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Although substantial progress was made during the last decade, all replication mechanisms in eucaryotes are not yet understood because:
(a) Replication is an event which occurs much more rarely than in bacteria. Initiation of replication is therefore highly controlled; this complicates the understanding of this process;
(b) The DNA is not naked, in the cell, but in the form of chromatin, i.e. a combination of DNA and proteins. Chromatin is organized in nucleosome: about 200 pairs of bases of DNA are wound around an octamer of histones (basic proteins of chromatin). There is therefore a higher level of organization resulting in a greater complexity for the replication (see fig. 6-34).
Mainly 3 experimental approaches were followed.
First of all, enzymes identical to those implied in bacterial replication were sought out, identified and very often purified. It is however difficult to know whether the protein has not been degraded during the purification as long as the gene coding for this polypeptide has not been isolated. On the other hand, it is only in some cases, by using appropriate biological systems, that it has been possible to correlate an activity showed in vitro to a precise role in vivo.
Recently, the existence of conditional mutants in some yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) has given much information on the role of DNA polymerases as well as on the role of topoisomerases (see below).
The third approach consists in studying the replication of animal DNA viruses, like the monkey virus SV 40 (Simian Virus 40). As a matter of fact, except for one protein coded by their genome, antigen T, these viruses utilize the enzymatic machinery of the cell to replicate their DNA.
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Based on the results obtained by these three approaches we will now summarize the present knowledge on the subject.
1. Steps of Replication:
The long chromosomal DNA molecules (108 base pairs) probably replicate from multiple origins of replication. By means of electron microscopy one can indeed observe “replication eyes”, i.e. open regions of the DNA molecule, which may correspond to two replication forks progressing in opposite directions.
If radioactive precursors are incorporated in the DNA being synthesized, one can watch the course of the phenomenon by autoradiography. These experiments confirm that there are numerous origins of replication, and that DNA synthesis is bidirectional from these origins.
In rapidly developing tissues, like the embryonic tissues, there are numerous origins of replication which decrease in number as the animal becomes adult. This therefore confirms that the control of DNA synthesis does take place at the initiation stage. The present view is that initiation at the origin of replication takes place using an RNA.
In the case of SV 40, initiation of replication implies the recognition of a specific sequence by a viral protein, antigen T, followed by a local unwinding of the double helix thanks to the DNA helicase activity of this protein. This situation is similar to the one described previously for E.coli.
On the strand of discontinuous synthesis, the Okazaki fragments are much smaller than in procaryotes (about 200 nucleotides). This difference in size could be due to the nucleosomal structure of chromatin. These Okazaki fragments contain at their 5′ end, a short RNA fragment (about 10 nucleotides) which is synthesized by the eucaryotic primase.
This primer is then elongated by DNA polymerase α. Unlike in E.coli, DNA polymerase and primase are combined into a complex which can be isolated in vitro (see below).
The elimination of RNA primers could be made by RNase H. The filling of gaps thus created would be accomplished either by DNA polymerase α, or DNA polymerase the fragments being then bound to one another by a polynucleotide ligase. According to results obtained recently with SV40, the replication of the strand of continuous synthesis would be carried out by the DNA polymerase δ.
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Very little information is available on termination; some experiments were carried out with SV40 whose replication is bidirectional. Examining molecules replicated to 90%, one observes that there exists a termination zone, in which the two replication forks stop at about 500 nucleotides from one another, probably due to the effect of topological constraints.
It then appears that an unwinding step (achieved by a topo-isomerase II) is required for the separation of the two daughter-strands. This has been confirmed in S.pombe thanks to a heat sensitive mutant of DNA topoisomerase II.
2. The DNA Polymerases:
There are probably four DNA polymerase in eucaryotes: DNA polymerase α, DNA polymerase β, DNA polymerase y and DNA polymerase δ. They can be distinguished by their physiochemical properties and sensitivity to various drugs.
DNA polymerase α or DNA polymerase I in S.cerevisiae, was purified in a native form. It comprises at least 4 sub-units. One of them is responsible for the polymerase activity. The primase activity is carried by one (or two) different sub-unit(s). One can therefore speak of a DNA polymerase α- primase complex, active in vitro.
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The existence of DNA polymerase δ (DNA polymerase III in yeast) was confirmed only recently. It is a multimeric enzyme which has an exonuclease activity 3′ → 5′. It is stimulated by cycline, a protein whose quantity is maximal during the DNA synthesis phase.
The role of DNA polymerase β has not yet been established accurately. This enzyme is found in all cells, including those at a terminal differentiation stage (neurone, spermatozoid). Moreover, it presents a constant level of activity during the phases of the cell cycle. It is generally admitted that it has a role in the phenomena of DNA repair, especially in those taking place outside a phase of DNA synthesis.
DNA polymerase γ is localized in the mitochondria. An exonuclease activity 3′ → 5′ is probably associated to it. It probably replicates the mitochondrial DNA, whose replication is chronologically and enzymatically autonomous from the replication of the nuclear DNA.
3. Other Proteins Involved in Replication:
As mentioned above, there are other proteins playing a role at the replication fork. They can be classified into proteins with enzymatic activity, proteins having a structural role and lastly modulator proteins, i.e. proteins which modify some enzymatic activities or make them specific.
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It is rather easy to identify proteins having an enzymatic activity capable of playing a role in replication. It was thus possible to isolate a topoisomerase of type I, one of type II, one RNase H, two DNA ligases and recently a DNA helicase.
However, although it could be shown that the activities of topoisomerase II or ligase I increase in parallel with the DNA synthesis, the role of these diverse enzymes in the replication process remains to be determined.
Numerous proteins having affinity for the single-stranded DNA were partially purified from various tissues, but it could not be proved that any of them played a role in replication.
The situation is even more complex for modulator proteins, because, they are a priori, present in very small quantities in the cell. However, it has been possible to isolate factors capable of increasing in vitro, the affinity between RNA primers and the DNA polymerase isolated from the same tissue.
4. Developments and Perspectives:
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As in the case of E.coli it is envisaged that the replication of the two chains of DNA in eucaryotes takes place in a coordinated manner thanks to a complex formed by the DNA polymerase α and DNA polymerase δ. This attractive hypothesis is however yet to be proved.
Cloning of genes coding for the catalytic sub-units of DNA polymerases has already been carried out for several enzymes obtained from different cells. It was thus possible to show some common sequences at the protein level, sequences which would probably correspond to the binding sites of the template, dNTPs etc. It was also possible to classify the various DNA polymerases in two families.
At present, the larger family comprises only the DNA polymerases which perform the replication of the genomic DNA with the exception of DNA polymerase III of E.coli which derives from a different ancestor.