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CHAPTER 18 Gene Mutations and DNA Repair Lou Gehrig at bat. Gehrig, who played baseball for the New York Yankees from 1923 to 1939, was diagnosed with amyotrophic lateral sclerosis, a disease that in some people is caused by an expanding nucleotide repeat mutation. Lou Gehrig and Expanding Nucleotide Repeats Lou Gehrig was the ﬁnest ﬁrst baseman ever to play major league baseball. A le -handed power hitter who grew up in New York City, Gehrig played for the New York Yankees from 1923 to 1939. Throughout his career, he lived in the shadow of his teammates Babe Ruth and Joe DiMaggio, but Gehrig was a great hitter in his own right: he compiled a lifetime batting average of.340 and drove in more than 100 runs every season for 13 years. During his career, he batted in 1991 runs and hit a total of 23 grand slams (home runs with bases loaded). But Gehrig’s greatest baseball record, which stood for more than 50 years and has been broken only once—by Cal Ripken, Jr., in 1995—is his record of playing 2130 consecutive games. In the 1938 baseball season, Gehrig fell into a strange slump. For the ﬁrst time since his rookie year, his batting average dropped below.300, and in the World Series that year, he managed only four hits—all singles. Nevertheless, he ﬁnished the season convinced that he was undergoing a temporary slump that he would overcome in the next season. He returned to training camp in 1939 with high spirits. When the season began, however, it was clear to everyone that something was terribly wrong. Gehrig had no 1196 power in his swing; he was awkward and clumsy at ﬁrst base. His condition worsened, and on May 2, he voluntarily removed himself from the lineup. The Yankees sent Gehrig to the Mayo Clinic for diagnosis. On June 20, his medical report was made public: Lou Gehrig was suffering from a rare, progressive disease known as amyotrophic lateral sclerosis (ALS). Within two years, he was dead. Since then, ALS has commonly been known as Lou Gehrig disease. Gehrig experienced symptoms typical of ALS: progressive weakness and wasting of skeletal muscles due to degeneration of the motor neurons. Most cases of ALS are sporadic, appearing in people with no family history of the disease. However, about 10% of cases run in families, and in these cases the disease is inherited as an autosomal dominant trait. ALS shares a number of features in common with another neurological disease called frontotemporal dementia (FTD); in fact, FTD occurs alongside ALS in some families, suggesting a common genetic basis underlying these two disorders. Mutations in several genes can cause familial cases of ALS and FTD, the most common of which occur in a gene on chromosome 9 called chromosome 9 open reading frame 72 (C9orf72). The alterations of C9orf72 that are associated with ALS and FTD belong to an unusual group of mutations called expanding nucleotide repeats, in which the number of copies of a set of nucleotides is increased. Most people have somewhere between 2 and 23 repeats of the nucleotide sequence GGGGCC in their C9orf72 gene, but this number is massively expanded in some people with ALS, who typically possess 700 to 1600 repeats of the sequence. How the expansion of the GGGGCC repeat in C9orf72 leads to symptoms of ALS and FTD is unknown, but recent research demonstrates that the repeats are translated into one or more proteins that are toxic to nerve cells. The repeats are translated in an unusual and intriguing way: the GGGGCC sequences on both the template and the nontemplate strands of the gene are transcribed into RNAs that are translated without a start codon. Because there is no start codon to set the reading frame, all three reading frames on both mRNAs are translated into proteins, resulting in ﬁve proteins, each with a different series of repeating dipeptides: glycine-alanine, glycine-proline, proline-alanine, glycine-arginine, and proline- arginine. To determine how the repeats might produce the disease, geneticists engineered a series of premature stop codons into the template and nontemplate strands of the C9orf72 gene so that RNA would be transcribed from the repeats but, because of the engineered stop codons, would not be translated into a protein. They inserted both the original repeat sequence and the engineered repeat sequence into fruit ﬂies. The unaltered repeats caused neurodegeneration and early death in the fruit ﬂies, but the engineered repeats had no effect. These results suggest that the toxicity of the repeat sequence was the result of the protein it encoded and not simply a product of the RNA alone (although the RNA may also be somewhat toxic). Further research suggested that proteins with the glycine-arginine and proline- arginine dipeptides are responsible for the neurodegeneration that occurs in ALS and FTD. The toxicity of these proteins may result from the fact that they mimic RNA-binding proteins and interfere with splicing of pre-mRNA and the processing of rRNA. This research provides important insight into the pathology of these diseases and suggests possible future targets for treatment. 