Mechanisms of DNA replication

Advances in recent years have led to exciting new ideas about the initiation, regulation and coordination of DNA replication. Structural studies have yielded fascinating glimpses of replisome action. In addition, the involvement of replication proteins in other cellular processes has blurred the lines between replication, repair and recombination.     

DNA replication and nuclear architecture

The model of in situ DNA replication provided by immunofluorescence and confocal imaging is compared with observations obtained by electron microscopic studies. Discrepancies between both types of observations call into question the replication focus as a persistent nuclear structure and as a replication entity where DNA replication takes place. Most electron microscopic analyses reveal that replication sites are confined to dispersed chromatin areas at the periphery of condensed chromatin, and the distribution of replication factors exhibits the same localization pattern. Moreover, rapid migration of newly synthesized DNA from the replication sites towards the interior of condensed chromatin regions obviously takes place during S-phase. It implies modifications of replication domains, hardly detectable by fluorescence microscopy. The confrontation of different observations carried out at light microscopic or electron microscopic levels of resolution lead to a conclusion that a combination of in vivo fluorescence analysis with a subsequent ultrastructural investigation performed on the same cells will represent an optimal approach in future studies of nuclear functions in situ.     

DNA replication and the nuclear membrane

To investigate the relationship between the nuclear membrane and DNA replication, Chinese hamster cells were labeled with tritiated thymidine and examined by electron microscope autoradiography. Unsynchronized cells were labeled for periods ranging from 0.5 to 20 minutes. There was no relative increase in the frequency of membrane-associated grains with the shorter labeling times, indicating that the replication point is not necessarily close to the nuclear membrane. When cells were synchronized to the beginning of the S period with mitotic selection and hydroxyurea, the percentage of membrane-associated grains was very low, indicating that DNA synthesis is not initiated at the nuclear membrane. When cells synchronized by mitotic selection were labeled at various times throughout the cell cycle, the percentage of peripheral grains was low in early S period and became progressively higher toward late S period as heterochromatin began to replicate. The labeling of Unsynchronized Microtus agrestis cells indicated that much of the peripheral labeling is due to the replication of intercallary heterochromatin. The results indicate that there is no association between the nuclear membrane and DNA replication.     

DNA replication and the nuclear envelope

Upon isolation of the nuclear membrane from cultured mouse leukemia L5178Y cells, approximately 1% of the total nuclear DNA was found to be attached to this structure. After pulse labeling of DNA and isolation of the nuclear membrane, the ratio of labeled DNA in the membrane fraction and in the rest of chromatin was compared. Results indicate that with a 3 min pulse, DNA in the membrane fraction showed slightly higher specific activity, but when the pulse was longer than 5 min there was no difference in the specific activities. Since the DNA fragment associated with the membrane fraction was found to be long enough to contain most of the DNA labeled during a 5 min pulse, the results obtained indicate that there is no preferential association of DNA to the nuclear envelope during initiation or elongation of DNA.     

Review: nuclear structure and DNA replication

DNA replication is a highly conserved process among eukaryotes where it occurs within a unique organelle-the nucleus. The importance of this structure is indicated by the fact that assembly of prereplication complexes on cellular chromatin is delayed until mitosis is completed and a nuclear structure has formed. Although nuclear structure is dispensable for DNA replication in vitro, it does appear to play a role in vivo by regulating the concentration of proteins required to initiate DNA replication, by facilitating the assembly or activity of DNA replication forks, and by determining where in the genome initiation of DNA replication occurs.     

Mechanisms and regulation of DNA replication initiation in eukaryotes

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation – from selecting replication start sites to replicative helicase loading and activation – and describe how these events are often distinctly regulated across different eukaryotic model organisms.     

