In this article we will discuss about DNA Replication:- 1. Subject-Matter of DNA Replication 2. Modes of DNA Replication 3. Mechanism.

Meaning of DNA Replication:

One of the most important properties of DNA functioning as the genetic material is that it can make exact copies of itself (autocatalytic function) forming the basis for transmission of hereditary characters it controls. This process is called replication.

Replication of a DNA molecule gives rise to two identical daughter molecules, fulfilling the criterion of autocatalytic function. Measurements of DNA content during the divisional cycle of cells shows that DNA replication takes place at a specific portion of interphase—the ‘S’ phase.

Every time a cell divides—just prior to cell division, the DNA of the cell must duplicate so that each of the two newly forming cells receives exactly the same complement of DNA and therefore the same set of genes—both quan­titatively and qualitatively—as was contained in the parent.

Replication of a DNA leads to the duplication of the entire chromosome once for every cell division cycle. As a result each chromosome exists as a pair of chromatids joined together by a centromere and they are separated equally during anaphase into newly forming daughter cells. Hence both chemical nature and the total amount of DNA in similar kind of cells remain constant every generation.

The mechanism of DNA replication is im­plicit in the Watson-Crick model of DNA. The two chains of a DNA are united by hydrogen bonds. When the hydrogen bonds break the two chains part and unwind. It starts from one end of DNA to the other end of DNA.

One by one each purine base separates from its partner pyrimidine base in each base pair, but the sugar-phosphate backbones do not break. It looks much like a zipper opening up. Each parental chain of the DNA then serves as template or mould for the synthesis of its complementary new chain on itself and forms two daughter identical DNA double helices (Fig. 20.1).

General View of DNA Replication

Modes of DNA Replication:

There are three possible modes of DNA replica­tion:

(i) Semiconservative;

(ii) Conservative; and

(iii) Dispersive.

i. Semiconservative:

The semiconser­vative mode of DNA replication was suggested (1953) by Watson and Crick along with the double-helix model of DNA. Here the replication of DNA involves the progressive separation of the two strands of DNA molecules by breaking up of hydrogen bonds between base pairs.

Each strand, acting as template, synthesizes their complementary new strand on itself taking raw materials from the nuclear sap. Thus two daughter DNA helices are formed. Each daughter DNA helix has one old or parental and one new strand.

It indicates that in each daughter DNA helix, one parental strand is retained and conserved while its complementary strand is new. Hence, according to this mode of DNA replication, the parental DNA is partially conserved in each new daughter DNA molecule. So this mode of DNA replication is called semiconservative replication [Fig. 20.2(a)].

Three Possible Modes of DNA Replication

Experimental Evidences for Semiconservative Replication:

Although three possible modes of DNA replica­tion were proposed, the semiconservative mode of DNA replication has been widely accepted. For this acceptance, experimental proof or evi­dence is needed to establish that DNA, in fact, duplicates in that manner.

At the same time it is necessary to rule out several other possibil­ities. Several experimental evidences has been presented to explain the mode of DNA repli­cation but the results of all experiments have proved conclusively that DNA replication is semiconservative.

The experimental evidences are:

(a) Meselson-Stahl Experiment;

(b) Cairns’ autoradiography experiment, and

(c) Taylor’s Experiment.

a. Meselson-Stahl Experiment:

The experimental evidence for semiconservative replication of DNA was first demonstrated by Mathew Meselson and Franklin Stahl in 1958. They grew cells of the bacterium Escherichia coli on a medium that contained 15N (a heavy isotope of 14N) in the form of ammonium chlo­ride (NH4C1).

The cells of E. coli were grown for 14 cell generation so that the cells used the 15 N to synthesise bases which were then incorporated into DNA. DNA having 15N has a detectable higher density (1.724 gm/cm2) than that having 14N (1.710 gm/cm2).