1197 THINK-PAIR-SHARE Propose some ways that the new information provided by research on the role of the GGGGCC repeat in ALS might be used to design potential treatments for the disease. Using the genetic code illustrated in Figure 15.10, show how translation of the GGGGCC repeat without a start codon results in the production of five proteins with diﬀerent dipeptide repeats. (Hint: Consider all reading frames of the two RNAs copied from this sequence.) The story of ALS and expanding nucleotide repeats illustrates the central importance of studying mutations: the analysis of mutants is o en a source of key insights into diseases and important biological processes. This chapter focuses on gene mutations—on how these errors in genetic instructions arise and how they are studied. We begin with a brief examination of the different types of mutations, including their phenotypic effects, how they can be suppressed, and their rates of occurrence. The next section explores how mutations can arise spontaneously during and a er the course of DNA replication, as well as how chemicals and radiation can induce them. A er discussing the analysis of mutations, we turn to transposable elements: DNA sequences that are capable of moving within the genome and that o en produce mutations when they do so. Finally, we take a look at DNA repair and some of the diseases that arise when DNA repair is defective. 1198 18.1 Mutations Are Inherited Alterations in the DNA Sequence DNA is a highly stable molecule that is replicated with amazing accuracy (as we saw in Chapters 10 and 12), but changes in DNA structure and errors of replication do take place. A mutation is deﬁned as an inherited change in the DNA sequence of genetic information; the descendants that inherit the change may be cells or organisms. The Importance of Mutations Mutations are both the sustainer of life and the cause of great suffering. On the one hand, mutation is the source of all genetic variation, the raw material of evolution. The ability of organisms to adapt to environmental change depends on the presence of genetic variation in natural populations, and genetic variation is produced by mutation. On the other hand, many mutations have detrimental effects, and mutation is the source of many diseases and disorders. Much of the study of genetics focuses on how genetic variants produced by mutation are inherited; genetic crosses are meaningless if all individual members of a species are identically homozygous for the same alleles. Much of Gregor Mendel’s success in unraveling the principles of inheritance can be traced to his use of carefully selected variants of the garden pea (see Chapter 3). Similarly, Thomas Hunt Morgan and his students discovered many basic principles of genetics by analyzing mutant fruit ﬂies (discussed in Chapter 4). Mutations are also useful for examining fundamental biological processes. Finding or creating mutations that affect different components of a biological system and studying their effects can o en lead to a better understanding of the system. This method, referred to as genetic dissection, is analogous to ﬁguring out how an automobile works by breaking different parts of a car and observing the effects; for example, smash the radiator and the engine overheats, revealing that the radiator cools the engine. The use of mutations to disrupt function can likewise be a source of insight into biological processes. For example, geneticists have begun to unravel the molecular details of development by studying mutations that interrupt various embryonic stages in Drosophila (see Chapter 22). Scientists have also used analysis of mutations to reveal the different parts of the lac operon (discussed in Chapter 16) and how they function in gene regulation. Although breaking “parts” to determine their function might seem like a crude approach to understanding a system, it is actually a very powerful one and has been used extensively in biochemistry, developmental biology, physiology, and behavioral science. But this method is not recommended for learning how your car works! THINK-PAIR-SHARE Question 1 CONCEPTS 1199 Mutations are heritable changes in DNA. They are essential to the study of genetics and are useful in many other biological fields. Categories of Mutations In multicellular organisms, we can distinguish between two broad categories of mutations: somatic mutations and germ-line mutations. Somatic mutations arise in somatic tissues, which do not produce gametes (Figure 18.1). When a somatic cell with a mutation divides (by mitosis), the mutation is passed on to the daughter cells, leading to a population of genetically identical cells (a clone). The earlier in development that a somatic mutation takes place, the larger the clone of cells that contain the mutation will be. 18.1 The two basic classes of mutations are somatic mutations and germ-line mutations. Description The illustration shows a green fish on the left. The center of the fish is labeled somatic tissue and the area