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I'm currently learning about DNA replication in both prokaryotic and eukaryotic cells. And my lecturer has mentioned that replication is a once in a lifetime activity. And I'm not sure what this is implying because I've searched up that DNA replication occurs during cell division (cell cycles), which occur repetitively as organisms develop.
This sounds like a difference in perspective of when exactly cells "die". If you consider that, in a cell division, a mother cell "gives birth" to two daughter cells, you could argue that the mother cell has "died". It makes sense to think about cell division in this way because it puts both daughter cells in the same level, without one being "special" due to it being the mother cell itself while the other is considered the "new cell".
If you face cell division like this, any cell (eukaryote or procaryote) will only really duplicate their DNA once: they duplicate their DNA -> they split into daughter cells, in which they "die" -> (eventually) each daughter cell duplicate their DNA -> and so on.
Like I said this is just a particular POV we can adpot when we try to understand and describe cell cycle and cell division (although it is a common one for researchers in the field).
DNA III: The Replication of DNA
The discovery that DNA is the material that forms our genes (see our DNA I: The Genetic Material module) opened the door to the modern field of molecular biology, sometimes called molecular genetics, in which scientists examine how DNA encodes all of the great complexities of living things. One of the first major advances of the new field of molecular biology was the deciphering of the DNA molecule's structure - the double helix (see our DNA II: The Structure of DNA module).
Part of the motivation behind scientists' extensive efforts to discover the structure of DNA was the long-held scientific principle that "structure begets function." In other words, what a cell or molecule does, and how it does it, is determined by its shape and structure. This makes sense even in our everyday experience. Consider a hammer or a screwdriver. These important tools can do what they do because of their unique shape. If we changed their shape, they wouldn't work very well. Shape drives function. The same is true for DNA.
The synthesis of DNA
As mentioned in our DNA II module, the moment James Watson and Francis Crick first gazed upon their newly built model of DNA, they could see clues about one of the major properties that they knew DNA must somehow exhibit: self-replication. The mystery of self-replication had confused scientists for many years. But one thing was certain: Every cell, whether a yeast, a bacterium, or a human cell, must be able to copy all of its genes, all of its DNA. This is because when a cell divides in two, both resulting cells are genetically identical to each other and to the original parent cell. The sheer number of times that the DNA in your body has been replicated (and accurately) is astounding.
You began life as a single cell, a zygote, the result of the fusion of a sperm and an egg. Since then, you have developed into an organism with somewhere between 10 and 100 trillion cells (>10,000,000,000,000). And, with certain rare exceptions, every single one of your trillions of cells has the same DNA sequence as the one cell did when you were just a zygote. How does all of this copying of DNA take place?
As mentioned, the structure of the double-stranded DNA molecule gave powerful hints as to how DNA might be accurately copied. Specifically, the complementary base-pairing of DNA follows a strict pattern that allows us to accurately predict what one strand of DNA looks like just by looking at the other, complementary strand. Put another way, if someone took a regular DNA molecule, pulled the two strands apart, and showed us only one strand, we could accurately list the series of nucleotides of the missing strand.
Watson and Crick saw this possibility when they ended their paper saying, "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." This possible copying mechanism is called semi-conservative DNA replication, because if a cell would duplicate its DNA in this manner, the DNA helix would split and half of both of the new double helices would retain DNA from the original strand (Figure 1). While this scheme makes good sense, it was just a logical guess at first. It wasn't until the late 1950s that Matthew Meselson and Franklin Stahl performed the scientific experiment that showed that the replication of DNA was indeed semi-conservative. (See our Meselson and Stahl: Models of DNA Replication.)
Figure 1: Schematic of DNA replication according to the rules of Watson-Crick base-pairing. In this model, the two strands of the original DNA molecule are first pried apart. Then, complementary nucleotides (A with T, G with C, etc.) are added opposite the nucleotides in both of the original strands. The result is two DNA molecules, both identical to the original strand (and thus to each other), and both with one old strand and one new strand.
In the 1950s, Meselson and Stahl, Watson and Crick, and many other scientists explored the properties of DNA using the intestinal bacterium Escherichia coli. Because a few rare strains of E. coli have been found to cause gastrointestinal illness, E. coli is frequently associated with outbreaks of food poisoning. But actually, most strains of E. coli are harmless and our large intestines are filled with this bacterium. E. coli was among the first routinely used "model organisms," a species that is chosen for extensive study in the laboratory because it offers certain practical advantages that make research easier. E. coli, in particular, is among the fastest growing organisms on Earth, with a generation time of under 20 minutes in ideal conditions. Since long before they knew what DNA was, scientists had noticed that the amount of DNA in an E. coli cell (and any other cell for that matter) doubles prior to cell division. The pool of DNA in the cell is then split equally between the two "daughter cells" that result, so that both have the same amount of DNA that the original bacterium had before replication. Because all of this happens in E. coli in about 20 minutes, it was the logical organism for early molecular biologists to select.
