In this article we will discuss about the mechanism for the replication of DNA.
The mechanism for the synthesis of DNA proposed by Watson and Crick offered the important advantage of explaining how new DNA molecules could be exact replicates of the old. According to Watson and Crick, each single strand is a template on mold for its complement, and a new helix has one old strand and one that is newly synthesized.
This type of replication is called semiconservative, in contrast to the conservative type in which two new strands are synthesized in the form of a double helix while the old double helix remains unchanged. A third type of replication—dispersive—would be possible if the double helical strands were to break down along their length into small pieces.
After each individual piece replicates, it would then be randomly reconnected with newly synthesized pieces to form a patchwork single string of “dispersed” old and new pieces.
The Experimental Support in Favour of Semi-Conservative Replication:
A good experimental support for the correctness of the explanation came from the work of Meselson and Stahal in 1958 on E.Coli. The bacterium when grown in a medium with a nitrogen source containing only 15N. This heavy isotope becomes incorporated in the new DNA formed.
This can be located because the DNA is now denser than 14N and it can be separated by centrifugation and its sedimentation rate can be measured. The bacteria are then transferred to a medium containing only 14N and the DNA is analysed at each successive doubling of the chromosomes.
In such analyses three kinds of DNA were recognised—15N DNA, 14N DNA and 15N/14N DNA. The result thus shows complete agreement to the model of Watson and Crick.
Synthesis of DNA is Escherichia Coli:
1. The template primer concept:
Primer should be restricted to the DNA chain from which growth occurs at its 3′- hydroxyl terminus, and priming to designate this function. The primer terminus is the terminal nucleotide of the primer bearing the 3′- hydroxyl group.
The term template applies to the DNA chain that furnishes directions for the sequence of nucleotides, and the term directing, not priming, designates this 2nd function. Thus the template-primes embraces two different functions and meanings.
2. Initiation of DNA chains by RNA primers:
The starting points for DNA replication may, therefore, be recognized not by a DNA polymerase, but by a form of RNA polymerase. The group of enzymes role is the growth of complementary RNA polymerase— a number of ribo-nucleotides are sequentially linked together until a stop signal is read.
The enzyme then detaches leaving a short RNA chain, then serves as a primer onto which DNA polymerase III adds deoxyribonu- cleotides. The best evidence that RNA may act as a primer in DNA replication comes from nearest neighbour frequency analysis of nascent DNA-J^NA segments synthesized—in vivo—from a number of mammalian cells.
Proteins of DNA Replications:
DNA polymerase:
The studies of DNA synthesis in an in vitro system containing the enzyme, each of the four different deoxyri- bonucleotide triphosphates, Mg++, and a small quantity of primer DNA. The presence of exonuclease activity in the enzyme suggested that mistakes in copying could be due to partial breakdown of DNA strands of the primer with subsequent repair by the polymerase.
Branching could be accounted for by copying mistakes that were due to switching of the elongating strand from a template strand to its complementary strand. Binding studies of DNA polymerase with various kinds of DNA reveal that the enzyme does not bind along intact helical sequences, but will bind at the ends of such a DNA or at “nicks” produced in the helical molecule. Binding alone, however, is not sufficient for active synthesis in phase ØX174 DNA.
When DNA polymerase is added to a closed circular duplex DNA (plasmid), no binding occurs. In addition to binding to DNA, the enzyme also binds the four deoxyribonucleotide triphosphates at a single site; for which all four triphosphates compete.
Polymerase I:
Polymerase 1 is an enzyme roughly 100,000 daltons in size. There is only one site on polymerase which recognizes the substrate, DNA, but before the enzyme can recognize DNA, the DNA molecule must be nicked or single stranded. Once Pol. 1 attaches to the recognition point, it repairs the molecule all the way from the starting end to the 3′ end.
After the repair is completed, the only action needed to make the molecule active is that of DNA ligase, which makes a circular molecule of the linear one simply by joining the two ends.
Polymerase II:
A second repair polymerase, Pol. II, repairs large regions of DNA molecules in the 5′-3′ direction, but it lacks exonuclease activity.
Polymerase III:
The “real” replication enzyme, Pol. Ill polymerizes in the 5′-3′ direction, but relatively little is known about it.
