Molecular Biology Lecture IV - DNA Replication, Damage, and Repair PDF

Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady DNA Replication, Damage, and Repair 1- The Cell Cycle The ability of organisms to produce more of their own kind is the one characteristic that best distinguishes living things from nonlivi...

Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady DNA Replication, Damage, and Repair 1- The Cell Cycle The ability of organisms to produce more of their own kind is the one characteristic that best distinguishes living things from nonliving matter. A multicellular organism starts out as a single cell that divides into two. Those two cells then divide into four. Cell division continues throughout an organism’s life, for growth or to replace worn-out or damaged cells. Each time a cell divides in this way, it is crucial that the daughter cells be genetically identical to the parent cell. This unique capacity to breed, like all biological functions, has a cellular basis. The continuity of life is based on the reproduction of cells, or cell division. Cell division plays several important roles in life. When a prokaryotic cell divides, it is actually reproducing because the process gives rise to a new organism (another cell). The same is true of any unicellular eukaryote, such as amoeba. As for multicellular eukaryotes, cell division enables each of these organisms to develop from a single cell, the fertilized egg. And cell division continues to function in renewal and repair in fully grown multicellular eukaryotes, replacing cells that die from accidents or normal wear and tear. For example, dividing cells in your bone marrow continuously make new blood cells. The reproduction of a cell, with all of its complexity, cannot occur by a mere pinching in half; a cell is not like a soap bubble that simply enlarges and splits in two. In both prokaryotes and eukaryotes, a crucial function of most cell divisions is the distribution of identical genetic material, DNA, to two daughter cells. (The exception is meiosis, the special type of eukaryotic cell division that can produce sperm and eggs.) What is most remarkable about cell division is the accuracy with which the DNA is passed from one generation of cells to the next. A dividing cell replicates its DNA, distributes the two copies to opposite ends of the cell, and then splits into daughter cells. Cellular Organization of the Genetic Material A cell’s DNA, its genetic information, is called its genome. Although a prokaryotic genome is often a single DNA molecule, eukaryotic genomes usually consist of a number of DNA molecules. The overall length of DNA in a eukaryotic cell is enormous. A typical human cell, for example, has about 2 m of DNA, a length about 250,000 times greater than the cell’s diameter. Before the cell can divide to form genetically identical daughter cells, all of this DNA must be copied, or replicated, and then the two copies must be separated so that each daughter cell ends up with a complete genome. The replication and distribution of so much DNA are manageable because the 39  Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady DNA molecules are packaged into structures called chromosomes (from the Greek chroma, color, and soma, body), so named because they take up certain dyes used in microscopy. Each eukaryotic chromosome consists of one very long, linear DNA molecule associated with many proteins (Seminar Slide 31). The DNA molecule carries several hundred to a few thousand genes. The associated proteins maintain the structure of the chromosome and help control the activity of the genes. Together, the entire complex of DNA and proteins that is the building material of chromosomes is referred to as chromatin. Every eukaryotic species has a characteristic number of chromosomes in each cell’s nucleus. For example, the nuclei of human somatic cells (all body cells except the reproductive cells) each contain 46 chromosomes, made up of two sets of 23, one set inherited from each parent. Reproductive cells, or gametes, such as sperm and eggs, have half as many chromosomes as somatic cells; in our example, human gametes have one set of 23 chromosomes. The number of chromosomes in somatic cells varies widely among species: 18 in cabbage plants, 48 in chimpanzees, 56 in elephants, 90 in hedgehogs, and 148 in one species of alga. We’ll now consider how these chromosomes behave during cell division. I- DNA Replication Hereditary information in DNA directs the development of your biochemical, anatomical, physiological, and, to some extent, behavioral traits. Your resemblance to your parents has its basis in the accurate replication of DNA prior to meiosis and therefore its transmission from your parents’ generation to yours. Replication prior to mitosis ensures faithful transmission of genetic information from a parent cell to two daughter cells. Of all nature’s molecules, nucleic acids are unique in their ability to dictate their own replication from monomers. The relationship between structure and function is evident in the double helix: The specific complementary pairing of nitrogenous bases in DNA has a functional significance. In this lecture, you will learn about the basic principle of DNA replication, damage, mutation, and repair, the copying of DNA, as well as some important details of the process. 