Therefore, they are called heavy and light DNA, respec­tively. Heavy and light DNA can be separated readily through equilibrium density gradient centrifugation where they form distinct band in the centrifuge tube (Fig. 20.3).

Experiment of Meselson and stahi demonstrating semiconservative replication of DNA

The cells of E. coli were then sub-cultured and grown to a medium containing normal 14 N. The transfer of cell to medium containing normal 14 N was followed by extraction of DNA from cells after zero, one, two, three etc. cell generations (one cell generation represents the time during which all the cells undergo one cell division).

The extracted DNAs in each cell generation were analysed in cesium chloride (CsCl) density gradient.

DNA that containing 15N in both strand (heavy DNA) forms a band in the CsCl density gradient at a higher density position than that contains 14N in both strand (light DNA). After one generation of growth in 14N medium, the DNA bands at an intermediate (hybrid DNA) density.

Such hybrid DNA contains 15N in one strand and 14N in the other strand. After two generations of growth in 14N medium, half of the DNA bands are seen at the hybrid density and half band at light density.

It may be pointed out that according to the conservative mode of replication, no interme­diate band will appear after one generation. Only heavy and light bands would be formed. Similarly, the dispersive mode of replication would make only one band having identical density. No light or intermediate bands would be formed.

Therefore, the result of Meselson and Stahl’s experiment clearly explains the semiconserva­tive mode of DNA replication, while the expec­tation of other mode of replication (conserva­tive, dispersive) are not fulfilled.

b. Cairns’ Autoradiography of Replicating Bacterial Chromosome:

Semiconservative mode of replication of bac­terial DNA or chromosome was also demon­strated by J. Cairns in 1963 using the tech­nique called autoradiography for detecting and localizing the incorporated radioactive isotopes present in the preparation of bacterial DNA by exposure to a photographic emulsion. Using autoradiography replication of DNA can easily be followed.

For the experiment, Cairns grew E. coli in medium containing tritiated de-oxy-ribonucleoside of thymidine (3H thymidine). Thymidine is found exclusively into DNA. It is not present in any other major component of the cell.

So it is obvious that when the bacterial DNA will replicate before cell division, it will absorb 3H thymidine from the medium to the cytoplasm of cell and, finally, incorporate into newly synthesizing DNA. After regular intervals Cairns collected the E. coli cells, lysed them very gently so as not to break the DNA and collected the intact DNA on membrane filters.

These filters were affixed to glass slides and then the slides were then covered by photographic emulsion or film which is very sensitive to β particles (the low-energy electrons emitted during decay of tritiated thymidine). The slides were stored in dark for sufficient long period.

During this storage the particles emitted by tritiated thymidine exposed the film. The films were then developed to see the autoradiographs of E. coli DNA or chromosome. This autoradiograph showed the regions of the presence of tritiated thymidine and thus indirectly the presence of labelled DNA.

In the diagram (Fig. 20.5) loops A and B have completed a second replication in 3H thymidine and loop C remains to be replicated the second time. Cairn drawing [Fig. 20.5(a)] shows radioactive strands of DNA as solid lines and non radioactive strands as dashed lines.

It is also noted that in the autoradiograph, loop B is with two radioactive strands and exhibits about twice the grain density of loop A with only one radioactive strand. X and Y indicates the replication forks, the branching point at which DNA synthesis occurs.

Cairns’ autoradiogram of circular chromo­some grown in medium containing 3H thymine show the presence of replication eyes or bub­bles. These so-called ϴ structures indicate that duplex DNA replicates by the progressive separation of its two parental strands accom­panied by the synthesis of their complementary strands to yield two semi-conservatively duplex daughter strands.

DNA replication involving ϴ structures is known as ϴ -replication. The separation of the two complementary strands of a DNA molecule would require rotation of the double helix by 360° for each turn of helix. This necessitates the existence of some kind of swivel in the chromosome.

