- 1 DNA Replication Mechanisms – DNA Replication in Prokaryotes and Eukaryotes
- 1.1 General Properties of Replication
- 1.2 Experimental Evidence in Support of Semi-Conservative Replication
- 1.3 Experiments of Meselson and Stahl
- 1.4 Replication of DNA in E coli cell
- 1.5 Requirement for Replication
- 1.6 Mechanism of Replication
- 1.7 Rolling Circle Replication
- 1.8 Replication in Eukaryotic Cells
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DNA Replication Mechanisms – DNA Replication in Prokaryotes and Eukaryotes
Prior to division, a cell duplicates its DNA material present in the nucleus. The mechanism which facilitates DNA duplication in a cell is known as replication. Replication on the other hand promotes the inheritance of characters or heredity. By the process of replication when the DNA amount in a cell is doubled, the genetic material then may be distributed equally in daughter cells formed by division. Actually, in replication, one DNA molecule may produce exactly two copies of the same DNA molecule. In all types of organisms, the mode of replication is identical. The double-stranded DNA molecule replicates in a semi-conservative fashion. In the daughter DNA molecules, it is found that of the two strands of the DNA molecule, one is from the old maternal DNA, and the other strand is newly formed. The process of replication is accomplished with the involvement of several enzymes and a number of protein factors. How replication occurs in the living cell is mostly understood from the observations on E. coli. Some special features are found in eukaryotic replication. However, basically, the mechanism of replication is similar in all types of organisms and it exhibits some basic properties.
General Properties of Replication
- Replication occurs by semi-conservative method when two polynucleotide strands of the DNA are separated during replication and each of the strands acts as a template for polymerizing nucleotides over it, thereby, forming two daughter DNA molecules, each having one old polynucleotide strand and one strand newly synthesized.
- Over one strand of the DNA molecule polymerization reaction occurs in a continuous fashion, while over the other strand, it occurs with pause causing fragmentary synthesis.
- The former template strand is called the leading strand and the latter is called the lagging strand. Such a mode of replication is called semi-discontinuous replication.
- On the template strand polymerization reaction continues from a 5′ to 3′ direction.
- Replication cannot be started anywhere over the chromosome or DNA, rather it always starts from one origin of replication.
- From the origin, the replication continues along both directions and therefore, replication is said to be bi-directional.
- Over the template strand to initiate the process of replication, a short RNA primer is required to be formed.
Experimental Evidence in Support of Semi-Conservative Replication
Semi-conservative replication denotes a pattern of replication in which two polynucleotide strands of the existing DNA molecule are separated and on the separated strands, complementary nucleotides are polymerized to give two double-stranded molecules which become the exact replica of the mother DNA molecule. Watson and Crick (1953) first predicted this replication pattern.
However, during the contemporary period, Delbruck suggested a conservative and dispersive mode of replication. Conservative mode of replication suggests that during replication the mother DNA molecule remains intact and a copy of the same is formed using the mother DNA as a template. This means that of the two DNA molecules produced after replication, one represents the old DNA and the other is newly formed. According to the dispersive mode of replication, the mother DNA molecule prior to replication gets fragmented and each fragment may produce its own copy. Finally, the copy fragments and maternal DNA fragments are assembled to form the complete DNA molecule.
However, in this reassembly, a mixed association of old and new fragments is very much possible. Therefore, according to the dispersive mode of replication, the two daughter DNA molecules produced are a mixture of old and new DNA fragments. Though there are predictions on three modes of replication of DNA, replication actually occurs in a semi-conservative fashion.
Experiments of Meselson and Stahl
M. S. Meselson and F. W. Stahl (1958) carried out an experiment on E. coli and from this experiment, they could be able to prove that replication occurs in a semi-conservative fashion. In their experimental protocol, they took the help of three techniques, namely
- DNA labeling by heavy nitrogen (N15)
- Equilibrium density gradient ultracentrifugation
- Photography with the use of UV light.
