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Unit 17: lesson 3.
- DNA replication and RNA transcription and translation
- Leading and lagging strands in DNA replication
- Speed and precision of DNA replication
- Molecular structure of DNA
- Molecular mechanism of DNA replication
Mode of DNA replication: Meselson-Stahl experiment
- DNA proofreading and repair
- Telomeres and telomerase
- There were three models for how organisms might replicate their DNA: semi-conservative, conservative, and dispersive.
- The semi-conservative model, in which each strand of DNA serves as a template to make a new, complementary strand, seemed most likely based on DNA's structure.
- The models were tested by Meselson and Stahl, who labeled the DNA of bacteria across generations using isotopes of nitrogen.
- From the patterns of DNA labeling they saw, Meselson and Stahl confirmed that DNA is replicated semi-conservatively.
Mode of DNA replication
The three models for dna replication.
- Conservative. Replication produces one helix made entirely of old DNA and one helix made entirely of new DNA.
- Semi-conservative. Replication produces two helices that contain one old and one new DNA strand.
- Dispersive. Replication produces two helices in which the individual strands are patchworks of old and new DNA.
- Semi-conservative replication. In this model, the two strands of DNA unwind from each other, and each acts as a template for synthesis of a new, complementary strand. This results in two DNA molecules with one original strand and one new strand.
- Conservative replication. In this model, DNA replication results in one molecule that consists of both original DNA strands (identical to the original DNA molecule) and another molecule that consists of two new strands (with exactly the same sequences as the original molecule).
- Dispersive replication. In the dispersive model, DNA replication results in two DNA molecules that are mixtures, or “hybrids,” of parental and daughter DNA. In this model, each individual strand is a patchwork of original and new DNA.
Meselson and Stahl cracked the puzzle
The meselson-stahl experiment, results of the experiment, generation 0, generation 1, generation 2.
- Generation 0 (see above). 100% of DNA in nitrogen-15 band.
- Generation 1. 100% of DNA in a band intermediate in position between nitrogen-14 and nitrogen-15 bands.
- Generation 2. 50% of DNA in a band intermediate in position between nitrogen-14 and nitrogen-15 bands. 50% of DNA in nitrogen-14 band.
- Generation 3. 25% of DNA in a band intermediate in position between nitrogen-14 and nitrogen-15 bands. 75% of DNA in nitrogen-14 band.
- Generation 4. 12% of DNA in a band intermediate in position between nitrogen-14 and nitrogen-15 bands. 88% of DNA in nitrogen-14 band.
Generations 3 and 4
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DNA Replication Steps and Process
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Why Replicate DNA?
DNA is the genetic material that defines every cell. Before a cell duplicates and is divided into new daughter cells through either mitosis or meiosis , biomolecules and organelles must be copied to be distributed among the cells. DNA, found within the nucleus , must be replicated in order to ensure that each new cell receives the correct number of chromosomes . The process of DNA duplication is called DNA replication . Replication follows several steps that involve multiple proteins called replication enzymes and RNA . In eukaryotic cells, such as animal cells and plant cells , DNA replication occurs in the S phase of interphase during the cell cycle . The process of DNA replication is vital for cell growth, repair, and reproduction in organisms.
- Deoxyribonucleic acid, commonly known as DNA, is a nucleic acid that has three main components: a deoxyribose sugar, a phosphate, and a nitrogenous base.
- Since DNA contains the genetic material for an organism, it is important that it be copied when a cell divides into daughter cells. The process that copies DNA is called replication.
- Replication involves the production of identical helices of DNA from one double-stranded molecule of DNA.
- Enzymes are vital to DNA replication since they catalyze very important steps in the process.
- The overall DNA replication process is extremely important for both cell growth and reproduction in organisms. It is also vital in the cell repair process.
DNA or deoxyribonucleic acid is a type of molecule known as a nucleic acid . It consists of a 5-carbon deoxyribose sugar, a phosphate, and a nitrogenous base. Double-stranded DNA consists of two spiral nucleic acid chains that are twisted into a double helix shape. This twisting allows DNA to be more compact. In order to fit within the nucleus, DNA is packed into tightly coiled structures called chromatin . Chromatin condenses to form chromosomes during cell division. Prior to DNA replication, the chromatin loosens giving cell replication machinery access to the DNA strands.