An increase in mitochondrial DNA promotes nuclear DNA replication in yeast

Coordination between cellular metabolism and DNA replication determines when cells initiate division. It has been assumed that metabolism only plays a permissive role in cell division. While blocking metabolism arrests cell division, it is not known whether an up-regulation of metabolic reactions accelerates cell cycle transitions. Here, we show that increasing the amount of mitochondrial DNA accelerates overall cell proliferation and promotes nuclear DNA replication, in a nutrient-dependent manner. The Sir2p NAD⁺-dependent de-acetylase antagonizes this mitochondrial role. We found that cells with increased mitochondrial DNA have reduced Sir2p levels bound at origins of DNA replication in the nucleus, accompanied with increased levels of K9, K14-acetylated histone H3 at those origins. Our results demonstrate an active role of mitochondrial processes in the control of cell division. They also suggest that cellular metabolism may impact on chromatin modifications to regulate the activity of origins of DNA replication.     

Isolation of a stimulatory factor for nuclear DNA replication

Aqueous extracts of isolated nuclei and intact plasmodia of Physarum contain a heat-stable stimulator of nuclear DNA replication. The stimulatory factor is present throughout the mitotic cycle, and its activity is unaffected by prior exposure of plasmodia to cycloheximide. The stimulatory substance has been partially purified by heat treatment, precipitation with ethanol, chromatography on DEAE cellulose, and gel filtration. The purified material contains both carbohydrate and protein, and exhibits a molecular weight of about 30 000. The active substance increases the rate and overall extent of DNA replication in S-phase nuclei, but does not trigger the initiation of DNA synthesis in nuclei isolated from G2-phase plasmodia. The stimulatory material contains little or no deoxyribonuclease or DNA polymerase activity, and it does not affect DNA polymerase activity assayed using a purified DNA template.     

Simian virus 40 DNA replication in nuclear monolayers.

Simian virus 40 DNA replication has been studied in nuclear monolayers prepared by treatment of monolayers of BSC-1 monkey kidney cells with Nonidet P-40. These nuclear monolayers incorporated [3H]TTP into two types of viral replicative intermediates that sediment as 25-26S and 22-23S species, respectively, in neutral sucrose gradients. The 22-23S species behaves, in dye buoyant density equilibrium gradients, as a late replicative intermediate. Examination of both species in alkaline sucrose gradients revealed the presence of two types of newly synthesized strands: (i) 4-7S strands and (ii) full-length, or nearly full-length, 10-16S strands. At low TTP concentrations (less than 0.5 muM), the two size classes were found in approximately equal amounts. However, at 10 to 50 muM TTP, the proportion of the longer strands increased, with a corresponding decrease in the relative amount of the 4-7S species. Thus, the joining of small, Okazaki-like fragments to the growing chain appears to require a much higher concentration of TTP than the synthesis of the fragments themselves. Replicating simian virus 40 DNA synthesized in the nuclear monolayers is is associated with "M bands", as previously demonstrated for replicating simian virus 40 DNA in cultured whole cells.     

Viral cyclin-cyclin-dependent kinase 6 complexes initiate nuclear DNA replication

The cyclins encoded by Kaposi sarcoma-associated herpesvirus and herpesvirus saimiri are homologs of human D-type cyclins. However, when complexed to cdk6, they have several activities that distinguish them from D-type cyclin-cdk6 complexes, including resistance to cyclin-dependent kinase inhibitors and an enhanced substrate range. We find that viral cyclins interact with and phosphorylate proteins involved in replication initiation. Using mammalian in vitro replication systems, we show that viral cyclin-cdk6 complexes can directly trigger the initiation of DNA synthesis in isolated late-G(1)-phase nuclei. Viral cyclin-cdk6 complexes share this capacity with cyclin A-cdk2, demonstrating that in addition to functioning as G(1)-phase cyclin-cdk complexes, they function as S-phase cyclin-cdk complexes.     

