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In this article we will discuss about:- 1. Definition of DNA Replication 2. Enzymes Involved In DNA Replication 3. Mechanism 4. Models 5. Watson and Crick’s Model.
Contents:
- Definition of DNA Replication
- Enzymes Involved In DNA Replication
- Mechanism of DNA Replication
- Models for DNA Replication
- Watson and Crick’s Model for DNA Replication
1. Definition of Replication of DNA:
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One of the most important properties of DNA is that it forms its additional identical copies. The process of forming its replica copy is called replication. Replication is the basis of evolution of all morphologically complex forms of life.
Howard and Pelc (1953) demonstrated that in eukaryotes replication occurs during interphase between mitotic cycles and also during interphase of meiosis. During interphase of cell division the number of DNA molecules doubles which at anaphase is separated into two daughter cells, and thus equal number of chromosomes is maintained.
However, replication does not occur during entire anaphase but is confined only to synthesis (vS) phase. There is a post-mitotic gap (G1) between the telophase and S phase. A second premitotic gap (G2) is between the S phase and prophase. Only S phase involves replication process.
The Gl phase is most variable and in many eukaryotic cells it is completed within 3 to 4 hours or even months depending on physiological conditions. Mostly DNA synthesis is accomplished in 7 to 8 hours. In bacteria growing at log phase, DNA synthesis occurs from the time a cell originates to give rise to two daughter cells. It is noteworthy that bacteria divide only through binary fission.
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In general, DNA carries out two important functions such as hetero-catalytic function and autocatalytic function. The hetero-catalytic function is protein synthesis directed by DNA, and autocatalytic function is the synthesis of its own DNA into replica copies. However, replication of DNA in prokaryotes differs from that of eukaryotes.
2. Enzymes Involved In DNA Replication:
Both the prokaryotic and eukaryotic cells contain three types of nuclear enzymes that are essential for DNA replication. These enzymes are nucleases, polymerases and ligases.
(i) Nucleases:
The polynucleotide is held together by phosphodiester bonds. The nucleases hydrolyse the polynucleotide chain into the nucleotides. It attacks either at 3′ OH end or 5′ phosphate end of the chain. The nucleases are of two types (Fig. 5.17-B).
(a) Exonucleases:
The nuclease that attacks on outer free end of the polynucleotide chain is called exonuclease. It breaks phosphodiester bond either in direction (A) or in 3’→ 5′ direction (B). The enzyme moves in either cases stepwise along the chain and removes nucleotides one by one. Thus, the whole chain is digested.
(b) Endooudeases:
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The endonucleases attack within the inner portion of one or the double strands. Therefore, a nick is made on double stranded DNA molecule. However, if the polypeptide chain is single stranded (e.g. in DNA viruses), the attack of endonuclease will render the chain into two pieces.
On double stranded DNA the nick contains two free ends that in turn act as template for DNA replication. Apart from this, the nicked double helix is distorted due to rotation of free molecules around its intact strand.
(ii) DNA Polymerases:
DNA polymerases carryout the process of polymerization of nucleotides and formation of polynucleotide chain. This enzyme is called replicase when it replicates the DNA molecules and inherited by daughter cells. In 1959, for the first time A. Romberg discovered an enzyme in E. coli which polymerized the deoxyribonucleotide triphosphate on a DNA template and produced complementary strand of DNA.
This enzyme was called DNA polymerase. Later on it was named as Komberg polymerase or Romberg enzyme after the name of discoverer, for demonstrating in vitro polymerization of DNA. For the catalysis of polymerization, it requires the four deoxyribonucleotide triphosphates e.g. dATP, dGTP, dTTP and dCTP, a DNA template, a primer for initiation of catalytic activity and Mg++ (Fig. 5.18).
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In prokaryotes, three types of DNA polymerases e.g. polymerase I (Poly-I), polymerase II(Pol II), and DNA polymerase III (Pol III) are found, whereas in eukaryotes three or four polymerases termed as α, β and ү polymerases and mitochondria (mt) DNA polymerase are present.
The molecular weight of α and ү polymerases are over 100,000 and that of β-polymerase is 30,000-50,000. The α and β polymerases are located in the nucleus. The β-polymerase copies a poly (A) or poly (C) template. The ү-polymerase copies many poly-ribonucleotides such as poly (A), poly (C), etc. The mtDNA polymerase is like ү-polymerase.