Why did molecular biologists choose E. coli for laboratory studies?
In vitro DNA replication
While Meselson and Stahl and others were testing the possible hypothetical models of DNA replication, other scientists set out to understand its molecular mechanism by re-creating it in a test tube. This process is called in vitro reconstitution and is often used in the field of biochemistry as a way of simplifying a complex cellular event so that it happens in isolation and can thus be observed and manipulated at will. The scientists who were first able to reconstitute DNA replication in a test tube were Arthur Kornberg and his wife Sylvy and the research team that they led. They achieved this incredible feat through a painstaking process of successive chemical purification of different proteins and other components from large batches of E. coli bacteria. By separating and purifying individual components, the Kornberg research team made several important discoveries about how DNA replication occurs.
These discoveries all began with the development of a critically important technique – the DNA synthesis assay. An assay is a quantitative laboratory measurement of a certain biological or chemical process, usually in a test tube (in vitro). The DNA synthesis assay is a technique for measuring the synthesis of new DNA molecules. The Kornberg laboratory was the first to develop this assay, and the assay itself is quite simple. First, DNA polymers are easily separated from free nucleotides because DNA is not soluble in solutions that contain trichloroacetic acid (TCA), while free nucleotides are. If a scientist adds TCA to a liquid mixture of DNA and free nucleotides, the DNA will precipitate out, while the nucleotides will remain dissolved in the liquid. The DNA precipitate can then be easily separated from the liquid by centrifugation.
The second important feature of the DNA synthesis assay is its use of radioactively labeled nucleotides. A scientist can add radioactive nucleotides when preparing a DNA synthesis assay, and then later, if DNA synthesis has occurred, some of the radioactive label will be incorporated into the TCA-insoluble DNA. This provides evidence that some of the labeled nucleotides were polymerized into a new DNA molecule. This DNA synthesis assay is very simple to execute and also very quantitative, which means that it gives very reliable and reproducible numerical values that can be used to calculate how much DNA was made and how fast the synthesis took place.
Armed with this assay, the Kornberg laboratory was the first to report the synthesis of DNA outside of a living cell. The popular press of the time announced that Arthur Kornberg had "created life in a test tube."
Of course, this was hardly the case, but the new ability to synthesize DNA in vitro captured the attention of the general population and is recognized as one of the crucial successes paving the way for the emergence of genetic engineering in the 1970s and 80s. Initially, the laboratory synthesis of DNA was extremely slow (much slower than it occurs in a cell), and it occurred only when crude extracts of E. coli were added to the test tubes. Crude extracts contain all the contents of the cells – all proteins, nucleotides, DNA, RNA, lipids, carbohydrates, etc. Nevertheless, the DNA synthesis assay was a good starting point in which Kornberg and others could begin to dissect the process of DNA replication in detail.
The first discovery and arguably the most important occurred in 1955: Kornberg's research team purified the enzyme from the crude extract that is chiefly responsible for the synthesis of DNA – DNA polymerase. When purified DNA polymerase is added to the DNA synthesis assay, the synthesis of DNA occurs hundreds of times more rapidly than when it is not added. However, the in vitro synthesis of DNA still required the addition of small amounts of crude cell extract. This is because DNA polymerase does not make DNA all by itself – there are many other factors required and not all of these were known at the time. The Kornberg lab and others around the world worked to purify other important components from the crude extract, in the hopes that one day they could make DNA using only the necessary factors and no crude extract.
Some of these required components were obvious, while others were unexpected. For example, it was very quickly discovered that nucleotides were required for the synthesis of DNA, which isn't very surprising because it was well known, even in the 1950s, that nucleotides are the building blocks of DNA. However, only nucleotides in the tri-phosphate form could be used as DNA building blocks (Figure 2). Later studies demonstrated why this is so - the breaking of the high-energy terminal phosphate bond of each new nucleotide added to a growing DNA molecule provides the energy for the polymerization reaction.
Figure 2: Only nucleotide tri-phosphates can be used for DNA synthesis. Although nucleotides can exist with one, two, or three phosphates attached to the 5' carbon of the pentose sugar, Kornberg found that only triphosphate nucleotide can be used as building blocks for DNA synthesis. Later work demonstrated that the reason for this requirement is that the breaking of the high-energy covalent bond between the phosphates provides the energy for forming the covalent bonds between adjacent nucleotides of DNA.
