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The replication of the DNA can be schematically divided in three steps: initiation, elongation and termination.
Step # 1. The Initiation Step:
As seen above, no DNA polymerase can perform any de novo synthesis (contrary to RNA polymerases which can initiate by placing a complementary ribonucleoside-5′-triphosphate opposite to the DNA to be transcribed). The primer 3’OH which will serve as start point for the synthesis of DNA must therefore be supplied to the DNA polymerase during the initiation step.
On the other hand, replication starts at a precise point of the DNA called origin of replication. When the DNA is double-stranded, it is first necessary to open out the 2 strands in order to carry out the actual initiation step. We will again examine this mechanism.
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The 3’OH can be supplied in various ways. In a very large number of cases it is a short RNA fragment either synthesized by a RNA polymerase or a DNA primase (E.coli, B.subtilis, phages M13, ϕX 174, T4, T7, eucaryotic cells, polyoma virus or Simian virus SV40) or vestige of a larger RNA (virus of hepatitis B).
In the case of single-stranded DNA, the primer can also be formed by one end doubling back upon itself (hair-pin structure); this is postulated for the parvoviruses (see fig. 6-31). Lastly, it may be a deoxyribonucleoside-5′-monophosphate bound covalently to a protein; the 2 best known cases are, on the one hand, the adenoviruses, and on the other, a phage of B.subtilis called ϕ29.
Step # 2. The Elongation Step:
The elongation steps poses more or less problems depending on whether the DNA is single-stranded or double-stranded. The most complex — and most interesting — situation arises when the 2 strands of a double-stranded DNA must be replicated simultaneously. A replication fork is then formed.
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We have seen in the foregoing, that the 2 chains of the double helix are of opposite polarities. But we know that polymerization takes place strictly in the direction 5′ → 3′. This implies that the DNA synthesis can take place on one strand continuously in the direction 5′ → 3′, but, in order to copy the other strand, it can take place only in the direction opposite of that of the progression of the fork.
To solve this dilemma it was tried to find a mechanism which would allow progression in the direction 3′ → 5′, for example the polymerization of nucleosides 3′ triphosphates. One could never find such precursors, nor a DNA polymerase capable of polymerizing nucleosides 3′ triphosphates synthesized chemically.
The question therefore remained unanswered: how does synthesis take place in the direction 3′ → 5′. The answer was given by experiments carried out on the replication of phage T4, by the Okazaki team; by adding a precursor of DNA synthesis, like thymidine (deoxythymidine) in a highly radioactive form and for a very short time, it is possible to label only the DNA which has just been synthesized.
This method shows that the newly synthesized DNA is in the form of nucleotide chains of 1 000 to 2 000 residues (Okazaki fragments). These fragments are then bound to one another to form a continuous chain in the direction 5′ → 3′. The same type of experiment could be reproduced with various phages, bacteria and eucaryotic cells. In eucaryotes however, these fragments are definitely shorter in size (100 to 200 nucleotides).
Therefore, the replication fork has, in fact, an asymmetric structure. One of the chains is synthesized in the direction 5′ → 3′ in a continuous manner (leading strand). The “other, although advancing as a whole in the direction 3′ → 5′, consists of fragments synthesized individually in the direction 5′ → 3′; this is a discontinuous synthesis (lagging strand).
Although these results eliminate one difficulty they require a solution to the problem of the primer (initiation of polymerization). Here again, it could be shown that the primer was a short oligoribonucleotide, synthesized by DNA primase. This primer will then be eliminated, the gap thus created being filled by a DNA polymerase.
The elongation step necessitates the coordinated action of numerous proteins.
Step # 3. The Termination Step:
Studies on the synthesis of DNA were first focussed on the elongation step, then, more recently, on the initiation step which in fact, permits the control of the process. Little is therefore known presently on the termination of replication.
There are two important problems:
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i. How does the replication cycle terminate? Why replication does not continue? Why newly synthesized molecules do not serve immediately as templates for a new replication cycle?
ii. How newly synthesized DNA chains separate? This point seems easy to solve for a linear genome but it poses major topological problems for circular genomes.
