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Read this article to learn two things to know about the replication of DNA. The two things to know about DNA replication are:
(1) Variations in Semiconservative Mode of Replication and (2) Proteins of DNA Replication. The six proteins which help in DNA replication are: (1) DNA Helicases (2) DNA Single-stranded Binding Proteins (3) DNA Gyrase (4) DNA Polymerase (5) Primase and (6) DNA Ligase.
Introduction:
DNA replication is a fundamental process occurring in all living organisms to copy their DNA. Watson and Crick with the proposal of DNA structure, in 1953 gave the view that the replication occurred by the gradual separation of the double helix, like the separation of two halves of a zipper.
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According to their proposal the replication of DNA is a “semi-conservative” process, in which each strand of the parent double-stranded DNA molecule serves as template for the production of the complementary strand. Hence, after DNA replication, two identical DNA molecules are produced from a single parental double-stranded DNA molecule i.e. each daughter duplex contains one stand from the parent structure.
Two other alternate schemes of replication were:
(i) Conservative replication:
In it the two original strands would remain together, as would the two newly synthesized strands. As a result, one of the daughter duplexes would contain only parental DNA, while the other daughter duplex would contain only newly synthesized DNA.
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(ii) Dispersive replication:
In it the integrity of each of the parental strands would be disrupted (Fig. 3.1). As a result, the daughter duplexes would contain strands that were composites of old and new DNA; i.e. neither the parental strands nor the parental duplex is conserved.
To decide among these three possibilities of replication Matthew Meselson and Franklin Stahl conducted an experiment in 1958, which confirmed that DNA replication was semi conservative. The experiment utilized the properties of nitrogen. Nitrogen is a major constituent of DNA. N is by far the most abundant isotope of nitrogen, but DNA with the heavier N isotope is also viable. The N isotope is not radioactive, only heavier than common nitrogen.
During the experiment E. coli were grown for several generations in a medium containing N as the sole nitrogen source. As a result, the nitrogen containing bases of the DNA of these cells contained only the heavy nitrogen isotope. After that, E. coli cells with only N in their DNA were put back into N containing medium and were allowed to divide in some samples for only once and in some for several generations (Fig. 3.2).
When DNA is extracted from N grown cells and centrifuged on a salt density gradient, the DNA separates out at the point at which its density equals that of the salt solution. The DNA of the resulting cells had a higher density (was heavier). DNA was then extracted from one generation grown N cell and its density was compared to DNA from N DNA and N DNA.
It was found to have close to the intermediate density. This result was in concordance with semi conservative replication. If replication is semi conservative one would expect that the density of DNA molecules would gradually decrease during culture in the N containing medium. The density would decrease as light strand were synthesized in association with heavy strands.
After one generation, all the DNA molecules would be N-N hybrids and their buoyant density would be halfway between that expected for totally heavy and totally light DNA. As replication continues, after one generation the newly synthesized DNA would continue containing only light isotopes and two types of duplexes would appear in the gradients; those containing N-N hybrids and those containing only N. As the time of growth in the light medium increases, the number of N DNA increases. The results of these density-gradient experiments are shown in Figure 3.2.
Thing # 1. Variations in Semiconservative Mode of Replication:
DNA replication by formation of replication fork is the predominant system, being used by chromosomal DNA in eukaryotes and by the circular genomes of prokaryotes. But some circular molecules like, animal mitochondrial genomes, use a slightly different process called as displacement replication. In these molecules, the replication begins at a point called as D-loop.
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It is an approximately 500 bp long region where the double helix is disrupted by the presence of an RNA molecule base-paired to one of the DNA strand. This RNA molecule act as the starting point for synthesis of one of the daughter strand. This strand is synthesized by copying of one of the parent strand of the helix, the second strand being displaced and later on copied after synthesis of the first daughter strand. The advantage of displacement replication is not clearly understood.
In contrast, a special type of displacement process called rolling circle replication is an efficient mechanism for the rapid synthesis of multiple copies of a circular genome. This type of replication occurs in X and various other bacterio-phages. This replication initiates at a nick which is made in one of the parent poly-nucleotides. The free 3′ end that results in extended, displacing the 5’end of the polynucleotide.
Continued DNA synthesis “rolls off’ a complete copy of the genome, and further synthesis eventually results in a series of genomes linked head to tail. These genomes are single stranded and linear, but can be converted to double-stranded circular molecules by complementary strand synthesis, followed by cleavage at the junction points between genomes, and circularization of the resulting segments (Fig. 3.3).
Thing # 2. Proteins of DNA Replication:
DNA exists in the nucleus as a condensed, compact structure. To prepare DNA for replication, a number of proteins help in the unwinding and separation of the double-stranded DNA molecule. These proteins are required because DNA must be single-stranded before replication can proceed.
1. DNA Helicases:
These proteins bind to the double stranded DNA and stimulate the separation of the two strands. This helps in the formation of replication fork by hydrolysis of ATP. The energy liberated is used in the breakage of hydrogen bonds between the bases.
2. DNA Single-stranded Binding Proteins:
These proteins bind to the DNA as a tetramer and stabilize the single-stranded structure that is generated by the action of the helicases. Replication is 100 times faster when these proteins are attached to the single-stranded DNA.
3. DNA Gyrase:
This enzyme catalyzes the formation of negative super-coils that is thought to aid with the unwinding process. It creates a nick in one strand, turns the helix and make it straight ladder (as compared to twisted ladder). The nick is again sealed.
4. DNA Polymerase:
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DNA Polymerase I (Pol I) was the first enzyme discovered with polymerase activity, and it is the best characterized enzyme. Although this was the first enzyme to be discovered that had the required polymerase activities, it is not the primary enzyme involved with bacterial DNA replication. The enzyme involved in DNA synthesis in a major way is DNA Polymerase III (Pol III). Three activities are associated with DNA polymerase I;
(i) 5′ to 3′ elongation (polymerase activity)
(ii) 3′ to 5′ exonuclease (proof-reading activity)
(iii) 5′ to 3′ exonuclease (repair activity)
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The second two activities of DNA Pol I are important for replication, but DNA Polymerase III (Pol III) is the enzyme that performs the 5′-3′ polymerase function.
5. Primase:
The requirement for a free 3′ hydroxyl group is fulfilled by the RNA primers that are synthesized at the initiation sites by these enzymes.
6. DNA Ligase:
Nicks occur in the developing molecule because the RNA primer is removed and synthesis proceeds in a discontinuous manner on the lagging strand. The final replication product does not have any nicks because DNA ligase forms a covalent phosphodiester linkage between 3′-hydroxyl and 5′-phosphate groups.