at the end of the fins is labeled germ line tissue. An arrow from the initial fish bifurcates. The top arrow shows somatic mutation. A mutant cell is present in a fish in its somatic tissue. Mitosis occurs and a green fish obtained shows a large population of mutant cells in its somatic tissue. The bottom arrow from the initial fish shows germ-line mutation. A mutant cell is present in a fish in its germ line tissue. Sexual reproduction occurs and two fish obtained on the right. A red-fish’s cells all carry the mutation and a green fish has no cells that carry mutation. Captions throughout read, Somatic mutations occur in non reproductive cells and are passed to new cells through mitosis, creating a clone of cells having the mutant gene. Germ-line mutations occur in cells that give rise to gametes. Meiosis and sexual reproduction allow germ-line mutations to be passed to approximately half the members of the next generation who will carry the mutation in all their cells. Because of the huge number of cells present in a typical eukaryotic organism, somatic mutations are numerous. For example, there are about 1014 cells in the human body. Typically, a mutation arises once in every million cell divisions, so hundreds of millions of somatic mutations must arise in each person. Many somatic mutations have no obvious effect on the phenotype of the organism because the function of the mutant cell is taken over by a normal cell, or the mutant cell dies and is replaced by normal cells. 1200 However, cells with a somatic mutation that stimulates cell division can increase in number and spread; this type of mutation can give rise to cells with a selective advantage and is the basis for cancer (see Chapter 23). Somatic mutations are also associated with some other diseases, including hemimegalencephaly, in which just one hemisphere of the brain is enlarged, usually resulting in epilepsy. And somatic mutations can lead to mosaicism, in which different tissues within the body have different genetic information (see Chapter 6). It has long been assumed that somatic mutations accumulate with age, giving rise to cancer and aging, but measuring the frequency of somatic mutations has been difﬁcult. In recent years, the development of methods to accurately sequence whole genomes of single cells (see Chapter 20) and to correct for sequencing artifacts has made it possible to reliably estimate numbers of somatic mutations. The sequencing of individual neurons from the brains of people of different ages has demonstrated that the number of somatic mutations increases with age, and individuals with genetic diseases that interfere with DNA repair (see Section 18.5) have higher rates of somatic mutations than expected for their age. Germ-line mutations arise in cells that ultimately produce gametes. A germ-line mutation can be passed to future generations, producing offspring that carry the mutation in all their somatic and germ-line cells (see Figure 18.1). When we speak of mutations in multicellular organisms, we’re usually talking about germ-line mutations. Historically, mutations have been partitioned into those that affect a single gene, called gene mutations, and those that affect the number or structure of chromosomes, called chromosome mutations. This distinction arose because chromosome mutations could be observed directly, by looking at chromosomes with a microscope, whereas gene mutations could be detected only by observing their phenotypic effects. Now DNA sequencing allows direct observation of gene mutations, and chromosome mutations are distinguished from gene mutations somewhat arbitrarily on the basis of the size of the DNA lesion. Nevertheless, it is practical to use chromosome mutation for a large-scale genetic alteration that affects chromosome structure or the number of chromosomes and to use gene mutation for a relatively small DNA lesion that affects a single gene. This chapter focuses on gene mutations; chromosome mutations were discussed in Chapter 8. Types of Gene Mutations There are a number of ways to classify gene mutations. Some classiﬁcation schemes are based on the nature of the phenotypic effect, others are based on the causative agent of the mutation, and still others focus on the molecular nature of the defect. Here we will categorize mutations primarily on the basis of their molecular nature, but we will also encounter some terms that relate the causes and the phenotypic effects of mutations. BASE SUBSTITUTIONS The simplest type of gene mutation is a base substitution, the alteration of a single nucleotide in the DNA (Figure 18.2a). There are two types of base substitutions. In a transition, a purine is replaced by a 1201 different purine or, alternatively, a pyrimidine is replaced by a different pyrimidine (Figure 18.3). In a transversion, a purine is replaced by a pyrimidine or a pyrimidine is replaced by a purine. The number of possible transversions (see Figure 18.3) is twice the number of possible transitions, but transitions arise more frequently because transforming a purine into a different