1- Semiconservative DNA Replication Requires a Template, Nucleotides, and Enzymes The biochemistry and molecular biology of DNA replication are similar in prokaryotes and eukaryotes, even though there are more protein components in eukaryote replication machines. When growing rapidly, bacteria replicate their DNA continually, and they can begin a new round before the previous one is complete. In contrast, DNA replication in most eukaryotic cells occurs 40  Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady only during a specific part of the cell division cycle, called the DNA synthesis phase or S phase (Seminar Slide 33). In a mammalian cell, the S phase typically lasts for about 8 hours. By its end, each chromosome has been replicated to produce two complete copies, which remain joined together at their centromeres until the M phase (M for mitosis), which soon follows. During replication one double-stranded DNA molecule produces two identical copies. Each strand of the original double-stranded DNA molecule serves as a template for the production of the complementary strand, a process referred to as semiconservative replication. DNA replication requires three things: something to copy (the parental DNA molecules serve as a template), something to do the copying (enzymes copy the template), and building blocks to assemble into the copy (nucleoside triphosphates). The process of replication can be thought of as having a beginning where the process starts (initiation); a middle where the majority of building blocks are added (elongation); and an end (termination) where the process is finished. A number of enzymes work together to accomplish the task of assembling a new strand, but the enzyme that actually matches the existing DNA bases with complementary nucleotides and then links the nucleotides together to make the new strand is DNA polymerase (Seminar Slide 34). 2- DNA Replication Begins at an Origin of Replication The replication of a DNA molecule begins at special sites called origins of replication, short stretches of DNA having a specific sequence of nucleotides. The E. coli chromosome, like many other bacterial chromosomes, is circular and has a single origin, whereas eukaryotic DNA has many such sites on each chromosome. Proteins that initiate DNA replication recognize this sequence and attach to the DNA, separating the two strands and opening up a replication "bubble". In a sequence of replication origin, A:T pairs dominate, which are easier to separate than a C:G pairs, because they are connected only by two hydrogen bonds. Unwinding of DNA at the origin and synthesis of new strands leads to the formation of a replication fork. Replication proceeds on both DNA strands simultaneously, until the entire molecule is copied (Seminar Slide 35A). In contrast to a bacterial chromosome, a eukaryotic chromosome may have hundreds or even a few thousand replication origins. Multiple replication bubbles form and eventually fuse, thus speeding up the copying of the very long DNA molecules (Seminar Slide 35B). As in bacteria, eukaryotic DNA replication proceeds in both directions from each origin. Replication of the entire human genome takes approximately 8 hours, whereas E. coli needs 42 minutes. 41  Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady At each end of a replication bubble is a replication fork, a Y-shaped region where the parental strands of DNA are being unwound. Several kinds of proteins participate in the unwinding (Figure 11). Helicases are enzymes that untwist the double helix at the replication forks, separating the two parental strands and making them available as template strands. After parental strand separation, single-strand binding proteins bind to the unpaired DNA strands, stabilizing them and preventing the duplex restoration. During DNA replication, the untwisting of the double helix causes tighter twisting (excessive twisting) and strain ahead of the replication fork. Because the two strands of DNA already coil around each other, the formation of additional coils due to twisting is referred to as supercoiling. So, the DNA ahead of the replication bubble becomes positively supercoiled, while DNA behind the replication fork becomes entangled forming negative supercoil (Seminar Slide 36). To understand why, imagine what happens if you start to pull apart the twisted strands of a rope, the intact section rotates in response. If the end of the rope is fixed in place, the rope coils on itself as the untwisting continues from the other end. The same thing happens to DNA. Without a solution to the DNA twisting problem, replication would soon halt. However, this problem is solved by a specialized enzymes called topoisomerases. These enzymes prevent the DNA double helix ahead of the replication fork from getting too tightly wound as the DNA is opened up. The enzymes break one DNA strand, then they swivel (rotate) the free ends of the broken strand around the unbroken strand and ligate (rejoin) the broken ends, allowing the replication machinery to proceed ahead. Without topoisomerases, DNA cannot replicate correctly. Indeed, inhibitors of topoisomerases are used as anticancer drugs since they inhibit division of cancer cells. Unfortunately, these drugs inhibit the division of all cells in the body, including normal cells. Thus, for example hair loss is a common side effect of therapy (since hair are growing due to a permanent division of cells in hair follicles). 3- Synthesizing a New DNA Strand The unwound sections of parental DNA strands are now available to serve as templates for the synthesis of new complementary DNA strands. DNA polymerases are enzymes that carry out all forms of DNA replication. DNA polymerase synthesizes a new strand of DNA by extending the 3' end of an existing nucleotide chain, adding new nucleotides complementary to the template strand. In E. coli there are three types of DNA polymerases: I, II and III, but two appear to play the major roles in DNA replication: DNA polymerase III and DNA polymerase I. The situation in eukaryotes is more complicated, with at least 11 different DNA polymerases discovered so 42  Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady far; however, the general principles are the same. The majority of DNA in prokaryotic cells is synthesized by DNA polymerase III (pol III). The DNA polymerase monitors the ability of the incoming nucleotide to form an A:T or G:C base pair, rather than detecting the exact nucleotide that enters the active site (Seminar Slide 37). Only when