Another important point that the Kornberg laboratory noted was that the test tube DNA synthesis reactions required the presence of an intact copy-template DNA in order for DNA polymerase to make more DNA. In other words, even in a test tube, DNA polymerase cannot build "random" DNA molecules through the willy-nilly polymerization of nucleotides. It can only make copies of DNA molecules that already exist. Think of it this way - DNA polymerase is like a copy machine, NOT like a computer with new sentences can be created. A copy machine cannot print anything unless it has a template to work with. So when Kornberg added purified intact DNA molecules to the DNA synthesis assay, once again the speed of DNA polymerase increased dramatically. (Prior to this discovery, DNA synthesis was occurring only because tiny amounts of DNA template were present in the crude extract that is added to the assay mixture.)
DNA polymerase makes it possible to synthesize DNA molecules in a test tube, a key aspect of genetic engineering.
6 Process and Types Involved in Replication of DNA (With Diagram)
It has now been over 30 years since J. D. Watson, F. H. C. Crick, and M. H. F. Wilkins established the double-stranded, helical nature of the DNA molecule and suggested how DNA serves in its own replication.
According to their original model for DNA replication, the two polynucleotide chains of the “parent” double helix separate and each serves as a “template” for the synthesis of a new, complementary polynucleotide chain.
During strand separation, the nitrogen bases of each original strand are exposed and establish sites for the association of free nucleotides.
These nucleotides are then enzymatically linked together to form a new complementary strand. Because deoxyadenylic acid (dAMP) can form hydrogen bonds only with thymine of the template strand (and dGMP can bond only to cytosine, dCMP only to guanine, and dTMP only to adenine), the newly synthesized strand will be identical to the original complementary strand.
As a result, two new double helices are formed, each consisting of one polynucleotide strand from the parent double helix and a newly synthesized polynucleotide strand. Because each of the two double helices conserves only one of the parent polynucleotide strands, the process is said to be semiconservative.
Replication as a “Semiconservative” Process:
Although semiconservative replication of DNA was predicted by the original Watson-Crick model, it was not verified until the classic studies of M. S. Meselson and F. W. Stahl. At the time of their experiments, two other modes of replication were deemed equally feasible (Fig. 21-1):
(1) Conservative replication, in which both strands of the parent double helix would be conserved and the new DNA molecule would consist of two newly synthesized strands and
(2) Dispersive replication, in which replication would involve fragmentation of the parent double helix and the interspersing of pieces of the parent strands with newly synthesized pieces, thereby forming the two new double helices.
Meselson and Stahl verified the semiconservative nature of DNA replication in a series of elegant experiments using isotopically labeled DNA and a form of isopycnic density gradient centrifugation (see Chapter 12). They cultured Escherichia coli cells in a medium in which the nitrogen was 15 N (a “heavy” isotope of nitrogen, but not a radioisotope) instead of the commonly occurring and lighter 14 N.
In time, the purines and pyrimidines of DNA in new cells contained 15 N (where 14 N normally occurs) and thus the DNA molecules were denser. DNA in which the nitrogen atoms are 15 N can be distinguished from DNA containing 14 N because during isopycnic centrifugation, the two different DNAs band at different density positions in the centrifuge tube (Fig. 21-2).
Meselson and Stahl centrifuged DNA isolated from the cells for 2-3 days at very high rotational speeds in centrifuge tubes initially containing a uniform solution of CsCl. During centrifugation, density gradients were automatically formed in the tubes as a result of the equilibrium that was established between the sedimentation of CsCl toward the bottom of the tube and diffusion of the salt toward the top of the tube. This form of centrifugation, called equilibrium isopycnic centrifugation,
Depending on its content of 15 N and 14 N, the DNA bands at a specific position in the density gradient. Because the DNA synthesized by cells grown in 15 N would be denser than 14 N-containing DNA it would band further down the tube (Fig. 21-2).
Cells grown for some time in the presence of 15 N- medium were washed free of the medium and transferred to 14 N-containing medium and allowed to continue to grow for specific lengths of time (i.e., for various numbers of generation times). DNA isolated from cells grown for one generation of time in the 14 N medium had a density intermediate to that of the DNA from cells grown only in 15 N-containing medium (identified as generation 0 in Fig. 21-3) and that of DNA from cells grown only in 14 N-containing medium (the controls of Fig. 21-3).
Such a result immediately ruled out the possibility that DNA replication was conservative, as conservative replication would have yielded two DNA bands in the density gradient for generation 1 (i.e., F,) cells. The single band of intermediate density (identified as “hybrid” DNA in Fig. 21-3) consisted of DNA molecules in which one strand contained 15 N and the other contained 14 N.