When DNA is labelled with N15 it becomes heavy in contrast to natural N14 DNA and the two DNA may easily be separated in the Caesium Chloride gradient by ultracentrifugation. The two separated DNA in a centrifuge tube may be easily detected by UV illumination and photography, the site where DNA is present will appear in the form of a dark band in the photographic plate. In their experimental protocol, Meselson and Stahl initially allowed some E. coli cells to grow for several generations in the culture medium that was provided with N15. By this procedure, almost all the DNA molecules of the bacterial cells in the culture could be heavily labelled with N15. They isolated DNA from the labelled E. coli cells as well as from the natural unlabelled E. coli cells and both the DNAs were exposed to ultracentrifugation in 6M CsCI solution for a period of about 48-72 hours at 50000 rpm. With this exercise DNAs from two sources got separated in cesium chloride gradient during rotation within the centrifuge tube. The heavy N15 DNA was found to be localized towards the bottom of the centrifuge tube and rested at a gradient equivalent to 1.724g/cm3 and the light N14 DNA was found to be localized towards the upper part of the centrifuge tube and rested at the gradient of 1.710g/cm3.
In the second step of their experiment, they transferred some labelled E. coli cells to a culture medium that no longer contained N15. The cells were allowed to grow there for one generation and DNA from the first generation bacteria was isolated. This DNA was then centrifuged in 6M CsCI solution at 50000 rpm for 48-72 hours. The DNA now was found to be localized at the intermediate position in the CsCI gradient of the centrifuge tube. This suggested that the DNA of the first-generation bacterial cells was of intermediate buoyant density and the DNA was of hybrid nature. This finding also ruled out the possibility of the conservative mode of replication, but still, the result failed to differentiate between the dispersive and semi-conservative modes of replication.
Meselson and Stahl then allowed the labelled bacterial cells to grow for another generation in the non-N15 medium. The DNA from the second-generation bacteria was isolated and centrifuged in a previous manner within a 6M CsCI solution. Now the DNA material was found to be localized at two different regions of the centrifuge tube, one part at the upper region and the other part at the intermediate position. These two fractions of DNA were 50 : 50 in distribution as revealed by UV photography. This also suggested that half of the DNA localized at the upper part was of lighter buoyant density and the other half being localized at the intermediate position was of intermediate density.
They then allowed the E. coli cells to multiply for a generation further. DNA from the third-generation bacteria was isolated and centrifuged in the same procedure as before. Now also the DNA materials were found to be localized at two positions as before, but the quantitative distribution was 75 : 25. The upper fraction of DNA was % and the intermediate fraction was V4 by proportional distribution. Logical analysis of the experimental results in consideration of the distribution of DNA fractions in the CsCI gradient indicated only a possibility of the semi-conservative mode of replication. If the replication would have occurred in dispersive mode, always the DNA of the same density would have been produced and a single band indicating localization of DNA at a single site would be observed.
Replication of DNA in E coli cell
In the cell of E. coli, only one double-stranded DNA molecule is present. Generally, this DNA remains in a superhelical configuration. Before division, this DNA is replicated into two DNA molecules.
Requirement for Replication
1. DNA template:
The existing DNA of the cell acts as a template in replication and new DNA molecules are synthesized and added to a primer based on the mother DNA molecule. The primer part of the primer: Template junction is one of the substrates of the process.
2. Nucleoside Triphosphates:
These are the precursors and one of the substrates for replication in the cell. There are pools of dATP, dGTP dCTP, and dTTP in the cell. These four types of dNTPs are taken from the cellular pool for their polymerization over the template strand. In the polymerization reaction, each of the dNTP molecules is converted into dNMP with the liberation of two pyrophosphates.
(a) DNA Polymerase: Studies of the atomic structure of various DNA polymerases attached to primer-template junction create a molecular concept that indicates that DNA substrate sets in a large C left that resembles a partially closed right hand. Based on this hand analogy, the three domains of DNA polymerase are the thumb, fingers, and palm.
Structure: Palm Domain Structure Composed of a β sheet containing primary elements of the catalytic site.
Function: Binds two divalent metal ions (Mg2+ or Zn2+) one metal ion reduces the affinity of 3′-OH for its Hydrogen resulting in 3’O- for the nucleophilic attack of α-phosphate of incoming dNTP. The other metal ion takes care of the negative charges of β and γ phosphate of dNTP and stabilizes the pyrophosphate produced. Monitors base pairing of recently added nucleotides.
Finger Domain: Helps in Catalysis.
Thumb Domain: Maintains correct position of primer and active site and close association between DNA polymerase is its substrate.
This enzyme is needed for the polymerization of nucleotides in E. coli cells. There are three types of DNA polymerases, namely DNA Pol. I (Kornberg enzyme as it was isolated by Arthur Kornberg and associated) DNA Pol. II and DNA Pol III. Of these three enzymes DNA Pol. Ill is the principal enzyme. However DNA polymerase I acts as an auxiliary to the DNA Pol. III. The DNA Pol. II is required for repair synthesis.
