Chapter 15 - DNA and the Gene: Synthesis and Repair
Chapter 15 - DNA and the Gene: Synthesis and Repair BIOL 2311
Popular in Biology 2311
Popular in Biology
verified elite notetaker
This 11 page Class Notes was uploaded by Ming-Han Lu on Saturday July 23, 2016. The Class Notes belongs to BIOL 2311 at University of Texas at Dallas taught by Dr. Mehmet Candas in Summer 2016. Since its upload, it has received 21 views. For similar materials see Biology 2311 in Biology at University of Texas at Dallas.
Reviews for Chapter 15 - DNA and the Gene: Synthesis and Repair
Report this Material
What is Karma?
Karma is the currency of StudySoup.
Date Created: 07/23/16
Chapter 15 – DNA and the Gene: Synthesis and Repair MingHan Lu Since Mendel’s time, the predominant research strategy in genetics had been to conduct a series of experimental crosses, crate a genetic model to explain the types and proportions of phenotypes that resulted, and then test the model’s predictions through reciprocal crosses, testcrosses, or other techniques. This strategy led to virtually all the discoveries of classical genetics, including Mendel’s rules, sex linkage, linkage, and quantitative inheritance. Scientists knew that genes and chromosomes were replicated during the cell cycle, but no one really knew how the copying really occurred. 15.1 What Are Genes Made of? The chromosome theory of inheritance proposed that chromosomes contain genes. Chromosomes are a complex of DNA and proteins. Many scientists believed that genes were made of protein because hundreds, if not thousands, of complex and highly regulated chemical reactions occur in even the simplest living cells. The amount of information required to specify and coordinate these reactions is mindboggling. With their almost limitless variation in structure and function, proteins are complex enough to contain this much information o Also many thought that DNA was just a simple molecule (shown through incorrect evidence). At this point, many were convinced that proteins did the job instead of DNA, but we’re still not sure yet! The HersheyChase Experiment Alfred Hershey and Martha Chase took up the question of whether genes were made of protein or DNA by studying how a virus called T2 infects and replicates within the bacterium Escherichia coli. o T2 infections begin when the virus attaches to the cell wall of E. Coli and injects its genes to the cell’s interior. These genes then direct the production of a new generation of virus particles inside the infected cell, which acts as a host for the virus. o During the infection, the exterior protein coat, or capsid, of the original, parent virus is left behind. The capsid remains attached to the exterior of the host cell. T2 is made up almost exclusively of protein and DNA… So which one was it? Strategy for determining the composition of the viral substance that enters the cell and acts as the hereditary material was based on two facts: 1. Proteins contain sulfur but not phosphorus. 2. DNA contain phosphorus but not sulfur. Biologists found that virtually all the radioactive protein was outside cells in the emptied capsids, while virtually all the radioactive DNA was inside the host cells. o DNA must be the hereditary material; DNA contained all the information for life’s complexity! Chapter 15 – DNA and the Gene: Synthesis and Repair MingHan Lu So no, protein doesn’t make up the gene, but DNA makes up the gene. The Secondary Structure of DNA Watson and Crick proposed a model for the secondary structure of DNA. Each deoxyribonucleotide consists of a deoxyribose sugar, a phosphate group, and a nitrogenous base. o Deoxyribonucleotides link together into a polymer when a phosphodiester bund forms between a hydroxyl group on the 3’ carbon of one deoxyribose and the phosphate group attached toe the 5’ carbon of another deoxyribose. The primary structure of each strand of DNA has two major features: 1. A “backbone” made up of the sugar and phosphate groups of deoxyribonucleotides and 2. A series of bases that project from the backbone. o Each strand of DNA has a directionality, or polarity: One end has an exposed hydroxyl group on the 3’ carbon of a deoxyribose, while the other has an exposed phosphate group on a 5’ carbon. Thus, the molecule has distinctly different 3’ and 5’ ends. Watson and Crick lined up two of these long strands in opposite directions (antiparallel fashion). o They realized that antiparallel strands will twist around each other into a spiral or helix because certain bases fit together snugly in pairs inside the spiral and form HYDROGEN BONDS. The doublestranded molecule that results is called a double helix. Chapter 15 – DNA and the Gene: Synthesis and Repair MingHan Lu The doublehelical DNA is stabilized by HYDROGEN BONDS that form between the bases adenine (A) and thymine (T) and between the bases guanine (G) and cytosine (C), along with the hydrophobic interactions that the bases experience inside the helix. o This hydrogen bonding of particular base pairs is called complementary base pairing. 