a correct base pair is formed are the 3'- OH of the primer and the α-phosphate of the incoming nucleoside triphosphate in the optimum position for catalysis to occur. Incorrect base pairing leads to dramatically lower rates of nucleotide addition as a result of unfavorable alignment of these substrates (Seminar Slide 38). In fact, the rate of incorporation of an incorrect nucleotide is as much as 10,000-fold slower than when base pairing is correct. The enzyme can add about 1,000 nucleotides per second and it is extremely precise; it has 3' → 5' exonuclease activity to check continuously the correctness of the replication. Recall nucleases that can only degrade from a DNA end are called exonucleases; nucleases that can cut within a DNA strand are called endonucleases. Thus, the enzyme proofread (check) each nucleotide against its template as soon as it is added to the growing strand. If it detects a wrong (incorrectly paired) nucleotide has been added, it will use its exonuclease activity to remove that nucleotide right away, after which it incorporates the right nucleotide and continues resuming DNA synthesis (Seminar Slides 38 & 39). When a mismatched base pair is present in the polymerase active site, the new strand:template junction is destabilized, creating several base pairs of unpaired DNA. The incorrect nucleotide is removed by the exonuclease (an additional nucleotide may also be removed). The removal of the mismatched base allows the new strand:template junction to re-form and rebind the polymerase active site, enabling DNA synthesis to continue. The addition of a proofreading exonuclease greatly increases the accuracy of DNA synthesis. On average, DNA polymerase inserts one incorrect nucleotide for every 105 nucleotides added. Proofreading exonucleases decrease the appearance of incorrect base pairs to 1 in every 107 nucleotides added. This error rate is still significantly short of the actual rate of mutation observed in a typical cell (approximately one mistake in every 1010 nucleotides added). This additional level of accuracy is provided by the pos-treplication mismatch repair process. A major limit to DNA polymerase accuracy is the occasional (about one in 10 5 times) flickering of the bases into the “wrong” tautomeric form (imino or enol). These alternate forms of the bases permit incorrect base pairs to be correctly positioned for catalysis (Seminar Slide 40). When the nucleotide returns to its “correct” state, the incorporated nucleotide is mismatched with the template and must be eliminated. Removal of these incorrectly base-paired nucleotides is 43  Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady mediated by a proofreading exonuclease activity of the DNA polymerases, the enzymes degrade DNA starting from a 3' DNA end (i.e., from the growing end of the new DNA strand). DNA polymerases also show an impressive ability to distinguish between ribonucleoside and deoxyribonucleoside triphosphates (rNTPs and dNTPs). Although rNTPs are present at approximately 10-fold higher concentration in the cell, they are incorporated at a rate that is more than 1000-fold lower than dNTPs. This discrimination is mediated by the steric exclusion of rNTPs from the DNA polymerase active site. In DNA polymerase, the nucleotide-binding pocket cannot accommodate a 2'-OH (in case of rNTPs) on the in-coming nucleotide. This space is occupied by two amino acids that make van der Waals contacts with the sugar ring. Changing these amino acids to other amino acids with smaller side chains (e.g., by changing a glutamate to an alanine) results in a DNA polymerase with significantly reduced discrimination between dNTPs and rNTPs. Nucleotides that meet some but not all of the requirements for use by DNA polymerase can inhibit DNA synthesis by terminating elongation. Such nucleotides represent an important class of drugs used to treat cancer and viral infections. 4- DNA Synthesis Requires an RNA Primer The DNA polymerases cannot initiate the synthesis of a polynudeotide; meaning that they can not start making a DNA chain from scratch, but require a pre-existing chain of nucleotides. They can only add nucleotides to the 3' end of an already existing DNA strand. In strict sense, they use the free -OH group found at the 3' end of a pre-existing nucleotide as a "hook," adding a nucleotide to this group in the polymerization reaction. How, then, does pol III add the first nucleotide at a new replication fork? Thereby, to start the synthesis of DNA a short RNA molecule (primer) is required. The primer is about 5-12 nucleotides in length, and is complementary to one strand of the template. It is synthesized by a specific enzyme, primase, which is the RNA polymerase. It should be noted, RNA polymerases, unlike DNA polymerases, can synthesize new strand without a primer. Primase starts an RNA chain from a single RNA nucleotide, adding RNA nucleotides one at a time, using the parental DNA strand as a template. The completed primer, generally 5 to 10 nucleotides long, is thus base-paired to the template strand. The new DNA strand will start from the 3' end of the RNA primer. The DNA pol III adds a DNA nucleotide to the RNA primer and then continues adding DNA nucleotides, complementary to the parental DNA template strand, to the growing end of the new DNA strand (Seminar Slide 41). The speed of the replication fork progression, formed by unwinded DNA, is about 1,000 bp per 44  Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady second. In eukaryotes, it moves much slower, only 50-100 bp per second. This is due to the association of DNA with histones. The pol III utilizes deoxyribonucleoside triphosphates to synthesize the growing DNA strand. These nucleosides are chemically reactive; as each monomer joins the growing end of a DNA strand, two phosphate groups are lost as a pyrophosphate molecule. Subsequent hydrolysis of the pyrophosphate to two molecules of inorganic phosphate is an exergonic reaction that helps drive the polymerization reaction (Seminar Slide 34). 