The DNA Pol. Ill of E. coli cell is a large molecule with a molecular weight of more than 600 Dalton. It is a complex protein composed of about 10 protein units. Two molecules of the same enzyme act on the DNA template during replication. The nature and function of the different components of the enzyme are different. In an E. coli cell 10-20 molecules of the enzyme may be available. But a number of DNA Pol. I is about 400. The comparative account of the different polymerases may be given in tabular form.
Comparison of DNA Polymerases of E. coli:
|Function||DNA Pol. I||DNA Pol. II||DNA Pol. III|
|Nucleotide Polymerization along 5′ to 3′||+||+||+|
|(a) 3′ to 5′ direction||+||+||+|
|(b) 5′ to 3′ direction||+||+||+|
|Atomic weight (Dalton)||103000||88000||90000|
|Number of sub-units||1||≥ 4||≥ 10|
|Addition of nucleotides/sec||16 – 20||~7||25 – 1000|
|Number per cell||400||30 – 50||10 – 20|
Structure and Function of DNA Pol. III of E. coli:
|Protein sub-unit||Genic Source||Atomic Wt. (Mr.)||Function|
|α||Pol C||132000||Nucleotide Polymerization|
|ξ||dna Q||27000||3’to 5′|
|θ||dna E||10000||Exonuclease Activity|
|τ||dna X||71000||Formation of stable bond with template and formation of dimer|
|γ||dna X||52000||Complex Formation|
|β||dna N||37000||To bind the enzyme like a ring with the template|
(b) Topoisomerase: Two types of topoisomerases are present in the E. coli cell, namely Topoisomerase I and Topoisomerase II. These two enzymes may bring about configurational changes in DNA molecules. The first enzyme may produce a nick in one strand of the double-stranded DNA molecule and by producing such a nick they can release any strain developed on the DNA molecule during the unwinding process. DNA topoisomerase II brings about the excision of two strands of DNA and releases strain. The activity of this enzyme is dependent on ATP. DNA gyrase is an example of Topoisomerase II.
(c) Helicase: This enzyme helps to unwind and separate two strands of the DNA molecule. It starts unwinding as well as the separation of strands from the replication origin and proceeds anteriorly promoting the polymerization of nucleotides. The molecular weight of the enzyme is about 300000 Dalton In a cell there are about six molecules of helicase. This is also known as dna B protein.
(d) DNA ligase: This enzyme helps in joining the Okazaki fragments.
(e) Primase: This is required for the synthesis of RNA primer. In E. coli cells there are about 50 primase molecules.
(f) Protein factors: The important protein factor needed for replication is SSB protein or single-stranded DNA binding protein. When helicase separates the two strands of the DNA, this protein binds with the single strand of the DNA and helps to keep them in a separated state for a considerable time period, so that polymerization of nucleotides may occur over the DNA strands by DNA polymerases. Besides SSB, several other factors needed during replication are Nus A, dna B, dna A, dna C, etc.
Mechanism of Replication
DNA replication is completed in three steps, namely the initiation phase, elongation phase, and termination phase.
1. Initiation of Replication:
Site of Replication: Initiation of replication in E. coli cells starts from the ori C region. The region contains about 245 base pair sequences. In this region, there are 3 repeats of 13 base pairs called 13 mers and 4 repeats of 9 base pairs sequence called 9 mers.
Role of Enzymes: Before replication starts about, 20 dnaA proteins bind with the 9 mers of the ori C region. Following this ATP and Hu protein accelerate the initiation reaction when dnaA protein causes denaturation along the 13 bp repeat region. After this helicase or DNA B binds with the denatured region. DnaB binding needs initially the help of dnaC. DnaB being bound with the open ends of the denatured DNA segment may then carry out the unwinding of DNA along both directions from the ori C region. Because of this, at both sides of the ori C, Y-shaped forks are generated.
When helicase unwinds the DNA forwardly from the replication fork considerable strain may be generated anterior to the fork region. This strain is neutralized by DNA gyrase. Four molecules of SSB protein together bind with each of the separated DNA strands and hold them in separate conditions. Actually, several tetramers of SSB protein may bind with the single DNA strand and a considerable portion of the DNA strand may be available for nucleotide polymerization.
While the two polynucleotide strands at the ori C region get separated, primase acts over the strands to produce a short RNA primer. About 10-50 nucleotides together form a primer on the DNA strand providing 3′ -OH end for the first nucleotide to be polymerized. Following this elongation starts.