A tetrapyrrole-regulated ubiquitin ligase controls algal nuclear DNA replication

In plant cells, organelle DNA replication (ODR) is coordinated with nuclear DNA replication (NDR), with ODR preceding NDR during cell cycle progression. We previously showed that the occurrence of ODR is signalled by a tetrapyrrole compound, most likely Mg-protoporphyrin IX (Mg-ProtoIX), resulting in the activation of cyclin-dependent kinase A (CDKA) and consequent initiation of NDR (refs 1, 2, 3). Here we identify an F-box protein of SCF-type E3 ubiquitin ligase (Fbx3) in the red alga Cyanidioschyzon merolae, which inhibits CDKA by ubiquitylating the relevant cyclin and inducing its degradation. Mg-ProtoIX binds to Fbx3 and inhibits cyclin ubiquitylation. Thus, these observations indicate that Fbx3 serves as the receptor for the plastid-to-nucleus retrograde signal Mg-ProtoIX and thereby contributes to a checkpoint mechanism ensuring coordination of ODR and NDR.     

Initiation of DNA replication at a nuclear matrix-attached chromatin fraction

It is still unclear what nuclear components support initiation of DNA replication. To address this issue, we developed a cell-free replication system in which the nuclear matrix along with the residual matrix-attached chromatin was used as a substrate for DNA replication. We found out that initiation occurred at late G1 residual chromatin but not at early G1 chromatin and depended on cytosolic and nuclear factors present in S phase cells but not in G1 cells. Initiation of DNA replication occurred at discrete replication foci in a pattern typical for early S phase. To prove that the observed initiation takes place at legitimate DNA replication origins, the in vitro synthesized nascent DNA strands were isolated and analyzed. It was shown that they were enriched in sequences from the core origin region of the early firing, dihydrofolate reductase origin of replication ori-beta and not in distal to the origin sequences. A conclusion is drawn that initiation of DNA replication occurs at discrete sub-chromosomal structures attached to the nuclear matrix.     

Mechanisms of separation of the complementary strands of DNA during replication

This article is a perspective on the separation of the complementary strands of DNA during replication. Given the challenges of DNA strand separation and its vital importance, it is not surprising that cells have developed many strategies for promoting unlinking. We summarize seven different factors that contribute to strand separation and chromosome segregation. These are: (1) supercoiling promotes unlinking by condensation of DNA; (2) unlinking takes place throughout a replicating domain by the complementary action of topoisomerases on precatenanes and supercoils; (3) topological domains isolate the events near the replication fork and permit the supercoiling-dependent condensation of partially replicated DNA; (4) type-II topoisomerases use ATP to actively unlink DNA past the equilibrium position; (5) the effective DNA concentration in vivo is less than the global DNA concentration; (6) mechanical forces help unlink chromosomes; and (7) site-specific recombination promotes unlinking at the termination of replication by resolving circular dimeric chromosomes.This revised version was published online in October 2005 with corrections to the Cover Date.     

Oxygen dependence of nuclear DNA replication in Ehrlich ascites cells

Oxygen was excluded from cultured Ehrlich ascites cells for 5-7 h and then readmitted. During the anaerobic period and for about 1 h following reoxygenation the DNA synthesis of the cells was studied by determining the DNA synthesis rate from [3H] thymidine incorporation data, by evaluation of the thymidine (pulse labelling) index, by DNA fibre autoradiography, and by alkaline sucrose gradients in order to follow the maturation of the daughter chains. The DNA synthesis rate was found to decay to a few percent of the initial value within 5-7 h after deoxygenation. Immediately after reoxygenation it increased to exceed the control level within 0.5-1 h. The only partial process of the genome replication definitely responding to deoxygenation/reoxygenation was the initiation of new replicon units, while progress of the replication forks and maturation of the new daughter chains were not significantly affected. The coordination of replicon initiation within groups or clusters was maintained throughout. The interruption of replication at the level of initiation of clusters upon deoxygenation was interpreted as a regulatory response of the cells to ensure basic viability under unfavourable conditions.