5- Antiparallel Elongation As noted previously, the two strands of DNA in a double helix are antiparallel, meaning that they are oriented in opposite directions to each other, like a divided highway. Clearly, the two new strands formed during DNA replication must also be antiparallel to their template strands. Because pol III can add nucleotides only to the free 3' end of a primer or growing DNA chain, the new DNA strand can elongate only in the 5'→3' direction. Along one template strand, pol III can synthesize a complementary strand continuously by elongating the new DNA in the 5'→3' direction. DNA pol III simply nestles in the replication fork on that template strand and continuously adds nucleotides to the new complementary strand as the fork progresses. The DNA strand made by this mechanism is called the leading strand. Only one primer is required for DNA pol III to synthesize the leading strand (Figure 12). To elongate the other new strand of DNA in the mandatory 5'→3' direction, pol III must work along the other template strand in the direction away from the replication fork. The DNA strand elongating in this direction is called the lagging strand. In contrast to the leading strand, which elongates continuously, the lagging strand is synthesized discontinuously, as a series of segments, called Okazaki fragments. The fragments are about 1,000 to 2,000 nucleotides long in E coli and 100 to 200 nucleotides long in eukaryotes. As shown in Figure 12, only one primer is required on the leading strand. On the other hand, each Okazaki fragment on the lagging strand must be primed separately. On the lagging strand, a primase reads the template DNA and initiates synthesis of a short complementary RNA primers. Then, pol III extends the primed segments, forming Okazaki fragments. Thereafter, another DNA polymerase, DNA polymerase I (DNA pol I), replaces the RNA nucleotides of the primers with deoxyribonucleotides, adding them one by one onto the 3' end of the adjacent Okazaki fragment (fragment 2 in Figure 13). The replacement of the last RNA nucleotide with DNA leaves the sugar-phosphate backbone with a free 3' end, since DNA pol I cannot join the last nucleotide of this replacement DNA segment to 45  Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady the first DNA nucleotide of the directly front DNA fragment (fragment 1 in Figure 14). So, DNA ligase accomplishes this task, it joins the sugar-phosphate backbones of all the Okazaki fragments into a continuous DNA strand (Figure 14). 6- Replicating the Ends of DNA Molecules As explained above, during DNA replication, DNA polymerase requires an RNA primer in order to enable itself to copy DNA in a 5'→3' direction. For linear DNA, such as the DNA of eukaryotic chromosomes, the fact that a DNA polymerase can add nucleotides only to the 3' end of a preexisting polynucleotide leads to an apparent problem. Unlike bacterial chromosomes, the chromosomes of eukaryotes are linear, meaning that they have ends. These ends pose a problem for DNA replication when the replication fork reaches an end of a linear chromosome. Why is this the case? When DNA is being copied, on the leading strand, DNA polymerase can make a complementary DNA strand without any difficulty because it reads the template strand from 3' to 5'. However, there is a problem going in the other direction on the lagging strand. Synthesis of the lagging strand at a replication fork must occur discontinuously through producing short Okazaki fragments that eventually are connected to form an unbroken strand. However, every RNA primer synthesized during replication can be removed and replaced with DNA strands except the final RNA primer at the 5′ end of the newly synthesized strand (that match the 3′ end of the DNA template). This small section of RNA can only be removed, not replaced with DNA since DNA pol I will add new DNA nucleotide only if the there an existing strand 5′ to it (“behind” it) to extend. In order to change RNA to DNA, there must be another DNA strand in front of the RNA primer. However, there is no more DNA in the 5′ direction after the final RNA primer (there is no available 3'-OH end that deoxynucleotides can be added to), so DNA pol I cannot replace the RNA with DNA (Seminar Slide 42). Even when an Okazaki fragment can be started with an RNA primer bound to the very end of the template strand, once the primer is removed it cannot be replaced with DNA because there is no 3'-OH end available for DNA pol I to start polymerization of DNA nucleotides. As a result, there is a short stretch of DNA that does not get covered by an Okazaki fragment and both daughter DNA strands have an incomplete 5′ strand with 3′ overhang. In other words, the DNA at the very end of the chromosome cannot be fully copied in each round of replication, resulting in gradual shortening of the chromosome (Figure 15). As a result, repeated rounds of replication produce shorter and shorter DNA molecules with uneven (staggered) ends (Seminar Slide 42). Without a mechanism to deal with 46  Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady this problem, each daughter DNA would become shorter than the parental DNA, and eventually entire DNA would be lost. This shortening of DNA does not occur in prokaryotes because their DNA is circular and therefore has no ends. To prevent this shortening, the tips of linear eukaryotic chromosomes have specialized structures called telomeres. These telomeres protect the important genes from being eroded away as cells divide and as DNA strands shorten during replication. Other crucial function of telomeres is to protect chromosomes from fusing with each other. Telomeres need to be protected from a cell's DNA repair systems because they have single-stranded overhangs (staggered ends), which look like damaged DNA. Staggered ends of a DNA molecule can trigger signal