2. Elongation Phase:
It has already been mentioned that after the first dNTP is added with the 3′- OH end of the primer, the elongation phase starts when depending upon the nucleotide sequence of the template strand complementary dNTPs come and join sequentially with the initial nucleotide incorporated at the 3′-OH end of the primer. The addition of nucleotides occurs in a continuous fashion over the leading strand with the polymerization reaction furnished by DNA Pol. III. In the polymerization reaction dNTPs are finally added in the form of dNMP and from each molecule two pyrophosphates are liberated. The polymerization reaction also indicates chain growth from a 5′ to 3′ direction. Elongation reaction on the lagging strand is of special notable event in the process of replication. It has already been pointed out that over the lagging strand due to discontinuous polymerization of nucleotides, synthesis on this strand occurs fragmentarily.
Due to polymerization in such a fashion, short segments of nucleotides are synthesized over this DNA strand and these are called Okazaki fragments. For the formation of each Okazaki fragment, a short RNA primer is formed at the initial point. However, a question naturally comes why only the lagging strand synthesis occurs in a fragmentary fashion? The answer to this question lies on the point that during replication direction of polymerization is specific i.e., from 5′ end to 3′ end. It is also to be pointed out that the same DNA Pol. III (in dimeric form) carries out polymerization on both the leading and lagging strands.
During this exercise, while polymerization is initiated from the ori C region, the direction of polymerization is to be maintained from 5′ to 3′ direction. The points for incorporation of nucleotides on both the strands are to differ by location and they are not just opposite to each other. This problem is solved by a unique fashion by the lagging strand which forms a loop surrounding the DNA Pol. III. Formation of such a loop by the lagging strand can easily maintain the direction of nucleotide polymerization when the same enzyme exhibits a forward movement along the leading strand carrying out easy and continuous polymerization of nucleotides there along 5′ to 3′ direction.
Okazaki fragments formed in E. coli are of short dimension containing 1000 – 2000 nucleotides. However, after the synthesis of the short segments, the primers are removed by the exonuclease activity of the DNA Pol. I. The gaps generated due to this may be filled up by the activity of the same enzyme and the short segments are then ligated by the enzyme ligase. Ligase activity at this stage depends upon the hydrolysis of ATP. Thus finally over the lagging strand also a continuous newly synthesized polynucleotide strand is formed.
In E. coli cells, DNA is circular, and when at the ori C region two strands are separated, and at the two ends of a replication bubble, replication forks are formed. As replication proceeds, the size or dimension of the replication bubble increases, and at an intermediate stage the circular DNA takes the shape of the letter 0. Sometimes this type of replication is considered a 0 mode of replication.
3. Termination Phase:
In the last stage of elongation, the replication forks form both sides of ori C, and reach a point opposite to ori C. This marks the termination of replication. At this stage, the Tus (Termiones Utilization Substance) protein binds with the termination points and this protein destroys the activity of DNA B or helicase. Because of this further unwinding of DNA becomes impossible and the polymerization reaction is ceased. At the junction, two newly formed circular DNA molecules remain in catenated condition. The enzyme topoisomerase IV separates two newly formed DNA molecules from each other.
Rate of Replication
In E. coli cell replication fork may proceed at a rate of about 1000 nucleotides per second. The DNA of this organism contains about 4.4 × 106 base pairs and complete replication of it may take about 42 minutes.
Rolling Circle Replication
In circular DNA sometimes this type of replication may be noticed. During conjugation in bacteria and some viral circular DNA, this mode of replication occurs. As per this model, a nick is developed in one of two strands in the circular DNA at the initiation phase prior to replication by the action of some endonuclease. Then some specific replication protein binds with the 5‘ end of the nicked strand and the intact circular strand shows rotation like a swivel. As a result, a portion of nicked strand comes in a linear orientation. Then from its 3‘ end still paired with the circular strand polymerization of nucleotides may be started over the circular strand making it the template.
As the circular strand rotates more and more, so the 5′ end of the nicked strand goes away from the nicked region. With this new strand formation and elongation further progress with the formation and elongation further progress with the formation of a completely new polynucleotide strand over the mother circular strand. Along with this the separated linear DNA strand also carries out nucleotide polymerization over it and be comes ligated to produce another circular DNA molecule. Thus with the rolling circle mode, the mother DNA molecule may produce two daughter DNA molecules.