When the incubation in 14 N medium was carried out for two generations of time (i.e., generation 2), two DNA bands were formed—one at the same density position as the DNA from cells grown exclusively in 14 N medium (i.e., “light controls”) and one of intermediate density. Subsequent generations produced greater numbers of DNA molecules that banded at the “light” ( 14 N- containing DNA) position in the density gradient. These results are consistent only with the model of semiconservative replication.
Dispersive replication would have produced a single band for each generation and the band would have been found at successively lighter density positions in the gradient. Studies using other prokaryotes as well as eukaryotes indicate that semiconservative replication of DNA is probably the universal mechanism.
Replication by Addition of Nucleotides in the 5′→3′ Direction:
Each nucleotide of a DNA strand is joined to the next nucleotide by a phosphodiester bond that links the 3′ carbon of its deoxyribose to the 5′ carbon of the deoxyribose of the next nucleotide. At one end of the polynucleotide chain, there is a hydroxyl group attached to the 3′ carbon of the last nucleotide, and at the other end there is a phosphate group attached to the 5′ carbon.
The two chains of a double helix have opposite polarities and are said to be antiparallel, that is, each end of the double helix contains the 5′ end of one strand and the 3′ end of the other. During replication, attachment of a nucleotide to a growing strand always takes place at the terminal 3′ position of that strand. In other words, the forming polynucleotide chain “grows” from its 5′ end toward its 3′ end.
Unidirectional and Bidirectional Replication:
Replication starts at a point on the chromosome where the two parental strands begin to separate this point is called the origin. Addition of complementary nucleotides to form two new strands takes place along both parent strand templates starting from that point (Fig. 21-4).
In unidirectional replication, growth proceeds along both strands in the same direction leading from the origin. Along one of the parental template strands, synthesis of the new complementary strand takes place by the continuous addition of nucleotides to the available 3′ end of the forming strand. The growing strand is called the leading strand or continuous strand. The 5′ end of this strand is located at the origin and its 3′ end at the moving replication fork (i.e., the progressing point of separation of the parental strands).
The other polynucleotide strand being formed is called the lagging strand or discontinuous strand. The elongation of this strand takes place by a somewhat modified mechanism. In contrast to the leading strand, the lagging strand has its 3′ position at the origin and its 5′ position at the replication fork. If nucleotides were sequentially added to the end of the lagging strand at the replication fork, then this strand’s growth would proceed in a 3’→5′ direction.
This does not occur. Instead, growth takes place by the synthesis of a number of short polynucleotide chains between the replication fork and the origin. Each short chain is laid down in the direction 5′ to 3′ and these are later linked together and to the 5′ end of the lagging strand.
As a result, the overall direction of growth of the lagging strand is the same as that of the leading strand. The unusual growth pattern that characterizes the synthesis of the lagging strand explains why it is also referred to as the “discontinuous” strand.
In bidirectional replication (Fig. 21-5), two replication forks are formed at the origin and these move away from the origin in both directions as the parental double helix is separated. The synthesis of the complementary strands also occurs in both directions. Behind each fork there is a set of leading and lagging strands. As in the case of unidirectional replication, elongation of the two leading strands is continuous, whereas elongation of the two lagging strands is discontinuous.
It is to be noted that regardless of whether replication is unidirectional or bidirectional, the addition of nucleotides always occurs in the direction from 5′ to 3′, as new nucleotides are added to available 3′ ends of either the continuous strand or the discontinuous strand. Discontinuous synthesis of lagging strands was first demonstrated by R. Okazaki. Okazaki incubated E. coli cells in a medium containing 3 H-thymidine for very short periods of time (a pulse of only 15 seconds) and then examined the distribution of the radioisotope in newly synthesized DNA.
The radioisotope was found in a number of polynucleotides (1000-2000 nucleotides long), now referred to as Okazaki fragments (Figs. 21-4 and 21-5). When pulsed cells were transferred to unlabeled medium for varying lengths of time prior to analysis, the radioactive label was recovered in much longer stretches of DNA. This is because the Okazaki fragments produced during the short tritium pulse had been linked together and connected to the 5′ end of the lagging strand.
In eukaryotic cells, Okazaki fragments are usually smaller (about 100-200 nucleotides long). Bidirectional replication of DNA is the mechanism employed in all eukaryotic and most prokaryotic cells. Unidirectional replication is rare and appears to occur in only a limited number of prokaryotes.
Visualization of Replication in E. coli:
In 1963, J. Cairns developed a procedure employing a combination of microscopy and autoradiography that made it possible to visualize the replication of the chromosome of E. coli. Cairns plaped E. coli cells in a medium containing 3 H-thymidine for various periods of time so that the radioactive thymidine was incorporated into the DNA as the chromosome was replicated in successive generations of cells.