(a) Polymerase I (Pol I):
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The Kornberg polymerase is known as Pol I. It is a single peptide chain with a molecular weight of 109,000 D. It is the largest single chain of globular protein known so far. One atom of zinc (Zn) per chain is present, therefore, it is metalloenzyine. In E. coli, approximately 400 molecules of Pol I are present.
Early experiments carried out by Kornberg revealed that when artificially synthesized DNA template strands alternating A and T i.e. poly d(AT) were incubated with polymerase and four radio- labelled nucleoside triphosphate, radioactive DN A containing alternating A and T was synthesized.
Though sufficient amount of dGTP and dCTP was present in the solution but these were not synthesized into DNA because the DNA strand contained only poly dAT. This emphasizes that Pol I synthesizes only complimentary copy of the template.
Shape of Pol I has been studied through electron microscope. It is roughly spherical of about 65 A diameters (Fig. 5.18) which gets attached regularly to the DNA chain.
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Pol I possesses several attachment sites such as:
(i) A template site for attachment to the DNA template,
(ii) A primer site of about 100 nucleotides contemporary to a segment of RNA on which the growth of newly synthesized DNA occur,
(iii) A primer terminus site containing a terminal 3’OH group at the tip, and
(iv) A triphosphate site for matching the incoming nucleoside triphosphates according to complementary nucleotide of DNA template.
Function:
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Pol I plays a significant role in polymerization (synthetic) as well as degradation (exonucleolytic) process of nucleotides, Pol I is broken by trypsin into two fragments, a large fragment (MW 75,000) and a small fragment (MW 36,000). The large fragement shows 3′ → 5′ exonuclease activity, and the small fragement shows 5′ → 3′ exonuclease activity. In E.coli the following three types of functions of Pol I have been found.
Polymerization:
Polymerization is a process of synthesis in 5′ → 3′ direction of short segments of DNA chain from deoxyribonucleoside triphosphate monomers to the 3′ -OH end of a DNA strand. It is not the main polymerization enzyme because it cannot synthesize a long chain. It synthesizes only a small segment of DNA.
It binds only to a DNA and forms nick in dsDNA. Therefore, it takes part in repair synthesis. In E.coli Pol I polymerize the nucleotides at the rate of 1,000 nucleotides per minute at 37°C. The chief enzyme associated with polymerization is known as polymerase III.
Exonuclease activity:
3’ → 5′ exonuclease activity:
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Pol I catalyses the breaking of one or two DNA strands in 3’ → 5′ direction into the nucleotide components i.e. the nucleotides are set free in 3′ → 5′ direction which is reverse to polymerization direction.
Therefore, it is called 3′ → 5′ exonuclease activity. Pol I correct the errors made during the polymerization, and edits the mismatching nucleotides at the primer terminus before the start of strand synthesis. Therefore, the function of Pol I is termed as repair synthesis.
5′ → 3′ exonuclease activity:
Pol I also breaks the polynucleotide chain in 5′ → 3′ direction with the removal of nucleotide residues. Upon exposure of DNA to the ultraviolet light two adjacent pyrimidines such as thymines are covalently linked forming pyrimidine dimers. These dimers block the replication of DNA. Therefore, removal of pyrimidine dimers e.g. thymine dimers (T=T) is necessary.
Through 5′ → 3′ exonuclease activity, Pol I removes pyrimidine dimers. Secondly, DNA synthesis occurs on RNA primer in the form Okazaki fragments. Through 5′ → 3′ exonuclease activity Pol I remove RNA primer and seal the gap with deoxyribonucleotides. Its onward movement results in removal of ribonucleotides from the front portion followed by of deoxyribonucleotides behind it.
(b) Polymerase II (Pol II):
For several years Pol I was considered to be responsible for replicating in E.coli. but the work done during 1970s made it clear that Pol I is associated only with repair synthesis and the other enzymes, Pol II and Pol III are involved in polymerization process. Pol II is a single polypeptide chain (MW 90,000) that shows polymerization in 5’ → 3’ direction of a complementary chain.
It also shows exonuclease activity in 3’ → 5’ direction but not in 5’ → 3’ direction. The polymerization activity of Pol II is much less than Pol I in E.coli cells. About 50 nucleotides per minute are synthesized. E.coli cells contain about 40 Pol II molecules.