φ × 174 is the virus containing a single-stranded circular DNA molecule. Replication of the DNA of this virus occurs by rolling circle mode. But prior to replication, this DNA forms a complementary strand over it, so that a double-stranded circular DNA may be formed. This double-stranded circular DNA is called RF or Replicative Form. In this replicative form of DNA, one strand is of the virus particle which is known as the + strand, and the other is newly formed called the – strand.
During replication, a nick is developed on the + strand, and then to the 5′ end of this nicked strand, a protein called gene A binds. The intact circular – strand then rotates like a swivel and the 5′ end of the nicked strand goes apart forming a linear strand. D from the 3‘ end of the nicked strand polymerization of nucleotides continues over the circular – strand. In this case due to rotation of the circular – strand when the + strand becomes detached completely from the – strand, the 5′ & 3′ ends are ligated to form a circular + strand DNA as the virus particle. Further nick formation on the newly formed + strand over the – strand and its rotation eventually may form additionally a + form of circular DNA. With the continuation of the same process for a considerable time period, many copies of the + form of circular DNA may be synthesized within the host cell.
It is to be oriented out in this case that the + strands do not synthesize a – strand in this step. The circular + strand may be enclosed in the protein capsids already formed within the host cell. Thus the formation of progeny phage particles becomes easy for the virus within the host cell by way of the rolling circle mode of DNA replication.
Replication in Eukaryotic Cells
Though replication of DNA follows the same basic principle in all organisms, some differences may be noted during replication in eukaryotes. Because DNA in eukary¬otes is quite large and is intimately associated with chromosomal proteins and the process of its replication is a bit complicated. On such large DNA molecules, replication may be started from many sites of origin at a time. Hence, in eukaryotic DNA there are multiple replication origins. In yeast cell 400 such replication origins could be found. Besides replication is facilitated by at least five DNA polymerases as found in mammalian cells.
The knowledge so far achieved is from the studies on virus SV 40 (Simian Virus 40). This DNA virus infects the animal cells and utilizes all machinery for multiplication as well as DNA replication. The virus (SV40) at the inception of replication synthesizes a protein called T-antigen which binds with the replication origin and causes the DNA strand separation. After this, the separated strands individually bind with a protein factor called RFA that acts like the SSB protein of E. coli. On the separated strands then the DNA polymerase acts. Over the lagging strand DNA Pol. α and over the leading strand DNA Pol. δ polymerizes nucleotides. The polymerase in combination with primase carries out the replication of the lagging strand. On the other hand, the DNA Pol.δ in association with a protein factor called PCNA polymerizes over the leading strand.
In certain cases, functions of DNA Pol. δ may be carried out by DNA Pol. ε Besides these polymerases DNA Pol. β and DNA Pol. γ are also found in eukaryotic cells. DNA Pol. β promotes repair synthesis and DNA Pol. γ is involved in mitochondrial DNA replication. The involvement of another protein factor called RFC is also found in eukaryotic replication. Okazaki fragments in eukaryotic cells are smaller in contrast to those in prokaryotes and each fragment may measure from 100 – 200 nucleotides. The rate of polymerization of nucleotides in the replication of the eukaryotes is also slower in comparison to that in prokaryotes. In humans, the replication fork shows a progression at a rate of about 100 nucleotides per second. In consideration of this, the total DNA present in a cell may carry out duplication in about 9 hours.
Difference between Prokaryotic and Eukaryotic Replication:
|Basis of Difference||Prokaryotic Replication||Eukaryotic Replication|
|DNA involved||Replication occurs in single circular DNA.||Replication occurs in usually more than one linear DNA.|
|Replication Origin No.||There is only one replication origin.||There are more than one (many) replication origins.|
|Replication Bubble||During replication, only one replication bubble is formed.||During replication, many replication bubbles are formed.|
|DNA Polymerase||Three DNA polymerases are involved – DNA pol. I, DNA pol. II and DNA pol. III.||DNA pol. α, DNA pol. β, DNA pol. δ and DNA pol. γ are involved in the replication.|
|Okazaki fragment||Okazaki fragments are 1000-30000 nucleotides long.||Okazaki fragments are 100-300 nucleotides long.|
Difference between Leading Strand and Lagging Strand:
|Basis of difference||Leading Strand||Lagging Strand|
|Mode of chain growth||Chain elongation occurs continuously.||Chain elongation occurs discontinuously with the formation of Okazaki fragments.|
|Number of RNA primer||The chain grows from a single RNA primer.||Chain growth needs many RNA primers.|
|DNA ligase||Does not require DNA ligase.||Requires DNA ligase for joining.|
|Growth speed||Growth is rapid.||Growth slower.|