Cells were removed from the medium after various periods of incubation and gently lysed to release the chromosome from the cell (the shear forces created by harsh lysis break the chromosome into small pieces). The chromosomes were then transferred to glass slides and coated with a photographic emulsion sensitive to the low-energy beta particles emitted by the 3 H-thymidine.
After exposing the emulsion to the beta rays, the emulsion was developed and examined by light microscopy. Wherever decay of labeled thymidine had occurred in a chromosome, the emulsion was exposed and created visible grains.
A chromosome not engaged in replication appeared as a circular structure formed from a close succession of exposure spots. Chromosomes “caught in the act” of replication gave rise to what are called theta structures because they have the appearance of the Greek letter theta (i.e., 0) (Fig. 21-6). The theta structures reveal the positions of the replication forks in the circular chromosome.
The Replicon and the Replication Sequence:
The sequence of events that takes place during DNA replication is best understood for prokaryotes and appears to be as follows (Fig. 21-7). Parental strand separation begins at a site called the origin which contains a special nucleotide sequence and directs the association of a number of proteins. ATP-dependent unwinding enzymes (also called helicases) promote separation of the two parental strands and establish replication forks that will progressively move away from the origin (Fig. 21-7a) the helicases separate the parental strand at about 1000 base pairs per second.
Behind the replication fork, the single DNA strands are prevented from rewinding about one another (or forming double-stranded hairpin loops in each single strand) by the actions of a set of proteins called helix-destabilizing proteins or single-strand binding proteins (i.e., “SSBs”) (Fig. 21-7b). The action of a helicase introduces a positive supercoil into the duplex DNA ahead of the replication fork. Enzymes called topoisomerases relax the supercoil by attaching to the transiently supercoiled duplex, nicking one of the strands, and rotating it through the unbroken strand. The nick is then resealed.
Prior to DNA synthesis beginning at the origin, short RNA polynucleotides are formed that are complementary to the DNA template. These stretches of RNA are called primers and are also laid down in the 5′ to 3′ direction. DNA nucleotides are then added one at a time to the free 3′ ends of the RNA primers. Because growth of the lagging strand is discontinuous, several RNA primers and Okazaki fragments are formed. Note that an RNA primer must be formed for each Okazaki fragment to be laid down (Fig. 21-7c). The enzymes required for the synthesis of the RNA primers are a special class of RNA polymerases called RNA primases.
Elongation of the leading strand and synthesis of the Okazaki fragments are catalyzed by an enzyme called DNA polymerase III. The substrates of DNA polymerase III are the deoxynucleoside triphosphates (e.g., dATP, dGTP, dCTP, and dTTp). Addition of a nucleotide to the available 3′ position of the continuously growing leading strand or an Okazaki fragment of the lagging strand involves removal of pyrophosphate to yield a deoxynucleoside monophosphate (e.g., dAMP, dGMP, dCMP, and dTMP).
On completion of the Okazaki fragments, the RNA primers are excised by DNA polymerase I, which then fills the resulting gaps with DNA (Fig. 21-7d). After DNA polymerase I adds the final deoxyribonucleotide in the gap left by the excised primer, the enzyme DNA ligase forms the phosphodiester bond that links the free 3′ end of the primer replacement to the 5′ end of the Okazaki fragment (Fig. 21-7f).
DNA Polymerases and “Processivity”:
In general, three different DNA polymerase enzymes are found in cells. In prokaryotes, these are called DNA polymerase I, DNA polymerase II, and DNA polymerase III. As noted above, DNA polymerase I excises the RNA primers and fills the gaps with DNA, whereas DNA polymerase III adds nucleotides to the growing leading strand and to the 3′ ends of the RNA primers.
The function of DNA polymerase II remains unknown. In eukaryotic cells, the DNA polymerases are DNA polymerase a, DNA polymerase II, and DNA polymerase 7 their functions are compared with the pro- karyotic enzymes in Table 21-1. The rapidity and efficiency with which a DNA polymerase extends a growing chain is referred to as processivity. For example, the processivity of DNA polymerase III acting on the 3′ end of the leading strand is very high because the enzyme remains associated with the growing end of the chain and the template strand.
During unidirectional replication, the replication fork fully circles the chromosome and the resulting DNA molecules are separated. For prokaryotes in which replication is bidirectional, the replication forks proceed around the chromosome until they meet. In eukaryotic chromosomes, where there are many replicating units or replicons, all replicons are linked together before the chromatids can be -separated. The replicon consists of that segment of a chromosome that includes an origin and two termination points (i.e., points where replication ends).