The 3′ → 5′ exonuclease activity of Pol II shows that it also plays a role in repair synthesis or DNA damaged by U.V. light just like Pol I. In the absence of Pol I, it can elongate the Okazaki fragments. Therefore, Pol II is an alternative to Pol I.
(c) Polymerase III (Pol III):
DNA polymerase III is several times more active than Pol I and Pol II enzymes. It is the dimer of two polypeptide chains with molecular weight 1,40,000 and 40,000 D respectively. Pol III polymerises deoxyribonucleoside triphosphates in direction very efficiently. Therefore, Pol III is the main polymerization enzyme that can polymerize about 15,000 nucleotides per minutes in E. coll.
Like Pol II, it cannot polymerize efficiently if the template DNA is too long but can do when ATP and certain protein factors are present. Synthesis of a long template also occurs when an auxiliary protein DNA (co-polymerase II) is linked with Pol III and produced Pol III-co Pol II complex. In addition Pol III also shows 3’→ 5′ exonuclease activity like Pol II.
The 5’→ 3′ exonuclease activity is absent. All the polymerases e.g. Pol I, Pol II and Pol III show 3’→ 5′ exonuclease activity, whereas besides Pol I, the other two polymerases (Pol I and Pol II) lack 5’→ 3′ exonuclease activity. However, some workers have shown both 3’→ 5′ and 5’→ 3′ exonuclease activity in Pol III.
(iii) DNA Ligases:
The DNA ligases seal single strand nicks in DNA which has 5’→ 3′ termini. It catalyses the formation of phosphodiester bonds between 3′-OH and 5′-PO4 group of a nick, and turns into an intact DNA. There are two types of DNA ligases: E. coli DNA ligase and T4 DNA ligase. The E. coli DNA ligase requires nicotinamide adenine dinucleotide (NAD+) as cofactor, whereas T4 DNA ligase uses ATP as cofactor for joining reaction of the nick (Fig 5.19).
3. Mechanism of DNA Replication:
A. Romberg (1992) has nicely discussed the DNA replication. In E. coli DNA replication has been investigated most extensively. It was thought that in eukaryotes probably similar mechanism operates. However, it has been found that in E. coli replication always starts at a very unique site called the origin. In E. coli die replicating apparatus contains an enzyme complex at the point where DNA thread is attached.
Through this replicating point DNA thread moves and replication is accomplished. In eukaryotes enzyme moves along the DNA thread. It has earlier been described that E. coli possesses three types of DNA polymerases; each reads DNA template in 3’→ 5’ direction and catalyses the synthesis of DNA in 5’→ 3’ direction.
The polymerases read deoxyribonucleotide triphosphates (dATP, dGTP, dCTP, dTTP) as substrate and a DNA template.
To the 3′ end of growing point, the nucleotides are added after interaction of 3′-OH end of deoxyribose with alpha (first) phosphate group of substrate releasing pyrophosphate as below:
Before the replication begins, DNA double helix must be unwounded to give rise to single strand. Unwinding process occurs very rapidly to form a fork that rotates about 75-l(X) revolutions per second. The unwinding process is facilitated by helicases.
Some important genes and proteins associated with replication are given in Table 5.7:
Overall DNA replication is accomplished in the following stages (Fig 5.20):
(i) Unwinding of Double Helix:
Helicases are responsible for unwinding of double helix. They use energy from ATP to unwind short stretches of helix just ahead the replication fork. After separation of strand it is very necessary to keep them single stranded through single stranded DNA binding proteins (SSB). The SSB is a tetramer with each of four subunits of a molecular weight of 18,500 – 22,000 Dalton.
It may bind as a binding sites of 8-10 nucleotides (Fig. 5.20). However there is possibility of leading tension and formation of super coils in helix.
The relieving of tension and promotion of unwinding process are done by the enzyme topoisomerases which transiently break one of two strands in such a way that it remains unchanged. It ties or unties a knot in DNA strand. DNA gyrase is one of the E. coli topoisomerases that removes super coiling of DNA during replication. Thus there is formation of a ssDNA template.
(ii) DNA Replication:
DNA replication is accomplished in several steps. The first step is the RNA-primer synthesis on DNA template near origin of replication. Synthesis of RNA primer is very necessary because during DNA replication there is chance of more error in initial laying down of first few nucleotides to pre-existing DNA template. DNA Pol I and Pol II cannot synthesize DNA without an RNA primer; therefore a special RNA polymerase called primase synthesizes an about 10 nucleotide long short primer.