15.1 Testing Early Hypotheses about DNA Synthesis Watson and Crick suggested that the existing strands of DNA served as a template (pattern) for the production of new strands and that deoxyribonucleotides were added to the new strands according to complementary base pairing. o For example, if the template strand contained a T, then an A would be added to the new strand to pair with that T. Similarly, a G on the template strand would dictate the addition of a C on the new strand. Three Alternative Hypotheseses 1. Semiconservative replication – If the old, parental strands of DNA separated, each could then be used as a template for the synthesis of a new, daughter strand. This hypothesis is called semiconservative replication because each new daughter DNA molecule would consist of one old strand and one new strand. 2. Conservative replication – If the bases temporarily turned outward so that complementary strands no longer faced each other, they could serve as a template for the synthesis of an entirely new double helix all at once. This hypothesis, called conservative replication, would result in an intact parental molecule and a daughter DNA molecule consisting entirely of newly synthesized strands. Chapter 15 – DNA and the Gene: Synthesis and Repair MingHan Lu 3. Dispersive replication – If the parental double helix were cut wherever one strand crossed over another and DNA was synthesized in short sections by extending each of the cut parental strands to the next strand crossover, then there would be a mix of new and old segments along each replicated molecule. This possibility is called dispersive replication – stretches of old DNA would be interspersed with new DNA down the length of each daughter strand. The MeselsonStahl Experiment Like all organisms, bacterial cells copy their entire complement(all) of DNA, or their genome, before every cell division. They introduced different nitrogen isotopes because if different generations of DNA were produced, then the parental and daughter strands would have different densities. RESULTS: Each newly made DNA molecule comprises one old strand and one new strand— replication is semiconservative. 15.3 A Model for DNA Synthesis DNA inside a cell = ancient text that has been copied and handed down, generation after generation. o The DNA in living cells has been copied and passed down for billions of years. DNA is replicated by molecular scribes. They discovered an enzyme (remember that this is a protein!) that does it! DNA polymerase – it polymerizes deoxyribonucelotides into DNA. This protein catalyzes DNA synthesis. DNA polymerases can add deoxyribonucleotides only to the 3’ end of a growing DNA chain. As a result, DNA synthesis always proceeds in the 5’ 3’ direction. For DNA synthesis, the reaction is exergonic (it releases energy) because the monomers that are used in the DNA synthesis reaction are deoxyribonucleoside triphosphate (dNTPs). (The N in dNTP stands for any of the four bases found in DNA: adenine, thymine, guanine, or cytosine). o Because they have three closely spaced phosphate groups, dNTPs have high potential energy—high enough to make the formation of phosphodiester bonds in a growing DNA strand exergonic as two of the phosphates are cleaved off. How Does Replication Get Started? Initially, the replication bubble forms at a specific sequence of bases called the origin of replication. Bacterial chromosomes have only one origin of replication, and thus a single replication bubble forms. Eukaryotes have multiple origins of replication along each chromosome, and thus multiple replication bubbles. o DNA synthesis is bidirectional—that is, it occurs in both directions at the same time. Therefore, replication bubbles grow in two directions as DNA replication proceeds. Chapter 15 – DNA and the Gene: Synthesis and Repair MingHan Lu A specific set of proteins are responsible for recognizing sites where replication begins and opening the double helix at those points. These proteins are activated by the proteins that initiate S phase in the cell cycle. Once a replication bubble opens at the origin of replication, a different set of enzymes takes over to start DNA synthesis. o Active DNA synthesis takes place at the replication forks of each replication bubble. o The replication fork is the Yshaped region where the parentDNA double helix is split into two single strands and copied. How Is the Helix Opened and Stabilized? A large group of enzymes and specialized proteins converge(meet) on the point where the double helix opens. The enzyme called DNA helicase breaks the hydrogen bonds between the base pairs. o This reaction causes the two strands of DNA to separate. Singlestrand DNAbinding proteins (SSBPs) attach to the separated strands and prevent them from snapping back into a double helix. Working together, DNA helicase and singlestrand DNAbinding proteins open up the double helix and maintain the separation of both strands during copying. The “unzipping” process that occurs at the replication fork creates tension farther down the helix. DNA does not become tightly coiled ahead of the replication fork, because the twisting induced by helicase is relaxed by proteins called topoisomerases. A topoisomerase is an enzyme that cuts DNA, allows it to unwind, and rejoins it ahead of the advancing replication fork. o Basically, the tension is relieved by the topoisomerase. How Is the Leading Strand Synthesized? DNA polymerase (1) works only in the 5’ 3’ direction and (2) requires both a 3’ end to extend from and a singlestranded template. The singlestranded template dictates which deoxyribonucleotide should be added next. A primer – a strand a few nucleotides long that is bonded to the template strand— provides DNA