Preparation for Replication
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Step 1: Replication Fork Formation
Before DNA can be replicated, the double stranded molecule must be “unzipped” into two single strands. DNA has four bases called adenine (A) , thymine (T) , cytosine (C) and guanine (G) that form pairs between the two strands. Adenine only pairs with thymine and cytosine only binds with guanine. In order to unwind DNA, these interactions between base pairs must be broken. This is performed by an enzyme known as DNA helicase . DNA helicase disrupts the hydrogen bonding between base pairs to separate the strands into a Y shape known as the replication fork . This area will be the template for replication to begin.
DNA is directional in both strands, signified by a 5' and 3' end. This notation signifies which side group is attached the DNA backbone. The 5' end has a phosphate (P) group attached, while the 3' end has a hydroxyl (OH) group attached. This directionality is important for replication as it only progresses in the 5' to 3' direction. However, the replication fork is bi-directional; one strand is oriented in the 3' to 5' direction (leading strand) while the other is oriented 5' to 3' (lagging strand) . The two sides are therefore replicated with two different processes to accommodate the directional difference.
Step 2: primer binding.
The leading strand is the simplest to replicate. Once the DNA strands have been separated, a short piece of RNA called a primer binds to the 3' end of the strand. The primer always binds as the starting point for replication. Primers are generated by the enzyme DNA primase .
DNA Replication: Elongation
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Step 3: Elongation
Enzymes known as DNA polymerases are responsible creating the new strand by a process called elongation. There are five different known types of DNA polymerases in bacteria and human cells . In bacteria such as E. coli, polymerase III is the main replication enzyme, while polymerase I, II, IV and V are responsible for error checking and repair. DNA polymerase III binds to the strand at the site of the primer and begins adding new base pairs complementary to the strand during replication. In eukaryotic cells, polymerases alpha, delta, and epsilon are the primary polymerases involved in DNA replication. Because replication proceeds in the 5' to 3' direction on the leading strand, the newly formed strand is continuous.
The lagging strand begins replication by binding with multiple primers. Each primer is only several bases apart. DNA polymerase then adds pieces of DNA, called Okazaki fragments , to the strand between primers. This process of replication is discontinuous as the newly created fragments are disjointed.
Step 4: Termination
Once both the continuous and discontinuous strands are formed, an enzyme called exonuclease removes all RNA primers from the original strands. These primers are then replaced with appropriate bases. Another exonuclease “proofreads” the newly formed DNA to check, remove and replace any errors. Another enzyme called DNA ligase joins Okazaki fragments together forming a single unified strand. The ends of the linear DNA present a problem as DNA polymerase can only add nucleotides in the 5′ to 3′ direction. The ends of the parent strands consist of repeated DNA sequences called telomeres. Telomeres act as protective caps at the end of chromosomes to prevent nearby chromosomes from fusing. A special type of DNA polymerase enzyme called telomerase catalyzes the synthesis of telomere sequences at the ends of the DNA. Once completed, the parent strand and its complementary DNA strand coils into the familiar double helix shape. In the end, replication produces two DNA molecules , each with one strand from the parent molecule and one new strand.
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DNA replication would not occur without enzymes that catalyze various steps in the process. Enzymes that participate in the eukaryotic DNA replication process include:
- DNA helicase - unwinds and separates double stranded DNA as it moves along the DNA. It forms the replication fork by breaking hydrogen bonds between nucleotide pairs in DNA.
- DNA primase - a type of RNA polymerase that generates RNA primers. Primers are short RNA molecules that act as templates for the starting point of DNA replication.
- DNA polymerases - synthesize new DNA molecules by adding nucleotides to leading and lagging DNA strands.
- Topoisomerase or DNA Gyrase - unwinds and rewinds DNA strands to prevent the DNA from becoming tangled or supercoiled.
- Exonucleases - group of enzymes that remove nucleotide bases from the end of a DNA chain.
- DNA ligase - joins DNA fragments together by forming phosphodiester bonds between nucleotides.
DNA Replication Summary
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DNA replication is the production of identical DNA helices from a single double-stranded DNA molecule. Each molecule consists of a strand from the original molecule and a newly formed strand. Prior to replication, the DNA uncoils and strands separate. A replication fork is formed which serves as a template for replication. Primers bind to the DNA and DNA polymerases add new nucleotide sequences in the 5′ to 3′ direction.
This addition is continuous in the leading strand and fragmented in the lagging strand. Once elongation of the DNA strands is complete, the strands are checked for errors, repairs are made, and telomere sequences are added to the ends of the DNA.
- Reece, Jane B., and Neil A. Campbell. Campbell Biology . Benjamin Cummings, 2011.