transduction pathways leading to cell cycle arrest or cell death. Specific proteins associated with telomeric DNA prevent the staggered ends of the daughter molecule from activating the cell’s systems for monitoring DNA damage. Telomeres do not contain genes; instead, their DNA typically consists of multiple repetitions of short nucleotide sequences. As shown in Figure 15, and Seminar Slide 43, after each round of DNA replication, some telomeric sequences are lost at the 5′ end of the newly synthesized strand on each daughter DNA strand, but because these are noncoding sequences, their shortening after each cell division is not detrimental to the cell. However, even these sequences are not unlimited. After sufficient rounds of replication and reaching a certain telomere length, all the telomeric repeats are lost, and the cell may stop to divide and die. This is a normal process in somatic (body) cells. If the chromosomes of germ cells (which give rise to gametes) became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce. However, this does not occur; an enzyme called telomerase catalyzes the lengthening of telomeres in eukaryotic germ cells, thus restoring their original length and compensating for the shortening that occurs during DNA replication. The discovery of the enzyme telomerase helped in the understanding of how chromosome ends are maintained. The telomerase enzyme attaches to the end of a chromosome and adds complementary RNA bases to the 3′ end of the DNA template strand. Once the 3′ end of the lagging strand template is sufficiently elongated, DNA polymerase adds the complementary nucleotides to the ends of the chromosomes; thus, the ends of the chromosomes are replicated. Telomerase is typically active in germ cells and adult stem cells, but is not active in adult somatic cells. As a result, telomerase does not protect the DNA of adult somatic cells and their telomeres continually shorten as they undergo rounds of cell division. 47  Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady Telomeres do tend to be shorter in dividing somatic cells of older individuals and in cultured cells that have divided many times. It has been proposed that steady shortening of telomeres with each replication in somatic cells may have a role in aging process of certain tissues and even to aging of the organism as a whole and in the prevention of cancer. Normal shortening of telomeres may protect organisms from cancer by limiting the number of divisions that somatic cells can undergo. Cells from large tumors often have unusually short telomeres, as one would expect for cells that have undergone many cell divisions. Further shortening would presumably lead to self-destruction of the tumor cells. Intriguingly, researchers have found telomerase activity in cancerous somatic cells, suggesting that its ability to stabilize telomere length may allow these cancer cells to persist. Interestingly, scientists found that telomerase can reverse some age-related conditions in mice. These findings may contribute to the future of regenerative medicine. In the studies, the scientists used telomerase-deficient mice with tissue atrophy, stem cell depletion, organ failure, and impaired tissue injury responses. Telomerase reactivation in these mice caused extension of telomeres, reduced DNA damage, reversed neurodegeneration, and improved the function of the testes, spleen, and intestines. Thus, telomere reactivation may have potential for treating age-related diseases in humans. II- DNA Mutation, Damage, and Repair 1- Mutations and Their Consequences To function correctly, each cell depends on thousands of proteins to do their jobs in the right places at the right times. According to the central dogma of molecular biology, these proteins are encoded by genes. Consequently, any alteration in the nucleotide sequence of a gene can cause the protein to malfunction or to be missing entirely. If the DNA repair machineries in a cell failed to fix incorrectly paired nucleotide(s), a mutation would arise and get passed on to daughter cells. These mutations can be deleterious, causing disease, structural abnormalities, or other effects, and can also be advantageous and lead to an evolutionary advantage. Because an alteration in the DNA sequence affects all copies of the encoded protein, mutations can be particularly damaging to a cell or organism. In contrast, any alterations in the sequences of RNA or protein molecules that occur during their synthesis are less serious because many copies of each RNA and protein are synthesized. Therefore, maintaining a low mutation rate is essential for cell viability and health. In fact, effects of the mutation could be observed only when it occurs in gene exons or regulatory elements. Changes in the non-coding regions of DNA generally do not affect function. If a 48  Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady mutation occurs in germ-line cells (egg or sperm), it can be passed to an organism’s offspring. In contrast, if it occurs in somatic cells, it can be passed to all descendent somatic cells, but they are not passed to offspring. Many cancers are the result of accumulated somatic mutations. In dipliod organisms, which have two copies of each gene, mutations can be recessive or dominant. Recessive mutations inactivate the affected gene and lead to a loss of function. Thus, to give rise to a mutant phenotype, both alleles must carry the mutation (otherwise the correct allele will provide a proper function). In contrast, the phenotypic consequences of a dominant mutation are observed in a heterozygous individual carrying one mutant and one normal allele. Dominant mutations often lead to a gain of function. Mutations in one allele may also lead to a structural change in the protein that interferes with the function of the wild-type protein encoded by the other allele. These are referred to as dominant negative mutations. In fact, bad effects