polymerase with a free 3’ hydroxyl (OH) group that can combine with an incoming deoxyribonucleotide to form a phosphodiester bond. o Before DNA synthesis can get under way, an enzyme called primase synthesizes a short stretch of RNA that acts as a primer for DNA polymerase. o Primase is a type of RNA polymerase – an enzyme that catalyzes the polymerization of ribonucleotides into RNA. Chapter 15 – DNA and the Gene: Synthesis and Repair MingHan Lu Unlike DNA polymerases, primase and the other RNA polymerases do not require a primer to begin synthesis. Once a primer is present on a singlestranded template, DNA polymerase begins working in the 5’ 3’ direction and adds deoxyribonucleotides to complete the complementary strand. o Deoxyribonucleotide addition is catalyzed at an active site in a groove between the enzyme’s “thumb” and “fingers.” As DNA polymerase moves along the DNA molecule, a doughnutshaped structure behind it, called the sliding lamp, holds the enzyme n place on the the template strand. The enzyme’s product is called the leading strand, or continuous strand, because it leads into the replication fork and is synthesized continuously. How Is the Lagging Strand Synthesized? The other strand must be synthesized in a direction that runs away from the moving replication fork. The strand of DNA that extends in the direction away from the replication fork is called the lagging strand, or discontinuous strand, because it lags behind the synthesis of occurring at the fork. Chapter 15 – DNA and the Gene: Synthesis and Repair MingHan Lu o As the replication fork moves, it exposes gaps of singlestranded template DNA. The Discontinuous Replication Hypothesis Primase synthesizes new RNA primers for lagging strands as the moving replication fork opens singlestranded regions of DNA, and that DNA polymerase uses these primers to synthesize short laggingstrand DNA fragments that are linked together into a continuous strand. The Discovery of Okazaki Fragments Researchers succeeded in finding short DNA fragments when they purified DNA from the experimental cells, separated the two strands of DNA, and analyzed the size of the molecules by centrifugation. o A small number of labeled DNA fragments about 1000 base pairs long were present immediately after the pulse. These short DNAs came to be known as Okazaki fragments. These small DNAs became larger during the chase as they were linked together into longer pieces. Okazaki fragments are connected by DNA polymerase I when it attaches to the 3’ end of an Okazaki fragment. o As DNA polymerase I moves along in the 5’ 3’ direction, it removes that RNA primer ahead of it and replaces the ribonucleotides with the appropriate deoxyribonucleotides. Chapter 15 – DNA and the Gene: Synthesis and Repair MingHan Lu o Once the RNA primer is removed and replaced by DNA, an enzyme called DNA ligase catalyzes the formation of a phosphodiester bond between adjacent fragments. In eukaryotes, the mechanism for primer removal is different, but the mechanism of synthesizing short Okazaki fragments that are later joined into an unbroken chain of DNA is the same. Working together, the enzymes that open the replication fork and manage the synthesis of the leading and lagging strands. All of the enzymes are joined into the replisome, a large macromolecular machine. After the DNA polymerase on the lagging strand completes synthesis of an Okazaki fragment, it is released from the DNA and reassembles on the most recently synthesized primer. 15.4 Replicating the Ends of Linear Chromosomes Replication of chromosome ends requires a specialized DNA replication enzyme that has been the subject of intense research. The End Replication Problem The region at the end of a eukaryotic chromosome is called a telomere. Now there’s a problem! When the replication fork reaches the end of a linear chromosome, a eukaryotic DNA polymerase synthesizes the leading strand all the way to the end of the parent DNA template. As a result, leadingstrand synthesis results in a doublestranded copy of the DNA molecule. On the lagging strand, primase adds an RNA primer close to the tip of the chromosome. DNA polymerase synthesizes the final Okazaki fragment on the lagging strand. An enzyme that degrades ribonucleotides removes the primer. DNA polymerase is unable to add DNA near the tip of the chromosome, because it cannot synthesize DNA without a primer. As a result, the singlestranded DNA that is left stays single stranded. The singlestranded DNA at the end of the lagging strand is eventually degraded, which results in the shortening of the chromosome. If this process were to continue unabated, every chromosome would shorten by about 50 to 100 deoxyribonucleotides each time DNA replication occurred. Over time, linear chromosomes would vanish. Telomerase Solves the End Replication Problem 1. Telomeres do not contain genes but are made of short stretches of bases that are repeated over and over. 2. A remarkable enzyme called telomerase that carries its own template is involved in replicating telomeres. Chapter 15 – DNA and the Gene: Synthesis and Repair MingHan Lu Telomerase catalyzes the synthesis of DNA from an RNA template that it contains. Telomerase adds DNA onto the end of a chromosome to prevent it from getting shorter. How does telomerase work to maintain the ends of eukaryotic chromosomes? Step 1 The unreplicated segment of the