N. K. Sinha and A. Kornberg have suggested that the DNA polymerases, RNA primases, and helicases may be associated with each other to form a multienzyme complex, the replisome that carries out the synthesis of the leading and lagging strands in a coordinated fashion (Fig. 21-8). Such a complex would be highly processive and assure rapid replication of the DNA.
High Fidelity of Replication:
Despite the complexity of the process and the rapidity with which it proceeds, very few errors are made during DNA replication. For example, it is estimated that for every error that occurs, 10 9 base pairs are replicated faithfully. The high fidelity of DNA replication is attributable in part to the special properties of the DNA polymerases.
The DNA polymerases will add a nucleotide to the available 3′-OH end of a growing DNA strand (or RNA primer) only if the prior nucleotide is properly base-paired with the template nucleotide. If a mismatched nucleotide is present, growth of the strand is transiently halted while a segment of the strand containing the error is excised. With a corrected 3′ end reestablished, elongation by the DNA polymerase is resumed.
Much remains to be learned about the enzymes that catalyze the reactions of replication. So far, the major obstacle to such studies has been the difficulty of isolating and purifying the enzymes, either individually or in complexes. This is due in part to the fact that some of them may be associated with membranes. In prokaryotic cells, the replication forks are bound to the plasma membrane.
About two dozen different proteins have been shown to be involved with the replication of DNA in E. coli cells. Several of the E. coli proteins are involved with prepriming reactions, that is, the reactions that occur before the formation of RNA primers.
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DNA repair, any of several mechanisms by which a cell maintains the integrity of its genetic code. DNA repair ensures the survival of a species by enabling parental DNA to be inherited as faithfully as possible by offspring. It also preserves the health of an individual. Mutations in the genetic code can lead to cancer and other genetic diseases.
Successful DNA replication requires that the two purine bases, adenine (A) and guanine (G), pair with their pyrimidine counterparts, thymine (T) and cytosine (C). Different types of damage, however, can prevent correct base pairing, among them spontaneous mutations, replication errors, and chemical modification. Spontaneous mutations occur when DNA bases react with their environment, such as when water hydrolyzes a base and changes its structure, causing it to pair with an incorrect base. Replication errors are minimized when the DNA replication machinery “proofreads” its own synthesis, but sometimes mismatched base pairs escape proofreading. Chemical agents modify bases and interfere with DNA replication. Nitrosamines, which are found in products such as beer and pickled foods, can cause DNA alkylation (the addition of an alkyl group). Oxidizing agents and ionizing radiation create free radicals in the cell that oxidize bases, especially guanine. Ultraviolet (UV) rays can result in the production of damaging free radicals and can fuse adjacent pyrimidines, creating pyrimidine dimers that prevent DNA replication. Ionizing radiation and certain drugs, such as the chemotherapeutic agent bleomycin, can also block replication, by creating double-strand breaks in the DNA. (These agents can also create single-strand breaks, though this form of damage often is easier for cells to overcome.) Base analogs and intercalating agents can cause abnormal insertions and deletions in the sequence.
There are three types of repair mechanisms: direct reversal of the damage, excision repair, and postreplication repair. Direct reversal repair is specific to the damage. For example, in a process called photoreactivation, pyrimidine bases fused by UV light are separated by DNA photolyase (a light-driven enzyme). For direct reversal of alkylation events, a DNA methyltransferase or DNA glycosylase detects and removes the alkyl group. Excision repair can be specific or nonspecific. In base excision repair, DNA glycosylases specifically identify and remove the mismatched base. In nucleotide excision repair, the repair machinery recognizes a wide array of distortions in the double helix caused by mismatched bases in this form of repair, the entire distorted region is excised. Postreplication repair occurs downstream of the lesion, because replication is blocked at the actual site of damage. In order for replication to occur, short segments of DNA called Okazaki fragments are synthesized. The gap left at the damaged site is filled in through recombination repair, which uses the sequence from an undamaged sister chromosome to repair the damaged one, or through error-prone repair, which uses the damaged strand as a sequence template. Error-prone repair tends to be inaccurate and subject to mutation.
Often when DNA is damaged, the cell chooses to replicate over the lesion instead of waiting for repair ( translesion synthesis). Although this may lead to mutations, it is preferable to a complete halt in DNA replication, which leads to cell death. On the other hand, the importance of proper DNA repair is highlighted when repair fails. The oxidation of guanine by free radicals leads to G-T transversion, one of the most common mutations in human cancer.
Hereditary nonpolyposis colorectal cancer results from a mutation in the MSH2 and MLH1 proteins, which repair mismatches during replication. Xeroderma pigmentosum (XP) is another condition that results from failed DNA repair. Patients with XP are highly sensitive to light, exhibit premature skin aging, and are prone to malignant skin tumours because the XP proteins, many of which mediate nucleotide excision repair, can no longer function.