that trigger changes in DNA and incidence of mutations are surrounding us. For example, not to mention replication errors are actually happening in the cells of our bodies all the time, DNA molecules are constantly subjected to potentially harmful chemical and physical agents. These include at least but not limited to any energy-rich radiation, reactive chemicals present in consumable plasticweres as well as toxic products of cellular metabolism (e.g., reactive oxygen species). Besides, purine and pyrimidine bases in DNA often undergo spontaneous chemical transformations (tautomerism) under normal cellular conditions that happen even without environmental insults! Furthermore, biological factors such transposons, viruses and some bacteria can change nucleotides in ways that affect encoded genetic information. To overcome all these bad effects, each cell continuously monitors and repairs any damage in its genetic material and any changes in DNA are usually detected and fixed before they become mutations perpetuated through successive replications. Otherwise, if the damage cannot be fixed, the cell will undergo programmed cell death (apoptosis) to avoid passing on the faulty DNA. 2- Mechanisms of DNA Repair Although cells employ an arsenal of editing mechanisms to correct mistakes, they do happen. Cells have a variety of mechanisms to prevent mutations, or permanent changes in DNA sequence. These include proofreading, mismatch repair, excision repair, chemical reversal, and double- stranded break repair. 49  Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady (i) Proofreading and DNA Mismatch Repair Errors are natural part of the DNA replication. For example, the fact that bases can take several forms, known as tautomers, increases the chance of mispairing during DNA replication. Both the purine and pyrimidine bases in DNA exist in two different tautomers, or chemical forms, in which the protons occupy different positions in the molecule. Nucleotide bases shift from their common “keto” form to their rarer, tautomeric “enol” form. In common base pair arrangements, the common form of thymine (T) binds with the common form of adenine (A), and the common form of cytosine (C) binds with the common form of guanine (G). However, if and when a nucleotide base shifted into its rarer tautomeric form (the "imino" or "enol" form), a likely result would be base-pair mismatching. Rare base-pairing arrangements result when one nucleotide in a base pair is the rare form instead of the common form. For example, the rare enol form of cytosine binds to the common keto form of adenine instead of guanine. The rare enol form of guanine binds to the common keto form of thymine instead of cytosine (Seminar Slide 40). In addition, guanine can undergo a reaction that attaches a methyl group to an oxygen atom in the base. The methyl-bearing guanine, if not fixed, will pair with thymine rather than cytosine during DNA replication (Seminar Slide 44). During DNA replication, most DNA polymerases "check their work," with each base that they add, and fix the majority of mispaired bases in a process called proofreading (Seminar Slide 45A). Incorrectly paired or altered nucleotides sometimes evade proofreading by a DNA polymerase and can also arise after replication; this is the main source of mutations. In fact, maintenance of the genetic information encoded in DNA requires frequent repair of various kinds of damage to existing DNA. For example, Mismatch repair happens right after new DNA has been made, and its job is to remove and replace mis-paired bases, ones that were not fixed during proofreading. How does mismatch repair work? Most cellular systems for repairing incorrectly paired nucleotides, whether they are due to DNA damage or to replication errors, use a mechanism that takes advantage of the base-paired structure of DNA. First, a protein complex recognizes and binds to the mispaired base. The mismatch repair machinery can distinguish the newly synthesized strand from the template (which is treated as a correct one). A second complex cuts the DNA near the mismatch, and more enzymes chop out the incorrect nucleotide and a surrounding patch of DNA. A DNA polymerase then replaces the missing section with correct nucleotides, and a DNA ligase seals the gap (Seminar Slide 45B). Evidence for the importance of 50  Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady proofreading and repair mechanisms comes from human genetic disorders. In many cases, mutations in genes that encode proofreading and repair proteins are associated with heredity cancers such as colon cancer. Apparently, this defect allows cancer-causing mutations to accumulate in the DNA at a faster rate than normal. In addition to mispairing and point mutations (viz a single base pair is added/deleted or changed), DNA replication can lead to the introduction of small insertions or deletions. These mutations can occur when one strand of the DNA template loops out and becomes displaced during replication, or when DNA polymerase slips or stumbles during replication, events termed replication slippage. If a loop occurs in the template strand during replication, DNA polymerase may miss the looped-out nucleotides, and a small deletion in the new strand will be introduced. On the other hand, if DNA polymerase repeatedly introduces nucleotides that are not present in the template strand, an insertion of one or more nucleotides will occur, creating an unpaired loop on the newly synthesized strand (Seminar Slide 46). Insertions and deletions may lead to frameshift mutations or amino acid insertions or deletions in the gene product. Replication slippage can occur anywhere in the DNA but seems distinctly more common in regions containing tandemly repeated sequences (e.g., microsatellites). Indeed, repeat sequences are hot spots for DNA mutation and in some cases contribute to hereditary diseases, such as fragile-X syndrome and Huntington