purine or a pyrimidine into a different pyrimidine is easier than transforming a purine into a pyrimidine, or vice versa. ❯ TRY PROBLEM 19 18.2 Three basic types of gene mutations are base substitutions, insertions, and deletions. Description The illustration shows an original D N A sequence at the top. The sequence is as follows: G G G, A G T, G T A, G A T, C G T. Part a below shows base substitution. The new sequence is as follows: G G G, A G T, G C (highlighted with a note that reads, one codon changed) A, G A T, C G T. A caption reads, A base substitution alters a single codon. Part b below shows Nucleotide insertion. The new sequence is as follows: G G G, A G T, G T T (highlighted), A G A, T C G, T. Nucleotide T is inserted. Part c below shows Nucleotide deletion. The new sequence is as follows: G G G, A G T, G A (nucleotide T is deleted) G, A T C, G T. A caption corresponding to the highlighted T in part b and the T being removed from G A G reads, An insertion or a deletion alters the reading frame and may change many codons. 1202 18.3 A transition is the substitution of a purine for a purine or of a pyrimidine for a pyrimidine; a transversion is the substitution of a pyrimidine for a purine or of a purine for a pyrimidine. Description The first part shows two types of transitions. The first transition shows a purine, a five membered ring fused with a six membered ring, converting to another purine. The possible base changes include: A to G and G to A. The second transition below shows a pyrimidine, a six membered ring, converts to another pyrimidine. The possible base changes include: T to C and C to T. The second part shows two types of transversions. The first transversion shows a purine converting to a pyrimidine. The possible base changes include: A to C, A to T, G to C, and G to T. The second transversion below shows a pyrimidine converting to a purine. Pyrimidine is a six membered ring whereas Purine is a five membered ring fused with a six membered ring. The possible base changes include: C to A, C to G, T to A, and T to G. INSERTIONS AND DELETIONS Another class of gene mutations consists of insertions and deletions (collectively called indels): the addition or removal, respectively, of one or more nucleotide pairs (Figure 18.2b and c). Although base substitutions are o en assumed to be the most common type of mutation, molecular analysis has revealed that insertions and deletions are o en more frequent. Insertions and deletions within sequences that encode proteins may lead to frameshi mutations: changes in the reading frame (see pp. 445–446 in Chapter 15) of the gene. Frameshi mutations usually alter all amino acids encoded by the nucleotides following the mutation, so they generally have drastic effects on the phenotype. Some frameshi s also introduce premature stop codons, terminating protein synthesis early and resulting in a shortened (truncated) protein. Not all insertions and deletions lead to frameshi s, however; insertions and deletions consisting of any multiple of three nucleotides leave the reading frame intact, although the addition or removal of one or more amino acids may still affect the phenotype. Indels that do not affect the reading frame are called in-frame insertions and in-frame deletions. CONCEPTS Gene mutations are changes in a single gene. They can be base substitutions (in which a single pair of nucleotides is altered) or insertions or deletions (in which nucleotides are added or removed). A base substitution can be a transition (substitution of like bases) or a transversion (substitution of unlike bases). Insertions and deletions o en lead to a change in the reading frame of a gene. CONCEPT CHECK 1 1203 Which of the following changes is a transition base substitution? a. Adenine is replaced by thymine. b. Cytosine is replaced by adenine. c. Guanine is replaced by adenine. d. Three nucleotide pairs are inserted into DNA. EXPANDING NUCLEOTIDE REPEATS Mutations in which the number of copies of a set of nucleotides increases are called expanding nucleotide repeats. As discussed in the introduction to the chapter, this is the type of mutation that is responsible for some familial cases of ALS. Expanding nucleotide repeats were ﬁrst discovered in 1991 in a gene called FMR-1, which causes fragile-X syndrome, the most common hereditary cause of intellectual disability. The disorder is so named because when specially treated cells from people with the condition are examined under a microscope, the tip of each long arm of the X chromosome is attached by a slender- appearing part of the chromosome (Figure 18.4). The normal FMR-1 allele (not containing the mutation) has 54 or fewer copies of the sequence CGG, but in people with fragile-X syndrome, the allele may harbor hundreds or even thousands of copies. 