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Essay on DNA Replication | Genetics
In this essay we will discuss about:- 1. Definition of DNA Replication 2. Mechanism of DNA Replication 3. Evidences for Semi-Conservative DNA Replication 4. Models for Replication of Prokaryotic DNA.
Essay # Definition of DNA Replication :
DNA replicates by “unzipping” along the two strands, breaking the hydrogen bonds which link the pairs of nucleotides. Each half then serves as a template for nucleotides available in the cells which are joined together by DNA polymerase. The nucleotides are guanine, cytosine, adenine and thymine. DNA replication or DNA synthesis is the process of copying a double-stranded DNA molecule.
This process is important in all known forms of life and the general mechanisms of DNA replication are the same in prokaryotic and eukaryotic organisms. The process by which a DNA molecule makes its identical copies is referred to as DNA replication. In other words, it is the process of duplicating the DNA to make two identical copies. The main points related to DNA replication are briefly presented below.
1. Time of Replication:
The process of DNA replication takes place during cell division. The DNA replication takes place during S sub stage of interphase. In prokaryotes, DNA replication is initiated before the end of the cell cycle. Eukaryotic cells can only initiate DNA replication at the beginning of S phase.
2. Replication Site:
In humans and other eukaryotes, replication occurs in the cell nucleus, whereas in prokaryotes it occurs in the cytoplasm. Prokaryotes have only one active replication site, but eukaryotes have many.
3. Template Used:
The existing DNA is used as a template for the synthesis of new DNA strands. It is possible that during replication on strand of DNA can replicate continuously and the other discontinuously or in piece. The continuously replicating strand is known as leading strand and the discontinuously replicating strand is known as lagging strand.
When one strand of DNA replicates continuously and other discontinuously, it is called semi-discontinuous replication. Earlier it was thought that DNA replicates discontinuously. But now it is believed that DNA replication is semi-discontinuous.
Short segments of nucleotides are synthesized in the lagging strand of DNA as a result of discontinuous replication. These are called Okazaki after the name of discoverer. Okazaki fragments are about 1,500 bases in length in prokaryotes, and 150 bases in eukaryotes
4. Enzymes Involved:
The process of DNA replication takes place under the control of DNA polymerase. In other words, the process is catalized by the polymerase enzyme. In eukaryotes, four types of polymerase enzymes, viz. alpha, delta, gamma and epsilon are used.
DNA Polymerase alpha and delta replicate the DNA. The alpha is associated with initiation, and delta extends the nascent strands. DNA polymerase epsilon and beta are used for repair. DNA polymerase gamma is used for replication of mitochondrial DNA
In prokaryotes [E. coli], there are three major DNA polymerases: DNA polymerase I, II and III. DNA poly I is found in the highest concentration of all DNA polymerases; it is involved in DNA repair and assists with primary DNA replication. DNA poly II is exclusively involved in repair. DNA poly III is the major DNA polymerase. All DNA polymerases add to the 3′ OH of the existing polynucleotide.
Currently, six families of polymerases (A, B, C. D, X, Y) have been discovered. At least four different types of DNA polymerases are involved in the replication of DNA in animal cells (POLA, POLG, POLD and POLE).
5. Direction of Replication:
The synthesis of one new strand takes place in 5-3 and that of other in opposite (3-5) direction. The replication may take place either in one direction or in both the directions from the point of origin. When replication proceeds in one direction only, it is called unidirectional replication. When the replication proceeds in both the directions, it is called bidirectional replication.
6. Replication Type:
Based on the direction, the replication may be unidirectional or bidirectional. On the basis of continuity, the replication may be continuous or discontinuous.
7. Origin of Replication:
The point of initiation of DNA replication is known as origin. The progress of replication process is measured from the point of origin.
8. Rate of Replication:
In prokaryotic cells the rate of replication is 500 bases per second. In eukaryotic cells the rate of replication- is 50 bases per second. Eukaryotes have 100 to 3,000 times more DNA than prokaryotes.
9. Replication Models:
There are three models which explain the accurate replication of DNA. These are: (i) dispersive replication, (ii) conservative replication, and (iii) semiconservative replication (Fig. 17.1).
These are explained as follows:
(i) Dispersive Replication:
According to this model of replication the two strands of parental DNA break at several points resulting in several pieces of DNA. Each piece replicates and pieces are reunited randomly, resulting in formation of two copies of DNA from single copy. The new DNA molecules are hybrids which have new and DNA in patches (Fig. 17.2). This method of DNA replication is not accepted as it could not be proved experimentally.