Differences and Similarities between DNA and RNA
Both molecules are nucleic acids made up of nucleotides, supported by a phosphate backbone. They are both major players in the central dogma. RNA is transcribed from the DNA to make proteins. DNA carries all the information needed for DNA replication and transfer new information to new cells.
They are involved in the maintenance, replication, and expression of hereditary information. DNA holds the key to heredity. RNA helps DNA unlock this code and show us what this code is capable of achieving. Together these molecules ensure that the DNA is replicated, the code is translated, expressed and that things go where they should go.
DNA and RNA work hand in hand in biology. It is rare that one can speak of the one without bringing up the other. Simply put, they are connected by the central dogma. The central dogma is the process of DNA transcription and translation for the purpose of protein synthesis which then perform a multitude of tasks in organisms. Different types of proteins guide the gene expression. Therefore, even though the DNA is the same throughout- different things happen at different part of the body. In addition to this, it also tells stem cells what to differentiate to. This is due to strict regulatory mechanisms in place to control gene expression.
Both DNA and RNA have a negative backbone (because of the phosphate group). They both have four nucleotides each, three of which they share (Guanine, Cytosine, and Adenine) with one significant difference, DNA has Thymine while RNA has Uracil. DNA is double-stranded while RNA is single-stranded. Last but not least, DNA is found in the nucleus while RNA resides both in the nucleus and the cytoplasm. DNA is long-lived while RNA is regenerated with each reaction.
They are both central to cell function.
The single chromosome of a bacterium is a loop of double-stranded DNA. The number of bases can vary from one species to another. The well-known bacteria E. coli has 4.7 million base pairs that take about 40 minutes to replicate, implying a speed of over 1,000 bases per second.
Replication starts at a single fixed location and proceeds on each strand in opposite directions. The replication process includes a proofreading step that ensures a mistake rate no higher than one in one billion.
DNA replication - how many times and when does it occur? - Biology
DNA replication is a semi-conservative process that occurs during the S phase of interphase. It is the process by which an organism’s DNA is replicated to produce two identical copies.
It starts at the origin of replication. Prokaryotes have one origin of replication, whereas eukaryotes have multiple. At each origin of replication, there is a replication bubble that contains two replication forks. At each replication fork is the enzyme DNA helicase that unwinds the DNA’s double helix structure. It does this by breaking the hydrogen bonds between nitrogenous bases, separating the two DNA strands. Single strand binding proteins prevent separated strands from reattaching at the replication fork. The two separated strands of DNA are now called template strands.
DNA polymerase III is the enzyme that is used to build a complementary DNA strand using a template strand. It does this by attaching nucleoside triphosphates to the 3’ end of a nucleotide. DNA polymerase III also ensures that the nucleotides being attached have complementary bases to the template strand. However, DNA polymerase III cannot add nucleotides to the template strand. So the RNA primase creates RNA primers: short RNA sequences that are complementary to the DNA template strand. The DNA polymerase III then has available 3’ ends to add nucleotides to. However, DNA polymerase III can only do this in a 5’ to 3’ direction.
DNA is antiparallel that is, each strand goes in the opposite direction (as seen in the diagram below).
Because of this, the two template strands are also in different directions. In the diagram above, the strand on the right would be the leading strand. This means that after an RNA primer is placed at the 3’ end of this strand, DNA polymerase can build its complementary strand continuously in a 5’ to 3’ direction.
The strand on the right of the diagram is the lagging strand. The lagging strand appears to be growing in a 3’ to 5’ direction, but this is not the case. The lagging strand is divided into segments called Okazaki fragments that are being individually built in a 5’ to 3’ direction by DNA polymerase III. Each fragment begins with an RNA primer. As helicase unzips the DNA double helix, more and more primers are added.
Other enzymes are involved in DNA replication as well. DNA polymerase I removes RNA primers and replaces them with DNA nucleotides. DNA ligase forms phosphodiester bonds between the DNA nucleotides that DNA polymerase I adds and the ends of Okazaki fragments. Finally, enzymes proofread the DNA strands to check for mistakes, preventing mutation.
7.1.1 Nucleosomes help to supercoil the DNA.
Nucleosomes also help to regulate gene expression. Some DNA is wrapped around the histones in the nucleosome and is not accessible by the RNA polymerase so it cannot be transcribed
7.1.2 DNA structure suggested a mechanism for DNA replication.
DNA is double stranded and the two strands are joined together via complementary base pairing. Therefore, it stands to reason that during replication the two strands separate and then through complementary base pairing nucleotides join the separated strands. And this would make 2 new, identical strands of DNA.
