The following points highlight the three main enzymes of DNA replications. The enzymes are: 1. Primase 2. DNA Polymerase 3. DNA Ligases.
Enzyme # 1. Primase:
A primase is an enzyme which makes the RNA primers required for initiation of Okazaki pieces on the lagging strand. Primase activity needs the formation of a complex of primase and at least six other proteins. This complex is called the primo-some.
The primo-some contains pre-priming proteins—arbitrarily called proteins i, n, n’ and n”—as well as the product of genes dna B and dna C. The primo-some carries out the initial priming activity for leading strand wherein the synthesis takes place continuously in the overall 5′ to 3′ direction.
It also carries out the repeating priming of the synthesis of Okazaki fragments for the lagging strand where the synthesis occurs discontinuously in the overall 3′ to 5′ direction.
The primase shows a very strong preference to initiate with adenosine followed by guanosine and this suggests that initiation of Okazaki fragments may occur at particular sites on the lagging strand. However, the small phage P4, which needs only about 20 Okazaki fragments per round of replication, shows no preferential initiation sites.
The primase that is tightly associated with the eukaryotic DNA polymerase Q is made of two sub-units and shows no stringent sequence requirements. But it does not act at random.
Enzyme # 2. DNA Polymerase:
DNA polymerase is an enzyme that makes a new DNA on a template strand. Both prokaryotic and eukaryotic cells contain more than one species of DNA polymerase enzymes. Only some of these enzymes actually carry out replication and sometimes they are designated as DNA replicases. The others are involved in subsidiary roles in replication and/or participate repair synthesis of DNA to replace damaged sequences.
DNA polymerase catalyses the formation of a phosphodiester bond between the 3′ hydroxyl group at the growing end of a DNA chain (the primer) and the 5′ phosphate group of the incoming deoxyribonucleoside triphosphate (Fig. 20.8).
Growth is in the 5’→3′ direction and the order in which the deoxyribonucleotides are added is dictated by base pairing to a template DNA chain. Thus, besides four types of deoxyribonucleotides and Mg++ ions, the enzyme requires both primer and template DNA (Figs. 20.9 and 20.10). No DNA polymerase has been found which is able to initiate DNA chains.
DNA polymerases isolated from prokaryotes and eukaryotes differ from each other in several aspects; a brief account of these enzyme is given below:
(i) Prokaryotic DNA Polymerase:
There are three different types of prokaryotic DNA polymerases which are called DNA polymerase I, II and III. These enzymes have been isolated from prokaryotes. DNA polymerase I or Romberg enzyme was first to be isolated from E.coli by Arthur Kornberg etal and was used for DNA synthesis in 1956. Kornberg received (jointly with Severo Ochoa) the Nobel Prize for this work in 1959.
DNA polymerase is a protein of Mr109, 000 in the form of a single polypeptide chain. It contains only one sulphydryl group and one disulphide’ group—the residue at the N- terminus is methionine.
Most of the prokaryotic DNA polymerase I exhibits the following activates:
i. Polymerase activity.
ii. 3′ → 5′ exonuclease activity.
iii. 5′ → 3′ exonuclease activity.
iv. Excision of the RNA primers used in the initiation DNA synthesis.
DNA polymerase I is mainly responsible for the synthesis of new strand of DNA. This is the polymerase activity. The direction of synthesis of the new strand’ is always 5′ → 3′. But it is estimated that DNA polymerase incorporates wrong bases during DNA replication with a frequency of 10-5. This is not desirable.
Hence DNA polymerase has also 3′ 5′ exonuclease activity (Fig. 20.11) which enables it to proofread or edit the newly synthesised DNA strand and, thereby, correct the errors made during DNA replication. An exonuclease is an enzyme that degrades nucleic acids from the free ends.
Therefore, whenever the DNA chain being synthesised has a terminal mismatch, i.e., insertion of a wrong base in the new chain, the 3’→ 5′ exonuclease activity of DNA polymerase I in reverse direction clips off the wrong base and immediately the same enzyme, i.e., DNA polymerase I, reinitiates the synthesis of correct base in the growing new chain.
Therefore, due to this dual activity of DNA polymerase I, the chance of errors in DNA replication is reduced.
The 5′ → 3′ exonuclease activity of DNA polymerase I is also very important. It functions in the removal of the DNA segment damaged by the irradiation of ultraviolet ray and other agents. An endonuclease (degrades nucleic acid by making internal cut) must cleave the DNA strand close to the site of damage before 5′ → 3′ exonuclease action of the DNA polymerase I may take place.
The 5′ → 3′ exonuclease activity of DNA polymerase I also functions in the removal of RNA primers from DNA. The ribonucleotides are Immediately replaced by deoxyribonucleotides due to the 5′ → 3′ polymerase activity of the enzyme.
The prokaryotic DNA polymerase II was discovered in pol A– mutant of E.coli. Pol A is a gene responsible for the synthesis of polymerase I. Therefore, the mutant of pol A– are deficient in DNA polymerase I or Kornberg enzyme. But, in absence of DNA polymerase I, replication of DNA also takes place in such mutant type.
Therefore, it is obvious that DNA polymerase II plays a role in DNA replication of such mutant. DNA polymerase II has 5’→ 3′ polymerase activity but it uses gapped DNA template. This enzyme also has the 3′ → 5′ but not the 5′ → 3′ exonuclease activity. The function of E.coli DNA polymerase II in vivo is unknown.
Prokaryotic DNA polymerase III was also discovered in pol A– mutant. There is a strong evidence that unlike DNA polymerase I and II, polymerase III is essential for DNA synthesis. The best template for DNA polymerase III is double-stranded DNA with very small gaps containing 3′-OH priming ends. In the DNA polymerase II, the core enzyme is tightly associated with two small sub-units.