(a) DNA Pol. III appears to be a tetramer at the time of DNA synthesis is initiated. For synthesis initiation Pol. Ill must be complexed with a co-enzyme called co-polymerase III (Copol. 111). The Pol-III-Copol-III complex initiates replication of single-strand templates (Copol.III and ATP are necessary for initiation of but not for continuation of DNA synthesis).
(b) The entire complex at the time of synthesis initiation consists of a double-helical, circular molecule of DNA, or RNA primer, an unwinding protein, ATP and Pol. III-Co-Pol. III. Once synthesis begins, ATP and Co-Pol are not necessary.
Proteins of DNA Replication of Eukaryotes:
Multiple polymerases have been extracted from both cytoplasm and nuclei from higher eukaryotic cells. These have been classified as DNA polymerase α, β or γ on the basis of their molecular weight, their sensitivity to sulphihydryl inhibitors, and their reactions with DNA template and a variety of primer molecules.
DNA Polymerase α:
Large molecular weight (100,000), N-ethylmaleimide (NEM)- sensitive protein which is usually found associated with other proteins of the replication complex, in particular a DNA-dependent ATPase of 60,000 daltons.
DNA Polymerase β:
Small molecular weight enzyme of 43,000-45,000, the NEM resistant enzyme has been purified to homogeneity from a number of eukaryotic sources and is immunologically distinct from other DNA polymerases.
DNA Polymerase α:
Large molecular weight enzyme, sensitive to NEM, the enzyme utilizes synthetic primer templates.
Proteins that Remove Superhelical Turns:
The operation of swivel is needed to relieve the forque in unwinding a very long DNA duplex. Without it, replication of a fully covalent circle would be physically impossible. The function of a swivel can be performed in two ways: One is by the joint action of nuclease and ligase: nicking one strand of the duplex relieves the forque on twist; sealing the nick by ligase restores the covalent state of the duplex rod or circle. Assuming that the protein acts catalytically, it can be considered a DNA- untwisting enzyme, a “swivelase”.
DNA Replication on a Mammalian Chromosome:
A diagram of DNA synthesis on a hamster chromosomes based on the interpretation of auto-radiographs. There are many initiation sites, and DNA synthesis proceeds in both directions along the length of the chromosomes.
The autoradiography studies of the DNA replication at the terminal phase of the ‘S’ period suggest:
1. Individual chromosome has its own ‘S’ period.
2. Longer chromosomes are not necessarily late, nor shorter chromosome early in terminating DNA replication.
3. Chromosomal asynchrony is the rule;
4. Both the DNA termination and initiation patterns may be used as a potential tool for the identification of a specific chromosome, or in segment, therefore, pinpointing a karyotypic disorder;
5. The very fact that in cases of X- polysomics having 3-6Xs all but one X- chromosomes has so variable degree of lateness suggesting the differential degree of genetic activity in these additional Xs.
The role of movement of replication complex during synthesis of DNA is very similar in animal cells and their viruses. The rate is 0.1-2 µm (2-40 x 105 daltons; 1-15 x 103 nucleotide residues) of DNA per minute (adjusted to 37° C).
Notwithstanding the apparent uniformity of results, it is becoming clear that the role of form progression does vary in response to genetic controls, to temporal controls, and to the environment. DNA replication proceeds in a highly ordered and complex pattern on all chromosomes throughout S Phase.
DNA-Histone Complex:
Briefly it is now known that DNA of 150- 200 base pairs in length is packaged in nucle- osome sub-units, the precise size and conformation of which are determined by chromosomal proteins, in particular the histones.
The nucleosome can be further dissected into a core particle that comprises a double-stranded helical DNA segment of about 140 base pairs, in association with an octameric aggregate of equimolar amounts of histones H2A, H2B, H3 and H4.
In cells from different species the inter particle or linker DNA segment varies from very few to 60-80 base pairs. It is associated with histone H1 (or histone H5 or chromosomal protein Hg in the case of nucleated avian erytrocytes and trout testis cells, respectively). Nuclease digestion analyses suggest that the core particle-bound DNA has secondary and tertiary structure determined by 10 base-pair segments.
A functional model for chromatin structure has been proposed which suggest that the core particles are composed of two isologously paired hetero tetramere of one mole each of histones H2A, H2B, H3 and H4, arranged symmetrically about the interwoven helical DNA strands.
The model is appealing because it would permit the half nucleosomes to participate in DNA replication, in chromatin replication, and in subsequent chromosome segregation without requiring major reorganization of DNA-protein associations.