epigenetic changes in chromatin structure. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) uses the cellular licensing machinery to regulate replication during latent infection of human cells. We found that the minimal replicator sequence of OriP, referred to as the dyad symmetry (DS), is flanked by nucleosomes. These nucleosomes were subject to cell cycle-dependent chromatin remodeling and histone modifications. Restriction enzyme accessibility assay indicated that the DS-bounded nucleosomes were remodeled in late G1. Remarkably, histone H3 acetylation of DS-bounded nucleosomes decreased during late G1, coinciding with nucleosome remodeling and MCM3 loading, and preceding the onset of DNA replication. The ATP-dependent chromatin-remodeling factor SNF2h was also recruited to DS in late G1, and formed a stable complex with HDAC2 at DS. siRNA depletion of SNF2h reduced G1-specific nucleosome remodeling, histone deacetylation, and MCM3 loading at DS. We conclude that an SNF2h-HDAC1/2 complex coordinates G1-specific chromatin remodeling and histone deacetylation with the DNA replication initiation process at OriP.     

Initiation of DNA replication in yeast chromosomes

A family of DNA fragments from the yeast genome has properties that suggest that chromosome replication starts at specific DNA sequences. These elements (autonomously replicating sequences: ARS) have a bipartite structure: a small (less than 20 base pairs) AT-rich region essential for function, flanked by larger regions important for maximal activity of the replicator. In an attempt to identify proteins involved in initiation of replication, yeast mutants that show an enhanced ability to replicate minichromosomes with defective ARSS have been isolated.     

Initiation of DNA replication in higher eukaryotes

In this review, of problems concerning initiation of DNA replication in higher eukaryotes is discussed, with special emphasis on the methods of replication origin mapping and biological tests for the activity of DNA replication origins in higher eukaryotes. Protein factors interacting with replication origins are considered in detail. The main events of replication initiation in higher eukaryotes are briefly analyzed. New data on the control of replication timing of large genomic regions are discussed.     

Initiation of DNA replication in human chromosomes

DNA replication within the first 10 min of the S phase was studied using synchronized human diploid cells. It appeared that every chromosome in the human genome, including late-replicating X, had segment(s) which initiated DNA replication within the first 10 min of the S phase. The position, the shape and the size of these segments corresponded to those of Q(G)-negative bands suggesting that each of them constitutes a basic unit of initiation of DNA replication.     

Initiation of genome replication: assembly and disassembly of replication-competent chromatin

Considerable progress has been made in research on the initiation of eukaryotic genome replication. This has generated a number of recent review articles. Here, we briefly summarize the major conclusions described in these articles and also include the results of more recent primary articles. The consensus view that has emerged is that a pre-replication complex assembles during the G1 phase of the cell cycle, making chromatin competent for replication. The complex consists of Orc proteins, Cdc6p, and the family of Mcm proteins. Chromatin, thus 'licenced' for replication, is guided into the S phase by the activation of cell-cycle-regulated protein kinases. Upon entry into S phase, the pre-replication complex is partially dissolved, first by the dissociation of Cdc6p and then by the gradual release of Mcm proteins. This appears to be accompanied by a recruitment of chain elongation factors and the establishment of replication forks.     

Initiation of DNA replication: a new hint from archaea

In this issue, Robinson and coworkers provide new insights into the mechanisms of initiation of chromosome replication in Archea. This and other studies, focused on model organisms, will certainly help to understand how the replication process has evolved in Eukaryotes.     

Initiation of DNA replication in nuclei from quiescent cells requires permeabilization of the nuclear membrane

We have investigated the replication capacity of intact nuclei from quiescent cells using Xenopus egg extract. Nuclei, with intact nuclear membranes, were isolated from both exponentially growing and contact- inhibited BALB/c 3T3 fibroblasts by treatment of the cells with streptolysin-O. Flow cytometry showed that > 90% of all contact- inhibited cells and approximately 50% of the exponential cells were in G0/G1-phase at the time of nuclear isolation. Intact nuclei were assayed for replication in the extract by incorporation of [alpha- 32P]dATP or biotin-dUTP into nascent DNA. Most nuclei from exponential cells replicated in the egg extract, consistent with previous results showing that intact G1 nuclei from HeLa cells replicate in this system. In contrast, few nuclei from quiescent cells replicated in parallel incubations. However, when the nuclear membranes of these intact quiescent nuclei were permeabilized with lysophosphatidylcholine prior to addition to the extract, nearly all the nuclei replicated under complete cell cycle control in a subsequent incubation. The ability of LPC-treated quiescent nuclei to undergo DNA replication was reversed by resealing permeable nuclear membranes with Xenopus egg membranes prior to extract incubation demonstrating that the effect of LPC treatment is at the level of the nuclear membrane. These results indicate that nuclei from G1-phase cells lose their capacity to initiate DNA replication following density-dependent growth arrest and suggest that changes in nuclear membrane permeability may be required for the initiation of replication upon re-entry of the quiescent cell into the cell cycle.