disease. (ii) DNA Damage Repair Mechanisms In fact, bad effects can happen to DNA at almost any point in a cell's lifetime, not just during replication. Although DNA is a highly stable material, as required for the storage of genetic information, it is a complex organic molecule that is susceptible, even under normal cell conditions, to spontaneous changes that would lead to mutations if left unrepaired. For example, the DNA of each human cell loses about 18,000 purine bases (adenine and guanine) every day because their N-glycosyl linkages to deoxyribose hydrolyze, a spontaneous reaction called depurination. Similarly, a spontaneous deamination of cytosine to uracil in DNA occurs at a rate of about 100 bases per cell per day (Seminar Slide 47). DNA bases are also occasionally damaged by an encounter with reactive metabolites produced in the cell, including reactive forms of oxygen and the high-energy methyl donor S-adenosylmethionine, or by exposure to chemicals in the environment. Likewise, ultraviolet radiation from the sun can produce a covalent linkage between two adjacent pyrimidine bases in DNA to form, for example, thymine 51  Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady dimers (Seminar Slide 47). If left uncorrected when the DNA is replicated, most of these changes would be expected to lead either to the deletion of one or more base pairs or to a base- pair substitution in the daughter DNA chain (Figure 16). The mutations would then be propagated throughout subsequent cell generations. Such a high rate of random changes in the DNA sequence would have disastrous consequences. Really, Cells have multiple mechanisms to detect and correct many types of DNA damage using different repair pathways that act upon different kinds of lesions. Therefore, most changes in DNA are quickly repaired. Those that are not repaired and get passed on to daughter cells, only when these mechanisms fail. Thus, mutation is, in fact, a consequence of the failure of DNA repair. If DNA gets damaged, it can be repaired by various mechanisms that detect and correct damage throughout the cell cycle. The base excision repair and nucleotide Excision repair are the major pathways exist to repair single stranded DNA damage (Figure 17). In both, the damage is excised, the original DNA sequence is restored by a DNA polymerase that uses the undamaged strand as its template, and a remaining break in the double helix is sealed by DNA ligase. In nucleotide Excision repair, damage to one or a few bases of DNA is often fixed by removal (excision) and replacement of the damaged region, whilst in base excision repair, just the damaged base is removed. (a) Base Excision Repair and Nucleotide Excision Repair Base excision repair is a mechanism used to detect and remove certain types of damaged bases. For example, a chemical reaction called deamination can convert a cytosine base into uracil, a base typically found only in RNA. During DNA replication, uracil will pair with adenine rather than guanine, so an uncorrected cytosine-to-uracil change can lead to a mutation (Figure 16). Similarly, due to its redox potential, guanine is the most susceptible base to oxidation, forming mainly 8-oxoguanine. This lesion is highly mutagenic and if not repaired, can pair with adenine, causing a G:C to T:A transversion. The earliest steps in the repair of base lesions is lesion recognition and removal by a group of enzymes called glycosylase (Figure 17). Each glycosylase can recognize a specific type of altered base in DNA and catalyze its hydrolytic removal, forming apurinic/apyrimidinic site (AP site). The backbone of the DNA remains intact. AP site is then cleaved by an AP endonuclease. The resulting single-strand break can then be processed by either short-patch or long-patch base excision repair. 52  Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady Nucleotide excision repair is another pathway used to remove and replace damaged bases. This pathway can detect and correct types of damage that distort the DNA double helix. For instance, this pathway detects bases that have been modified with bulky chemical groups, like the ones that get attached to your DNA when it is exposed to chemicals in cigarette smoke. Nucleotide excision repair is also used to fix some types of damage caused by UV radiation, for instance, when you get a sunburn. When DNA is exposed to UV light (non-ionizing radiation), two neighboring pyrimidines (cytosines or thymines) may form a dimer (Figure 18) that distort the double helix and cause errors in DNA replication. The most common type of linkage, a thymine dimer, consists of two thymine bases that react with each other and become chemically linked. If the unrepaired dimer is not fixed, these dimers are mutagenic. This is because, if, for example, the cytosine dimer is formed by UV light, during replication in the new strand adenine could be incorporated (instead of guanine). In the next round of replication opposite to the adenine, thymine will be built, which will cause the mutation consisting in a change of C:C to T:T (Figure 16). While cytosine dimers can be repaired, mutations arising after replication is no longer detectable by the DNA repair system. Fortunately, most of these genetic lesions are corrected seconds after they are created, before they can do permanent damage. In nucleotide excision repair pathway, enzymes recognize bulky distortions in the shape of the DNA double helix. After the initial recognition step, the damaged nucleotide(s) are removed along with a short surrounding single-stranded DNA segment (24-32 nucleotides). A single-strand gap created in the DNA is subsequently filled in by DNA polymerase, which uses the undamaged strand as a template, and a DNA ligase seals the gap in the backbone of the strand (Figure 17). The importance of repairing this kind of damage is underscored by the disorder xeroderma pigmentosum, which in most cases is caused by an