The available evidence suggests that a cleavage or break of one phosphodiester bond in one strand of the double helix provides an axis of rotation to allow the unwinding. Cairns interpreted that semiconservative replication started at a site on the chromosome or DNA which he called the replication origin and proceeded in one direction till the entire DNA of E. coli is replicated.

But it is known at present that DNA replication actually proceeds bi-directionally, not in one direction. DNA replication begins in both directions from replication origin point and both the Y-shaped structures are replication forks the two replication forks move in opposite directions—sequentially around the circular DNA.

 

Equilibrium density gradient centrifugation

c. Taylor’s Experiment:

Semiconservative mode of replication of chro­mosome of higher plant was also demonstrated by J.H. Taylor etal in the root tip cells of Vicia faba using autoradiography technique. They observed that when tritiated thymidine treated root tips of Vicia faba were transferred to triti­ated thymidine-free medium, in the first gener­ation of duplication, both chromatids were la­belled.

In the second generation of duplication, in each chromosome, one of the two chromatids was found to be labelled while the other chro­matid was un-labelled. In the third generation of duplication, 50% chromosomes were made of one labelled and one un-labelled chromatids.

Taylor’s experiment is very important because it demonstrated the semiconservative mode of replication in chromosomes of a higher plant.

Distinction between undirectional and bidirectional DNA replication

ii. Conservative:

According to this mechanism, the replication of DNA may be conservative which means that the parental double helix is totally conserved and remains intact and somehow directs the synthesis of a daughter double helix made of two newly synthesised strand [Fig. 20.2(b)],

iii. Dispersive:

In dispersive replication, the old or parental DNA molecule breaks up into several pieces. Each piece then replicates. The old and new segments recombine randomly to yield daughter DNA molecules having both old and new segments along their entire length [Fig. 20.2(c)],

Mechanism of DNA Replication:

DNA replication is a multistep complex pro­cess. It is catalysed by the multi-enzyme complex, often called the replication apparatus, or the replisome, and needs the involvement of several other proteins. DNA replication always begins at certain unique and fixed points of DNA called origin.

Each prokaryotic chromo­some has a single origin but every eukaryotic chromosome has several origins (e.g., the giant salivary chromosome of Drosophila contains 7,000 origins), phage T2 has one primary and one secondary origin. In presence of primary origin, the secondary origin remains nonfunc­tional. But when the primary origin is deleted, then the secondary origin takes over as the functional origin.

Studies with various organisms show that the replication of DNA molecules in both prokaryotes and eukaryotes is bidirectional. However, bidirectional replication is not universal. The chromosome of coli-phage P2 which, like the lambda chromosome, is circular during replication, replicates unidirectional from a unique origin.

The double-stranded DNA does not repli­cate. Hence, before replication, two strands of DNA must be separated gradually from the point of replication origin in the form of replication eye or Y-shaped replication fork.

During separation, DNA synthesis also occurs usually bi-directionally or rarely unidirectional. DNA is replicated by enzymes known as DNA directed DNA polymerase or simply DNA polymerases. This enzyme utilizes free single- stranded DNA as a template on which the synthesis of daughter complementary strand takes place.

To fulfil the requirement of DNA polymerase activity, two enzymes and DNA helicase are required. DNA gyrase reduces the linking of DNA strands and DNA helicase first binds to the origin points and induces the unwinding of complementary strands of DNA double helix to make it single-stranded.

Certain proteins—called single-strand binding proteins (SSBPs)—bind tightly to single-stranded re­gion of DNA and help stabilise the extended single-stranded templates needed for the activ­ity of DNA polymerase. In a DNA duplex, two parental antiparallel strands run in opposite directions (one is 5′ → 3′ and other is 3’→ 5′)- But the two parent strands are replicated in different ways.