7.1.3 DNA polymerases can only add nucleotides to the 3’ end of a primer.
DNA polymerase cannot add bonds to the phosphate on the 5' end of DNA, so it creates bonds on the 3' end.
7.1.4 DNA replication is continuous on the leading strand and discontinuous on the lagging strand.
DNA replication occurs on both strand of the DNA, one strand is called the leading strand and the other is called the lagging. The leading strand is the one that moves in a 3' to 5' direction in the same way the helicase does.
7.1.5 DNA replication is carried out by a complex system of enzymes.
DNA polymerase III - Catalyzes the reaction that binds free floating nucleotides to make a new DNA strand
Helicase - Separates the original DNA strands
Gyrase/Topoisomerase - Helps uncoil the DNA helix
DNA Primase - Provides a site called the primase for the DNA polymerase III to begin adding nucleotides.
DNA Polymerase I - Replaces the RNA primer with DNA
Single Strand Binding Proteins - Prevents the DNA from re-annealing
Ligase - Joins the okazaki fragments together
7.1.6 Some regions of DNA do not code for proteins but have other important functions.
DNA that does not code for a protein is referred to as a non-coding sequence. Some non-coding sequences have important functions such as regulating gene expression by promoting and repressing the transcription of genes next to the.
Additionally, many DNA coding sequences are interrupted by non-coding sequences called introns. The introns are removed before translation but they are important in mRNA processing
On the ends of chromosomes are non-coding sequences called telomeres. During DNA replication the end of the molecule cannot be replicated so telomeres protect parts of the DNA from being lost during replication
Lastly, some non-coding sequences code for tRNA molecules instead of a protein.
7.1.7 Rosalind Franklin’s and Maurice Wilkins’ investigation of DNA structure by X-ray diffraction.
In 1950, Marice Wilkins developed a method of producing a way of imaging DNA molecule through X-Ray diffraction. Rosalind Franklin worked in the same lab as Wilkins and developed a high-resolution detector that took very clear images of DNA. Her findings were essential in the discovery of the double helix by Crick and Watson
7.1.8 Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication in preparation of samples for base sequencing.
DNA sequencing is a process by which the order of the nucleotides in DNA can be found. One of the preliminary steps for DNA sequencing is fragmenting the DNA into pieces. To do this ddNA is added (dideoxyribonucleic acid) which do not have a OH molecule on the 3' end of the ribose sugar, this means that when the DNA polymerase reaches the ddNA it cannot continue replicating.
Gel electrophoresis is the process by which the sequence of the DNA is found. The fragmented DNA is put into a gel and an electric current runs through it. DNA is a polar molecule and is affected by the electric current and moves down the gel. Lighter/smaller fragments of the DNA moves further while heavier/longer fragments move very little. The result is a pattern of bands that one can use to figure out the sequence of the DNA. But remember that the sequence of the original strand is complementary the one shown by the banding patterns since the DNA had replicated.
7.1.9 Tandem repeats are used in DNA profiling.
A variable number tandem repeat (VNTR) is a short sequence of nucleotides that can be used to create a profile of a person based on how many times the sequence is repeated. The VNTR is found at the same locus (location on the chromosome) among different people making it relatively simple to find. DNA profiling can be used to solve criminal cases and solve parental disputes among other things
7.1.10 Analysis of results of the Hershey and Chase experiment providing evidence that DNA is the genetic material.
Hershey and Chase conducted an experiment to see whether proteins or DNA were the genetic material of the cell. To do this, they infected an E.coli bacteria with the T2 Phage virus. Viruses consist of only DNA inside a protein coat so there were no other variables in the mix.
DNA contains phosphorus but not sulphur, and proteins can contain sulphur but not phosphorous. This distinction was used in the experiment using isotopes (different forms) of sulphur and phosphorous. They made two strains of the T2 Phage bacteria, one with a heavier phosphorus isotope in the DNA, and one with a heavier sulphur isotope in the proteins. They did this by putting the T2 phage in a solution with everything necessary to replicate and only the heavier version of the sulphur or the heavier version of the phosphorous. After the T2 phage replicated a couple of times it would mostly consist of the heavier isotope
Hershey and Chase caused the T2 phage virus to inject its genetic material into the E. Coli bacteria. And then they put that E. Coli in a test tube and put that test tube in a centrifuge to spin it really fast in a circle. Because of the quick, circular motion the heavier parts of the E. Coli moved to the bottom of the test tube while the lighter isotopes were at the top.
When the sulphur strain injected its genetic material the percentage of heavy isotopes vs light isotopes were negligible. However, when the phosphorous strain of the virus injected its genetic material into the bacteria 65% of the DNA in the E. Coli was of the heavy isotope based on the centrifuge results.
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