18.4 Fragile-X syndrome is associated with a characteristic constriction (fragile site) on the long arm of the X chromosome. Description The illustration shows an X shaped chromosome with almost equal arms. The illustration shows another X shaped chromosome with almost equal arms. Both the long arms of this chromosome have a tiny extension below them with a round lump at the end labeled, fragile site. Expanding nucleotide repeats have been found in almost 30 human diseases, several of which are listed in Table 18.1. Most of these diseases are caused by the expansion of a set of three nucleotides (called a trinucleotide), most o en CNG, where N can be any nucleotide. However, some diseases are caused by repeats of four, ﬁve, and even twelve nucleotides. The number of copies of the repeat o en correlates with the severity or age of onset of the disease. The number of copies of the repeat also correlates with its 1204 instability: when more repeats are present, the probability of expansion to even more repeats increases. This association between the number of copies of nucleotide repeats, the severity of the resulting disease, and the probability of expansion leads to a phenomenon known as anticipation (see Chapter 5), in which diseases caused by expanding nucleotide repeats become more severe in each generation. Less commonly, the number of nucleotide repeats may decrease within a family. Expanding nucleotide repeats have also been observed in some microbes and plants. TABLE 18.1 Examples of human genetic diseases caused by expanding nucleotide repeats Number of Copies of Repeat Disease Repeated Sequence Normal Range Disease Range Spinal and bulbar muscular atrophy CAG 11–33 40–62 Fragile-X syndrome CGG 6–54 50–1500 Jacobsen syndrome CGG 11 100–1000 Spinocerebellar ataxia (several types) CAG 4–44 21–130 Autosomal dominant cerebellar ataxia CAG 7–19 37–220 Myotonic dystrophy CTG 5–37 44–3000 Huntington disease CAG 9–37 37–121 Friedreich ataxia GAA 6–29 200–900 Dentatorubral-pallidoluysian atrophy CAG 7–25 49–75 Myoclonus epilepsy of the Unverricht–Lundborg type CCCCGCCCCGCG 2–3 12–13 Increases in the number of nucleotide repeats can produce disease symptoms in different ways. In several diseases (e.g., Huntington disease), the nucleotide expansion occurs within the coding part of a gene, producing a toxic protein that has extra glutamine (the amino acid encoded by CAG). In other diseases, the repeat is outside the coding region of a gene and affects its expression. For example, in fragile-X syndrome, the additional copies of the nucleotide repeat cause the DNA to become methylated, which turns off the transcription of an essential gene. In other cases, it appears that RNA transcribed from the repeats may itself be toxic or may be abnormally translated into short proteins that cause disease symptoms (see introduction to this chapter). Expansion of nucleotide repeats occurs in the course of DNA replication and appears to be related to the formation of hairpins and other special secondary structures that form in single-stranded DNA consisting of nucleotide repeats. Such structures may interfere with normal replication by causing strand slippage, misalignment of the sequences, or stalling of replication. One model of how hairpin formation in the repeats might result in their expansion is shown in Figure 18.5. Watching Animation 18.1 will help you understand how copies of nucleotide repeats increase in number. Other models of repeat expansion that occur through transcription and DNA repair have also been proposed. Many aspects of this phenomenon are not well understood, including why repeat expansion occurs in some people and not in others. 1205 1206 18.5 A model of how the number of copies of a nucleotide repeat may increase in replication. Description The illustration shows a D N A double strand at the top. The top strand shows the following sequence: G T C repeated eight times and labeled 1 through 8. The bottom strand with complementary bases is as follows: C A G repeated eight times. A caption reads, This D N A molecule has eight copies of a C A G repeat. The two strands separate and replicate. The bottom strand separates from the top strand. The top strand remains below. Replication occurs. The double strand obtained below has same top strand as in the beginning. The sequence of newly synthesized bottom strand is as follows: C A G repeated seven times. An arrow to the right side is present after this sequence. The bottom strand forms a hairpin loop below, where 5 C A G repeats are involved. As the 5 G T C repeats of the top strand do not have complementary bases, the bottom strand with hairpin loop gets elongated. The bottom strand now has 5 addition C A G repeats, labeled 9 through 13, corresponding to G T C repeats in first strand labeled 4 through 8. A caption reads, In the course of replication, a hairpin forms on the newly synthesized strand causing part of the template strand to be replicated twice and increasing the number of repeats on the newly synthesized strand. Both the strands separate, only the bottom strand remains below. A caption reads, The two strands of the new D N A molecule separate, and the strand with extra C A G repeats serves as a template for replication. Now the remaining bottom strand with C A G repeats, labeled 1 through 13, serves as a template and a complementary