Before priming, preprimer intermediate is formed with the help of six pre-priming proteins e.g. dnaB, dnaC, n, n’, n” and i proteins. For the synthesis of primer, primase needs several accessary proteins which combine with primase. The complex of primase- accessory protein is called primosome. Therefore, DNA Pol III holoenzyme starts synthesis of DNA in 5’→ 3′ direction at the end of RNA primer (Fig. 5.20B).
The second step is chain elongation. A new DNA strand starts synthesizing by addition of deoxyribonucieoside triphosphates to the 3′ end of last nucleotide of RNA primer. DNA synthesis occurs in 5’→ 3′ direction catalysed by the replisome. The replisome has two DNA Pol III holoenzyme complexes. It is a very large complex containing DNA Pol III and several proteins.
The ү and β-subunits bind the holoenzyme to the DNA template and primer. The a-subunit synthesizes the DNA. One polymerase continuously copies the leading strand (i.e. a strand growing in the direction of replication fork and showing continuous replication).
The lagging strand (i.e. a strand growing in opposite direction of replication fork and showing discontinuous replication of strand) loops around replisome continuously. There is formation of Y shaped replicating fork at the point where two strands are separated.
On leading strand DNA synthesis occurs continuously because there is always a free 3′-OH at the replication fork to which a new nucleotide is added. But on the opposite strand called lagging strand, DNA synthesis occurs discontinuously because there is no 3′ -OH at the replication fork to which a new nucleotide can link. On this strand there is free 3′ -OH at the opposite end away from growing point. Therefore, on lagging strand a small (11 bases long) RNA primer must be synthesised by primase to provide free 3′ -OH group.
The replication of DNA has two directions, one direction (unidirectional replication) and both the directions (bidirectional replication) from the point of origin. The bidirectional replication is found in most of the bacteria (e.g. E. coli. Bacillus subtilis. Salmonella typhimurium, etc.), whereas unidirectional replication occurs in E. coli bacteriophages (P2 and 186) and mtDNA of mouse LD cells.
Finally, about 1000-2000 nucleotides long fragment in bacteria and about 100 nucleotides long fragment in eukaryotic cells are synthesized. These fragments are called the Okazaki fragments after the name of a Japanese discoverer, R. Okazaki (Fig. 5.20 B).
(iii) Removal of RNA Primer and Completion of DNA Strand:
When the Okazaki fragments are formed; most of lagging strands become duplicated. The RNA primer is removed by DNA Pol I or RNase H. DNA polymerase I synthesizes a short segment of complementary DNA to seal the gap. Possibly Pol I remove the primer nucleotide at a time and replace it with suitable complementary deoxyribonucleotide (Fig. 5.20)
Fig. 5.20 : DNA replication in bacteria (diagrammatic) A. overall process of DNA replication; B. action of replisome, helicases and primosome, and looping of lagging strand around polymerase III; C, complrtion of Okazaki fragments, realease of lagging strand and sealing the gap by DNA ligase.
(iv) Joining of Fragments:
At the end, the fragments are joined by DNA ligase that forms a phosphodiester bond between 3′-OH end of growing strand and 5’ end of an Okazaki fragment (Fig. 5.20 C). Reaction of DNA ligases is given in Fig 5.19. In mutants defective ligase is produced; therefore, joining of Okazaki fragments is greatly improved.
E. coli DNA ligase derives energy from NAD. It is first adenylated by AMP moiety of NAD releasing the nicotinamide mononucleotide (NMN). The adenylated ligase reacts with ssDNA having a nick and forms phosphodiester bond.
The complete reaction is as below:
E. coli ligase + NAD → ligase – AMP + NMN
Ligase – AMP + DNA (with break) → phosphodiester + ligase + AMP
AMP + NMN → NAD
Obviously, DNA replication is a very complex process. If any error is made during replication, it leads to mutation. E. coli makes error about 10-6 per gene per generation. The DNA Pol I and Pol III act as proof reader of the newly formed DNA.
These move along new DNA synthesized, read mistakes formed due to improper base-pairing and correct those through exo-nuclease activity. Despite all these complexity, replication takes place rapidly in bacteria (750-1,000 base pairs per second) and much slower in eukaryotes (50-100 base pairs per second).
4. Models for DNA Replication:
The pattern of DNA replication in prokaryotes differs from that of eukaryotes. These differences are due to the nature of prokaryotic DNA. For example, the circular DNA of E. coli replicates at a replication point, the origin. In others the mechanism is different.