telomere at the 3’ end of the template for the lagging strand forms a singlestranded “overhang”. Step 2 Telomerase binds to the overhanging singlestranded DNA and begins DNA synthesis. The template for this reaction is a portion of the RNA held within telomerase. Step 3 Telomerase synthesizes DNA in the 5’ 3’ direction and catalyzes repeated additions of the same short DNA sequence to the end of the growing single strand. Step 4 Once the singlestranded overhang on the parent strand is lengthened, the normal enzymes of DNA synthesis use this strand as a template to synthesize a complementary strand. The result is that the lagging strand becomes slightly longer than it was originally. Telomerase Regulation Telomerase is active in only a limited number of cell types. In humans, active telomerase is found primarily in the cells that produce gametes. Most somatic cells, meaning cells that are not involved in gamete formation, lack telomerase activity. Chromosomes of somatic cells gradually shorten with each mitotic division, becoming progressively smaller as an individual ages. Telomere shortening has a role in limiting the number of cell divisions of somatic cells. Most cancer cells have active telomerase, which allows the unlimited divisions of cancer cells. 15.5 Repairing Mistakes and DNA Damage Level of accuracy is critical. Genes that contain errors are often defective. o Natural selection favors individuals with enzymes that copy DNA quickly and accurately. Correcting Mistakes in DNA Synthesis As DNA polymerase marches along a DNA template, hydrogen bonding occurs between incoming deoxyribonucleotides and the deoxyribonucleotides on the template strand. DNA polymerases are selective about the bases they add to a growing strand because (1) the correct base pairings (AT and GC) are energetically the most favorable, and (2) these correct pairings have a distinct shape. DNA Polymerase Proofreads Chapter 15 – DNA and the Gene: Synthesis and Repair MingHan Lu If a newly added deoxyribonucelotide is not correctly paired with a base on the complementary strand, the positioning of the incorrect deoxyribonucleotide provides a poor substrate for the DNA polymerase to extend. o DNA polymerase’s active site can detect these shapes and will add a new deoxyribonucelotide only when the previous base pair is correct. o DNA polymerase III can proofread. If the wrong base is added during DNA synthesis, the enzyme pauses, removes the mismatched deoxyribonucelotide that was added, and then proceeds again with synthesis. Mismatch Repair If the problem isn’t fixed by proofreading, the DNA polymerase leaves a mismatched base behind in the newly synthesized strand, a battery of enzymes springs into action to correct the problem. Mismatch repair occurs when mismatched bases are corrected after DNA synthesis is complete. o The proteins recognize the mismatched base, remove a section containing the incorrect base from the newly synthesized strand, and fill in the correct bases using the older strand as a template. This final layer of error detection and correction brings the overall error rate of DNA synthesis down to roughly one mistake per billion deoxyribonucleotides. Mutations in components of the mismatch repair system are observed in many common human cancers, where they plan an important role in cancer development and progression. Repairing Damaged DNA To fix problems caused by chemical attack, radiation, or other events, organisms have evolved a wide array of DNA damagerepair systems. o Nucleotide excision repair system that works on DNA damage caused by ultraviolet light and many different chemicals. UV can cause a covalent bond to form between adjacent pyrimidine bases within the same DNA strand thymine dimer (creates a kink in the structure of DNA, which blocks DNA replication causing cell death). Nucleotide excision repair fixes thymine dimers and many other types of damage that distort the DNA helix. STEP 1 – An enzyme recognizes the kink in the DNA helix. STEP 2 – Once a damaged region is recognized, another enzyme removes a segment of singlestranded DNA containing the defective sequence. STEP 3 – The intact DNA strand provides a template for synthesis of a corrected strand, and the 3’ hydroxyl of the DNA strand next to the gap serves as a primer. STEP 4 – DNA ligase links the newly synthesized DNA to the original undamaged DNA. Xeroderma Pigmentosum: A Case Study Chapter 15 – DNA and the Gene: Synthesis and Repair MingHan Lu Xeroderma pigmentosum (XP) is a rare autosomal recessive disease in humans. Sensitivity to UV light. James Cleaver proposed that they are extremely sensitive to sunlight because they are unable to repair damage induced by UV light. XP cells died off much more rapidly than normal skin cells. They were also unable to repair like normal patients. Hypothesis: if repair is defective in XP individuals, then their cells should incorporate little radioactive deoxyribonucleotide into their DNA. o Cells from unaffected individuals, in contrast, should incorporate large amounts of labeled deoxyribonucleotide into their DNA as it is repaired. That’s exactly what happened. XP patients: mutations in any of eight genes. Defects in DNA repair genes are frequently associated with cancer. If the overall mutation rate in a cell is elevated because of defects in DNA repair, then mutations that trigger cancer become more likely.