inherited defect in a nucleotide excision repair enzymes. Individuals with this disorder are hypersensitive to sunlight and develop severe sunburns from just a few minutes in the sun. If mutations in their skin cells caused by ultraviolet light are left uncorrected, the mutations cause skin cancer and those individual die by the age of 10 unless they avoid the sun. (b) Single-Stranded Break Repair Ionizing radiation may lead to the formation of single-stranded and double-stranded breaks in the sugar-phosphate backbone of DNA, as well as to the modification of bases, e.g., formation of pyrimidine dimers. Single-strand breaks, also called DNA nicks, are very common. They 53  Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady are easily reversed by DNA ligase but oxidative attack can cause deoxyribose residues to disintegrate, producing more complex strand breakage. A type of base-excision repair is employed: strand breaks are rapidly detected and briefly bound by a sensor molecule, poly(ADPribose), that initiates repair by attracting suitable repair proteins to the site (Figure 15). The gap is then filled using a DNA polymerase and DNA ligase. (c) Double-Stranded Break Repair Double-strand DNA breaks (DSBs, entire chromosome splits into two pieces) are normally rare in cells. However, they do occur as a result of chemical attack on DNA by endogenous reactive oxygen species (but at much lower frequencies than single-strand breaks) or by external some environmental factors, such as high-energy radiation. In these cases, DNA repair is required, but can sometimes be difficult to perform. For example, when the two complementary DNA strands are broken simultaneously at sites sufficiently close to each other, neither base pairing nor chromatin structure may be sufficient to hold the two broken ends opposite each other. The DNA termini will often have sustained base damage and the two broken ends are liable to become physically dissociated from each other, making alignment difficult. Unrepaired DSBs are highly dangerous to cells because large segments of chromosomes, and the hundreds of genes they contain, may be lost if the break is not repaired. The break can also lead to inactivation of a critically important gene, and the broken ends are liable to recombine with other DNA molecules, causing chromosome rearrangements that may be harmful or lethal to the cell. Cells respond to DSBs in different ways. Two major DNA repair mechanisms, non-homologous end joining and homologous recombination, can be deployed to repair a DSB (Seminar Slide 48), but if repair is incomplete, apoptosis is likely to be triggered. One pathway involved in double-strand break repair is homologous recombination repair (HR). This highly accurate repair mechanism requires a homologous intact DNA strand to be available to act as a template strand. Normally, therefore, it operates after DNA replication (and before mitosis), using a DNA strand from the undamaged sister chromatid as a template to guide repair. It is important in early embryogenesis, when many cells are rapidly proliferating, and in the repair of proliferating cells. The first step in this process involves the activity of an enzyme that recognizes the DSB and then digests back the 5′ ends of the broken DNA helix, leaving overhanging 3′ ends (Seminar Slide 49). One overhanging end searches for a region of sequence complementarity on the sister chromatid and then invades the homologous DNA 54  Molecular Biology (1), Lecture IV, by Prof. of Molecular Biology, Ayman I Elkady duplex, aligning the complementary sequences. Once aligned, DNA synthesis proceeds from the 3′ overhanging ends, using the undamaged homologous DNA strands as templates. The interaction of two sister chromatids is necessary because, when both strands of one helix are broken, there is no undamaged parental DNA strand available to use as a template DNA sequence during repair. After DNA repair synthesis, the resulting heteroduplex molecule is resolved and the two chromatids separate. DSB repair usually occurs during the late S or early G2 phase of the cell cycle, after DNA replication, a time when sister chromatids are available to be used as repair templates. Because an undamaged template is used during repair synthesis, HR repair is an accurate process. Recently, interest in DSB repair has grown because defects in these pathways are associated with X-ray hypersensitivity and immune deficiency. Such defects may also underlie familial disposition to breast and ovarian cancer. Several human disease syndromes, such as Fanconi anemia and ataxia telangiectasia, result from defects in DSB repair. A second pathway, called nonhomologous end joining (NHEJ), also repairs double- strand breaks. However, as the name implies, the mechanism does not recruit a homologous region of DNA during repair. This system is activated in G1, prior to DNA replication. This repair mechanism typically involves the loss, or sometimes addition, of a few nucleotides at the cut site. It can lead to translocations and telomere fusion. So, non-homologous end joining tends to produce a mutation, but this is better than the alternative (loss of an entire chromosome arm). End joining involves a complex of many proteins. Specific proteins bind to the exposed DNA ends and recruit a special DNA ligase, DNA ligase IV, to rejoin the broken ends. These proteins bind to the free ends of the broken DNA, trim the ends, and ligate them back together. Because some nucleotide sequences are lost in the process of end joining, it is an error-prone repair system. In addition, if more than one chromosome suffers a double-strand break, the wrong ends could be joined together, leading to abnormal chromosome structures. Unlike HR-mediated DNA repair, NHEJ is, in principle, always available to cells, but it is most important for the repair of differentiated cells and of proliferating cells in G1 phase before the DNA has replicated. 55