Synthesis of daughter DNA always proceeds in the 5’→3′ direction on both the parental strands. All known DNA polymerases of both prokaryotic and eukaryotic have an absolute requirement of a free 3′-OH group of preex­isting polynucleotide for the initiation of DNA replication, a primer (RNA or DNa but only RNA is used in vivo) and the four deoxynucleoside triphosphates (dATP, dTTP, dGTP and dCTP) which are the building blocks of DNA.

In case of 3’→ 5′ template strand of parental DNA, free 3′-OH group is available directly for the activity of DNA polymerase. Hence the daughter DNA is continuously synthesised in its 5′ →3′ direction as the replication fork advances. The replication of other parental template strand (5′ → 3′) of the DNA molecule is discontinuous. The replication of this (5′ → 3′) strand begins somewhat later than that of the 3′ → 5′ strand.

Consequently, a segment of the 5′ →3′ strand (with reference to origin) of a DNA molecule always replicates later than the homologous segment of the 5′ → 3′ strand. Therefore, the 3’→ 5′ strand of a DNA molecule is known as leading strand while the 5′ → 3′ strand is termed the lagging strand.

The leading strand is synthesised continuously and the lagging strand is syn­thesised discontinuously—this is called semi- discontinuous replication.

In the lagging strand (5’→ 3′), free 3′-OH group is not readily available for the activity of DNA polymerase. So the lagging strand transcribes a 10-60 nucleotide long-RNA primer (in 5′ →the origin (in the 3′ -> 5′ direction of the lagging strand).

The 3′-OH of this primer RNA provides the initial point for DNA polymerase to catalyse the replication of lagging strand. Obviously, the replication of lagging strand proceeds from the replication fork towards the origin, i.e., its direction is opposite to that of the leading strand (from origin towards the fork). However, the new daughter strand is synthesised from the 5′ → 3′ in the case of both leading strands.

Synthesis of the RNA primers is catalysed by a special class of enzymes called primases. Primase activity needs the formation of a complex of primase and at least six other proteins—the complex is called the primo some. In addition to primase the primo some contains pre-priming proteins which are tentatively designated as protein i, n, n’ and n” and the products of genes dnaB and dnaC.

The primo-some carries out the initial priming reaction for the lead­ing strand and the repeated priming of the synthesis of Okazaki fragments for the lagging strand. Okazaki fragments (Fig. 20.7) is named after R. Okazaki who first identified them.

The replication of lagging strand is discontinuous in that it has to be initiated several times and every time one Okazaki fragment is produced. Okazaki fragments are about 1,000-2,000 nu­cleotides long DNA fragments in E. coli while they are only 100-200 nucleotides long DNA fragments in eukaryotes.

Replication of DNA of E.Coli

In prokaryotes, synthesis of daughter DNA on leading strand and synthesis of Okazaki fragments on lagging strand are catalysed by DNA polymerase III—a complex enzyme con­taining seven different polypeptides—α, β, y, δ, ɛ, r, Q.

In lagging strand, Okazaki fragments are associated with RNA primers which are digested by DNA polymerase I in prokaryotes. This enzyme also catalyses the filling of the gaps so produced in the new strand.

The Okazaki fragments (after the gap filling by DNA polymerase I) are joined together by the DNA ligase which catalyses the formation of phosphodiester bonds between the adjacent Okazaki fragments. Thus, a complete daugh­ter DNA strand on lagging parental strand is formed.

From above description it is noted that DNA replication progresses in one direction from the origin and the same events also occur in the opposite direction of the origin. It is also pointed out that the replication of leading strand (with reference to origin) of a DNA molecule is continuous while that of lagging strand is discontinuous.

This concept is ap­plicable for prokaryote and eukaryote organism and is also tenable to both replication forks generated at each origin.

 

DNA Replication

 

Enzymes and proteins involved in DNA replication:

DNA replication is a complex process. It re­quires a great variety of enzymes and proteins.