(ii) Conservative Replication:
According to this model of DNA replication two DNA molecules are formed from parental DNA. One copy has both parental strands and the other contains both newly synthesized strands (Fig. 17.2). This method is also not accepted as there is no experimental proof in support of this model.
(iii) Semiconservative Replication:
This model of DNA replication was proposed by Watson and Crick. According to this model of DNA replication, both strands of parental DNA separate from each other. Each old strand synthesizes a new strand. Thus each of the two resulting DNA molecules has one parental and one new strand (Fig. 17.3). This model of DNA replication is universally accepted because there are several evidences in support of this mode.
Essay # Mechanism of DNA Replication :
The semi-conservative model (mechanism) of DNA replication consists of six important steps, viz:
(2) Binding of RNA primase,
(4) Removal of primers,
(5) Termination, and
(6) DNA repair.
These are briefly discussed as follows:
The first major step in the process of DNA, replication is the breaking of hydrogen bonds between bases of the two anti-parallel strands. The unwinding of the two strands is the starting point. The splitting happens in places of the chains which are rich in A-T.
That is because there are only two bonds between Adenine and Thymine, whereas there are three hydrogen bonds between Cytosine and Guanine. The Helicase enzyme splits the two strands. The initiation point where the splitting starts is called “origin of replication”. The structure that is created is known as “Replication Fork”.
2. Binding of RNA Primase:
Synthesis of RNA primer is essential for initiation of DNA replication. RNA primer is synthesized by DNA template near the origin with the help of RNA Primase. RNA Primase can attract RNA nucleotides which bind to the DNA nucleotides of the 3′-5′ strand due to the hydrogen bonds between the bases. RNA nucleotides are the primers (starters) for the binding of DNA nucleotides.
The elongation proceeds in both directions, viz. 5′-3′ and 3′-5′ template. The 3′-5′ proceeding daughter strand that uses a 5′-3′ template— is called leading strand because DNA Polymerase ‘a’ can “read” the template and continuously add nucleotides. The 3′-5′ template cannot be “read” by DNA Polymerase a. The replication of this template is complicated and the new strand is called lagging strand.
In the lagging strand the RNA Primase adds more RNA Primers. DNA polymerase a reads the template and lengthens the bubbles. The gap between two RNA primers is called “Okazaki” Fragments. The RNA Primers are necessary for DNA Polymerase a to bind Nucleotides to the 3′ end of them. The daughter strand is elongated with the binding of more DNA nucleotides.
4. Removal of Primers:
The RNA Primers are removed or degraded by DNA polymerase I. This enzyme also catalyzes the synthesis of short DNA segments to replace the primers. The gaps are filled with the action of DNA Polymerase which adds complementary nucleotides to the gaps.
The DNA Ligase enzyme adds phosphate in the remaining gaps of the phosphate-sugar backbone. Each new double helix is consisted of one old and one new chain. This is called semi-conservative replication.
The termination takes place when the DNA Polymerase reaches to an end of the strands. In other words, it is the separation of replicated linear DNA. After removal of the RNA primer, it is not possible for the DNA Polymerase to seal the gap because there is no primer.
Hence, the end of the parental strand where the last primer binds is not replicated. These ends of linear (chromosomal) DNA consist of noncoding DNA that contains repeat sequences and are called telomeres. A part of the telomere is removed in every cycle of DNA Replication.
6. DNA Repair:
The DNA replication is not completed without DNA repair. The possible errors caused during the DNA replication are repaired by DNA repair mechanism. Enzymes like nucleases remove the wrong nucleotides and the DNA Polymerase fills the gaps. Similar processes also happen during the steps of DNA Replication of prokaryotes though there are some differences.
Essay # Evidences for Semi-Conservative DNA Replication :
Various experiments have demonstrated the semi-conservative mode of DNA replication. Now it is universally accepted that DNA replicates in a semi-conservative manner. There are three important experiments which support that DNA replication is semi-conservative.
These experiments include:
(1) Meselson and Stahl experiment,
(2) Cairns experiment, and
(3) Taylor’s experiment.
1. Meselson and Stahl Experiment  :
Meselson and Stahl conducted their experiment with common bacteria of human intestine i.e. Escherichia coli.
They used heavy isotope of nitrogen for labelling DNA. The bacteria were grown on culture medium containing heavy isotope of Nitrogen [N15] for 14 generations (30 minutes per generation) to replace the normal nitrogen [N14] of E. coli with heavy nitrogen.
Then the bacteria were transferred to normal nitrogen medium. The density of DNA was determined after one, two and three generations. Principle Involved. It is possible to detect minute differences in density through density gradient centrifugation. District bands are formed in centrifuge tube for different density DNA.