Some of the models for DNA replication are discussed below:
(i) The Cairns Model for DNA Replication:
J.Caims (1963) was the first to visualize the replicating chromosome of E. coli through auto-radiographic study. This study revealed that the replicating DNA thread got fixed at a specific site called origin, and was moving in one direction and within a replicating fork where the original strands are synthesized (Fig. 5.21).
Further studies have shown that in circular DNA (Fig. 5.22A) the two strands denatured at origin site (B). There is bidirectional DNA synthesis i.e. after initiation there appeared two growing points travelling in opposite directions around the circular DNA molecule (C). Growing points proceed with unwinding of DNA double helix.
The process of unwinding creates a torque that is transmitted to the un-replicated part of the DNA molecule resulting in formation of super helix or super twist (D). Super coil prevents its further replication. A temporary nick is made on one of the strands by a swivelling protein (w) which contracts this effect.
The nick allows the parental strand for their free rotation on each other and finally freed. At the end swivelling protein seals the nick to continue the replication process (E). Replication process goes on and the two growing point converge on the terminus (F).
(ii) The Rolling Circle Model:
Gilbert and Dressier (1969) described the rolling circle model to explain reactivation in ssDNA viruses e.g. Ø × 174 and the transfer of E. coli sex factor.
(iii) Replication in Eukaryotic Chromosome:
DNA replication in eukaryotic chromosome is not well understood as compared with prokaryotic chromosome. However, the well accepted model for replication of eukaryotic DNA is the bidirectional model (Fig. 5.23).
DNA synthesis starts at a midpoint of replication unit which is called initiation point (O-origin) (Fig. 5.23 A-B), and progresses in both the directions until reaches the terminal point (T) (C). The replication fork meets at T point (D) on the entire chromosome. There may be thousands of initiation points.
5. Watson and Crick’s Model for DNA Replication:
Each chain of double helix acts as template and is evolved in replication of DNA. Watson and Crick proposed that the hydrogen bonds between the base pairs of two strands are broken and separated from each other.
Each purine and pyrimidine base of the strands forms hydrogen bonds with complementary free nucleotides to be involved in polymerization in the cell. The free nucleotides form phosphodiester bonds with deoxyribose residue resulting in formation of a new polynucleotide molecule (Fig. 5.15). This model of Watson and Crick for DNA replication was later on verified experimentally.
Experimental Evidence for Watson and Crick’s Model for DNA Replication:
M. Meselson and F. Stahl (1958) provided the experimental support for Watson and Crick’s model for semiconservative nature of DNA replication which is called Meselson-Stahl experiment. They grew E. coli cells in medium containing heavy isotopic nitrogen (15N) for several generations.
They obtained a population of E. coli that contained totally 15N – labelled DNA. Density was measured by density gradient centrifugation in CsCl containing ethidium bromide. Density of 15N- DNA was heavier (1.722 g/cc) than the normal DNA (1.708 g/cc). Again the cells of E. coli were grown on medium containing less dense isotopic nitrogen (14N) and were allowed to multiply several times.
After the first generation, DNA was extracted which was found to be hybrid of 15N-14N (Fig. 5.16). This strand is 15N and the other 14N. It was neither heavier than 15N nor lighter than 14N. In the cells of first generation 50% 14N – DNA, and 50% hybrid (15N – 14N) DNA was recorded. In the second generation the ratio of normal and hybrid DNA molecules was 3:1.
This was the semiconservative nature of DNA replication because in the first generation one of the parental strands is converted into progenies and the other complementary polynucleotide strand is replicated. Thus, the two strands were the hybrids.
One can postulate for conservative mode of DNA replication i.e. both the original DNA strands act as template for a new duplex but is not separated, and results in an old and new double helix in the first generation. No hybrid 15N – 14N-DNA is formed. Therefore, this model could not be supported by Meselson-Stahl experiment.
Also, this experiment does not support for dispersive mode of DNA replication in which model there is no pattern of replication. The parental strands break randomly at several points during replication. Each segment will replicate and rejoin randomly.
This results in varying amount of old and new DNA molecules in daughter cells. After first generation instead of a single 15N- 14N hybrid, a wide spectrum of DNA densities is detected. Therefore, the dispersive mode of replication is also ruled out through Meselson and Stahl experiment.