strand is synthesized above. The top strand is synthesized from right to left and has G T C repeats. The newly synthesized top strand elongates and now has G T C repeats, labeled 1 through 13 for the C A G repeats for the template strand below. A caption reads, The resulting D N A molecule contains five additional copies of the C A G repeat. CONCEPTS Expanding nucleotide repeats are mutations in which the number of copies of a set of repeated nucleotides increases over time. These mutations are associated with several human genetic diseases. Functional Eﬀects of Mutations Another way that mutations are classiﬁed is by their functional effects. At the most general level, we can distinguish a mutation on the basis of its phenotype compared with the wild-type phenotype. A mutation that alters the wild-type phenotype is called a forward mutation, whereas a reverse mutation (a reversion) changes a mutant phenotype back into the wild type. Geneticists use other terms to describe the effects of mutations on protein structure. A base substitution that results in a different amino acid in the protein is referred to as a missense mutation (Figure 18.6a). A nonsense mutation changes a sense codon (one that speciﬁes an amino acid) into a nonsense codon (one that terminates translation), as shown in Figure 18.6b. If a nonsense mutation occurs early in the mRNA sequence, the protein will be truncated and usually nonfunctional. 1207 18.6 Base substitutions can cause (a) missense, (b) nonsense, or (c) silent mutations. Description The top center of the illustration shows a D N A double strand. The top strand has the following sequence: T, C, A. The bottom strand has the following sequence: A, G, T. A downward arrow from the double strand divides into four arrows below. The first arrow, at bottom left, shows no mutation. A double D N A strand is present at the top. The sequence of the top strand is as follows: T, C, A. The sequence of the bottom strand is as follows: A, G, T. An m R N A is produced below with the following sequence: U, C, A. A protein is formed below with serine (Ser), at the center of the chain. A caption below reads, Wild-type protein produced. The second arrow on the right shows (a) Missense mutation. A double D N A strand is present at the top. The sequence of the top strand is as follows: T, T (highlighted), A. The sequence of the bottom strand is as follows: A, A (highlighted), T. An m R N A is produced below with the following sequence: U, U, A. A protein is formed below with leucine (Leu), at the center of the chain. A caption below reads, The new codon encodes a different amino acid; there is a change in amino acid sequence. The third arrow on the right shows (b) Non sense mutation. A double D N A strand is present at the top. The sequence of the top strand is as follows: T, A (highlighted), A. The sequence of the bottom strand is as follows: A, T (highlighted), T. An m R N A is produced below with the following sequence: U (highlighted), A (highlighted), A (highlighted). The m R N A is labeled, stop codon. A protein chain is formed below. A caption below reads, The new codon is a stop codon; there is premature termination of translation. The fourth arrow on the right shows (c) Silent mutation. A double D N A strand is present at the top. The sequence of the top strand is as follows: T, C, G (highlighted). The sequence of the bottom strand is as follows: A, G, C (highlighted). An m R N A is produced below with the following sequence: U, C, G. A protein is formed below with serine (Ser), at the center of the chain. A caption below reads, The new codon encodes the same amino acid; there is no change in amino acid sequence. Because of the redundancy of the genetic code, some different codons specify the same amino acid. A silent mutation changes a codon to a synonymous codon that speciﬁes the same amino acid (Figure 18.6c), altering the DNA sequence without changing the amino acid sequence of the protein. Not all silent mutations, however, are truly silent: some do have phenotypic effects. They may have phenotypic effects, for example, when different tRNAs (called isoaccepting tRNAs; see Chapter 15) bind to different synonymous codons. Because some isoaccepting tRNAs are more abundant than others, which synonymous codon is used may affect the rate of protein synthesis. The rate of protein synthesis, in turn, 1208 can inﬂuence the phenotype by affecting the amount of protein present in the cell and, in a few cases, the folding of the protein. Some silent mutations alter nucleotides that serve as binding sites for regulatory proteins or alter sequences near exon–intron junctions that affect mRNA splicing (see Chapter 14). Still other silent mutations can inﬂuence the binding of miRNAs to complementary sequences in the mRNA, which determines whether the mRNA is translated (see Chapter 14). A neutral mutation is a missense mutation that alters the amino acid sequence of a protein but does not signiﬁcantly change its function. Neutral mutations occur when one amino acid is replaced by another that is