Major enzymes and proteins involved in DNA replication are:

i. Enzymes known as helicases that separate DNA strands at replication fork;

ii. Topoisomerases;

iii. Proteins that prevent them from reannealing before they are replicated;

iv. Enzymes that synthesise RNA primers;

v. A DNA polymerase;

vi. An enzyme to remove RNA primers, and

vii. An enzyme to covalently link successive Okazaki fragments.

DNA Helicases (DNA-dependent ATPases):

DNA helicases have the property to unwind a DNA duplex. For this process the enzyme needs the energy obtained from the hydrolysis of ATP. Two molecules of ATP are hydrolysed per nucleotide unwound.

This is a high price to pay for unwinding DNA as it involves the release of 60 kJ of energy to break hydrogen bonds. DNA helicases are also required to initiate transcription. In E. coli, seven helicases have been partially characterised—helicase I, helicase II, helicase III and so on.

Topoisomerases:

All synthetic activities (transcription, replica­tion, etc.) involving double-stranded DNA require the strands to separate. It means that double-stranded DNA is to be converted into two single-stranded structures at the site of synthetic activity.

However, two strands of DNA do not simply lie side by side, they are inter-wind. Their separation, therefore, requires the strands to rotate about each other in space. This necessitates the existence of some kind of swivel in the DNA.

Present evidence also suggests that a transient single-strand cut (breakage of one phosphodiester bond in one strand of the double helix) provides an axis rotation to allow unwinding. DNA actually behaves as a closed structure lacking free ends.

Hence—after a transient single-strand cut and using the cut free end—one strand can be rotated about the other, after which the break is made good. Such a reaction converts one topological isomer into another.

Topological isomers are molecules of DNA that are identical except for a difference in linking number, the number of times one strand cross-over the other in space. DNA topoisomerases catalyse conversion of this type. Some topoisomerases can remove only negative supercoils from DNA; others can remove both negative and positive supercoils.

There are two types of topoisomerase:

Type I enzyme acts by making a transient break in one strand of DNA.

Type II enzymes act by introducing a transient double-strand break.

The best characterised Type I topoisomerase is the product of the top A gene of E. coli which relaxes highly negatively supercoiled DNA. This enzyme does not act on positively supercoiled DNA.

In prokaryotes, topoisomerase I bind to DNA and it forms a stable complex in which one strand of the DNA has been nicked and its 5′ phosphate is covalently linked to a tyrosine residue in the enzyme. The eukaryotic enzyme (topoisomerase) produces a 5’OH group (which can be phosphorylated to polynucleotide kinase) and a 3′ phosphate which is linked to a tyrosine in the enzyme.

Type I topoisomerase first bind to a region in which duplex DNA becomes separated into its single-strands; then it breaks one strand and pulls the other strand through the break and finally seals the break.

Topoisomerase Type II generally remove both negative and positive supercoils of DNA. The reaction needs ATP. One ATP is probably hydrolysed for use to drive the enzyme through conformational changes that provide the force required to push one DNA duplex through the break made in the other duplex.

The reaction of topoisomerase II probably represents a nonspecific recognition of DNA duplexes which are brought into apposition.

The enzyme makes double-stranded break in one duplex and unbroken duplex is passed through end of break. Finally, the break is sealed and the enzyme release DNA. In this way topoisomerase II relax both positive and negative supercoils.

There are at least two types of topoisomerase II. The eukaryotic enzymes and the enzyme from bacteriophage T4 are capable of relaxing supercoiled DNA. That is they catalyse the conversion of supercoiled DNA into relaxed, cyclic duplex.

Bacterial type II topoisomerases are known as DNA gyrases and they are additionally ca­pable of introducing negative super-helical turns into covalently closed, relaxed, cyclic duplex DNA molecules.

DNA gyrases which catalyse the formation of negative supercoils in DNA are essential for replication and are believed to play a key role in the unwinding process. A third type of topoisomerase II has been reported in archaebacteria. This reverse gyrase is able to convert a relaxed cyclic DNA duplex into a positively supercoiled molecule.