2. Rolling Circle Model of DNA Replication :
This model of circular DNA replication was proposed in 1968. This model explains mechanism of DNA replication in single stranded circular DNA of viruses, e.g. ɸX174, and the transfer of E. coli sex factor (plasmid). The ϕX174 chromosome consists of a single stranded DNA ring (Positive Strand). This model is most widely accepted.
The mechanism of replication consists of following important steps:
(i) Synthesis of New Strand:
First the chromosome becomes double stranded by synthesis of a negative strand. The original strand is positive. The negative strand is synthesized in side of parental positive strand.
(ii) Cut in Outer Strand:
The negative or inner strand remains a close circle and the positive strand is nicked at a specific site by endonuclease enzyme. This enzyme recognizes a particular sequence at this point. Thus a. linear strand with 3′- and 5′-ends is created.
(iii) Formation of Tail:
The original positive strand comes out in the form of a tail of a single linear strand as a consequence of rolling circle. The 5′-end of the broken strand becomes attached to the plasma membrane of the host bacterium.
Such replicating phage DNA is commonly found associated with bacterial membranes. The unbroken parental strand rolls and unwinds as synthesis proceeds, leaving a ‘tail’ which is attached to the membrane.
(iv) Synthesis of New Strand:
The synthesis of new strand takes place along the parental strand at the tail end in a 3-5 direction. The 3′-end serves as a primer for the synthesis of a new DNA strand under the catalytic action of DNA polymerase. The unbroken strand is used as the template for this purpose, and a complementary strand is synthesized. Thus the parental molecule itself is used as a primer for initiating replication.
New DNA is also synthesized in the tail region in discontinuous segments in the 5-3 direction. This synthesis is presumably preceded by the synthesis of an RNA primer under the catalytic action of RNA polymerase. The tail is cut-off by a specific endonuclease into a unit length progeny rod.
(v) Cutting of Tail:
Now the tail is cut-off into a linear segment by endonuclease. The linear segment becomes circular by joining two ends with the help of ligage enzyme. Thus a new circular molecule is formed which can become new rolling circle and replicate further.
Genetic information is preserved in the single stranded template ring which remains circular and serves as an endless template. There is no swiveling problem or creation of torque in the rolling circle model. As the strands unwind the 3′-end is free to rotate on the unbroken strand. The growing point itself thus serves as a swivel.
Evidence for the rolling circle model has been obtained from the replication of several viruses (M13, P2, T4, λ), replication resulting in transfer of genetic material during mating of bacteria, and the special DNA synthesis during oogenesis in Xenopus.
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DNA replication is semiconservative. Each strand in the double helix acts as a template for synthesis of a new, complementary strand. New DNA is made by enzymes called DNA polymerases, which require a template and a primer (starter) and synthesize DNA in the 5' to 3' direction.
Because eukaryotic genomes are quite complex, DNA replication is a very complicated process that involves several enzymes and other proteins. It occurs in three main stages: initiation, elongation, and termination. Initiation Eukaryotic DNA is bound to proteins known as histones to form structures called nucleosomes.
Dispersive replication. In the dispersive model, DNA replication results in two DNA molecules that are mixtures, or “hybrids,” of parental and daughter DNA. In this model, each individual strand is a patchwork of original and new DNA. Most biologists at the time would likely have put their money on the semi-conservative model.
Initiator proteins bind at replication origins and recruit DNA replication machinery proteins • DNA polymerase is responsible for catalyzing synthesis of new strands Replication forks form and involve a leading and a lagging strand • DNA is directional; two strands are antiparallel • DNA polymerase can only synthesize from 5’ to 3 ...
The process of DNA duplication is called DNA replication. Replication follows several steps that involve multiple proteins called replication enzymes and RNA. In eukaryotic cells, such as animal cells and plant cells, DNA replication occurs in the S phase of interphase during the cell cycle.
3. RNA nucleotides bind with complementary base sequences under the direction of the enzyme RNA primase. These RNA nucleotides act as a primer for DNA nucleotides.
PDF | On Sep 18, 2018, Tariku Simion published DNA Replication | Find, read and cite all the research you need on ResearchGate
California State University, Northridge
DNA replication or DNA synthesis is the process of copying a double-stranded DNA molecule. This process is important in all known forms of life and the general mechanisms of DNA replication are the same in prokaryotic and eukaryotic organisms. The process by which a DNA molecule makes its identical copies is referred to as DNA replication.