The core enzyme has both 3′ → 5′ exonuclease (which could be involved in proof-reading) and 5’→ 3′ exonuclease activities, although the latter is only manifest in vitro on duplex DNA with a single-stranded 5′ tail.
This enzyme has a higher affinity for nucleotide triphosphate than DNA polymerase I and II and catalyses the synthesis of DNA chains at very high rates, i.e., 10-15 times the rate of polymerase I. The major properties of the three DNA polymerases are summarised in Table 22.3.
A DNA polymerase molecule has four functional sites which are involved in polymerase activity.
These sites are:
(i) Template site,
(ii) Primer site,
(iii) Primer terminus site, and
(iv) Triphosphate site.
The template site binds to the DNA strand functioning as template during DNA replication and holds it in the correct orientation. The primer site is the site where the primer chains to which the nucleotides will be added are attached.
The primer terminus site ensures that the primer binding to the primer site has a free 3′- OH. A primer without a free 3′-OH is not able to bind to this site.
The triphosphate site is the site for binding deoxyribonucleotide 5′-triphosphate that is complementary to the corresponding nucleotide of the template and catalyses the formation of phosphodiester bond between the 5′ phosphate of this nucleotide and the 3′-OH of the terminal primer nucleotide. In addition, there is a 3’→ 5′ exonuclease site and a 5′-3′ exonuclease site of DNA polymerase I.
(ii) Eukaryotic DNA Polymerase:
In higher eukaryotes, there are at least four DNA polymerases known as α, β,y and δ and a fifth (ɛ) has recently been described. In yeast DNA, polymerase I corresponds to DNA polymerase a, polymerase II to e, polymerase III to 6 and polymerase m to S and they have renamed accordingly.
Polymerase α is present in the nuclei of the cell. DNA polymerase a shows optimal activity with a gapped DNA template but shows a remarkable ability to use single-stranded DNA by forming transient hairpins. It will not bind to duplex DNA.
The native, undegraded enzyme consists of a 180 K Da polymerase together with three sub- units—the 60 and 50 K Da sub-units of about 70, 60 and 50 K Da. Association of the 180 K Da polymerase with the 70KDa protein makes the 3’→ 5′ exonuclease activity of the larger sub-units comprise a primase activity which allows the enzyme to initiate replication on unprimed single-stranded cyclic DNAs.
Therefore, polymerase a have dual activity, i.e., both the polymerase and primase activity. The association of primase with DNA polymerase α is restricted to the DNA synthetic phase.
Polymerase β is also present in the nuclei. It shows optimal activity with native DNA activated by limited treatment with native DNA-ase I to make single-stranded nicks and short gaps bearing 3′-OH priming termini and also shows negligible activity with denatured DNA. DNA polymerase β is believed to play a role in repair of DNA.
Polymerase δ is present in the dividing cell and have got similar properties polymerase a, but having 3′ → 5′ exonuclease activity. The activity of polymerase δ is dependent on activity on two auxiliary proteins: cyclin and activator I.
Due to presence of approximately equal activities of DNA polymerase α and δ it has been proposed that they act as a dimer at the replication fork with the highly processive polymerase δ acting on the leading strand and the primease-associated polymerase a acting on the lagging strand.
Cyclin or PCNA (proliferating cell nuclear antigen) independent form of DNA polymerase 6 is known as polymerase e which has two active polymerase sub-units of 220 and 145 K Da. DNA polymerase e is also probably involved in replication and it has been proposed that it takes over from DNA polymerase a in the synthesis of Okazaki fragments.
Polymerase y is found in small amount in animal cells. It is also found in mitochondria and chloroplasts and is believed to be responsible for replication of the chromosome of these organelles. DNA polymerase 7 isolated from chick embryos is a tetramer having four identical sub-units. It has also a proof-reading exonuclease activity.
Enzyme # 3. DNA Ligases:
DNA ligase is an important enzyme involved in DNA replication. DNA ligases catalyse the formation of a phosphodiester bond between the free 5′ phosphate end of an oligo or polynucleotide and the 3′-OH group of a second oligo or polynucleotide next to it.
A ligase-AMP complex seems to be an obligatory intermediate and is formed by reaction with NAD in case of E.coli and B. subtilis and with ATP in mammalian and phage-infected cells.
The adenyl group is then transferred from the enzyme to the 5′ phosphoryl terminus of the DNA. The activated phosphoryl group is then attached by the 3′-hydroxyl terminus of the DNA to form a phosphodiester bond. DNA ligases join successive Okazaki fragments produced during discontinuous DNA replication and seal the nicks left behind by DNA polymerase.
Reverse Transcriptase:
The enzymes so far discussed are required for the synthesis of DNA on parental template strand of DNA. But in certain RNA virus or retrovirus, there is an enzyme—called RNA-dependent DNA polymerase or reverse transcriptase—which uses parental RNA strand as a template for the synthesis of DNA.
The immediate product of this enzyme activity is the formation of double-stranded RNA-DNA hybrid which is the result of the synthesis of a complementary strand of DNA using single- stranded viral RNA as template. This enzyme uses viral RNA as template.
This enzyme uses one of the tRNA molecules (e.g., tRNA trp) as primer to synthesise DNA on the template single-stranded RNA. Reverse transcriptase is the product of the Pol gene of retroviruses. Reverse transcriptase is also found in bacteria where it acts in the production of multi-copy, small single-stranded DNA molecules in eukaryotic cell-the telomerase is, by definition, also a reverse transcriptase.