chemically similar, or when the affected amino acid has little inﬂuence on protein function. For example, some neutral mutations occur in the genes that encode hemoglobin; although these mutations alter the amino acid sequence of hemoglobin, they do not affect its ability to transport oxygen. Loss-of-function mutations cause the complete or partial absence of normal protein function. A loss-of- function mutation is one that so alters the structure of the protein that the protein no longer works correctly, or one that occurs in regulatory regions that affect the transcription, translation, or splicing of the protein. Loss-of-function mutations are frequently recessive, in which case a diploid individual must be homozygous for the mutation before the effects of the loss of the functional protein can be exhibited. The mutations that cause cystic ﬁbrosis are loss-of-function mutations: these mutations produce a nonfunctional form of the CFTR protein, which normally regulates the movement of chloride ions into and out of the cell (see Chapter 5). In contrast, a gain-of-function mutation causes the cell to produce a protein or gene product whose function is not normally present. The result could be an entirely new gene product or one produced in an inappropriate tissue or at an inappropriate time in development. For example, a mutation in a gene that encodes a receptor for a growth factor might cause the mutated receptor to stimulate growth all the time, even in the absence of the growth factor. Gain-of-function mutations are frequently dominant in their expression because a single copy of the mutation leads to the presence of a new gene product. Other mutations are conditional mutations, which are expressed only under certain conditions. For example, some conditional mutations affect the phenotype only at elevated temperatures. Still others are lethal mutations, which cause premature death (see Chapter 5). ❯ TRY PROBLEM 23 THINK-PAIR-SHARE Question 2 Suppressor Mutations A suppressor mutation is a genetic change that hides or suppresses the effect of another mutation. This type of mutation is different from a reverse mutation, where the mutated site is changed back to the original wild-type sequence (Figure 18.7). A suppressor mutation occurs at a site distinct from the site of the original mutation; thus, an individual with a suppressor mutation is a double mutant, possessing both the original mutation and the suppressor mutation but exhibiting the phenotype of the nonmutated wild type. Geneticists distinguish between two classes of suppressor mutations: intragenic and intergenic. 1209 18.7 Relation of forward, reverse, and suppressor mutations. Description The illustration shows a wild type genotype on the left. The wild type genotype is as follows: big A superscript positive big B superscript positive. A downward arrow shows a fly with red, large eyes. A forward arrow from the wild type genotype big A superscript positive big B superscript positive shows a forward mutation big A superscript negative and results in mutation big A superscript negative big B superscript positive. A caption reads, A forward mutation changes the wild type into a mutant phenotype. A backward arrow from the mutation big A superscript negative big B superscript positive shows reverse of mutation big A superscript negative and leads to wild type genotype big A superscript positive big B superscript positive. A caption reads, A reverse mutation restores the wild-type gene and the phenotype. A downward arrow from the mutation leads to a fly with white, large eyes. A forward arrow from the mutation big A superscript negative big B superscript positive shows suppressor mutation big B superscript negative and leads to mutations big A superscript negative big B superscript negative. A downward arrow from the mutations big A superscript negative big B superscript negative leads to a fly with red, large eyes. A caption reads, A suppressor mutation occurs at a site different from that of the original mutation and produces an individual that has both the original mutation and the suppressor mutation but has the wild-type phenotype. INTRAGENIC SUPPRESSOR MUTATIONS An intragenic suppressor mutation takes place in the same gene that contains the mutation being suppressed. It may work in any of several ways. The suppressor may change a second nucleotide in the same codon altered by the original mutation, producing a codon that speciﬁes the same amino acid that was speciﬁed by the original, nonmutated codon (Figure 18.8). 1210 18.8 An intragenic suppressor mutation occurs in the gene containing the mutation being suppressed. Description The illustration starts with a D N A double strand at the left. The sequence of the top strand is as follows: T, T, A. The sequence of the bottom strand is as follows: A, A, T. An m R N A is produced below with the following sequence: U, U, A. A protein is formed below with leucine (Leu), at the center of the chain. A forward arrow from the D N A double strand represents mutation and points to another D N A double strand, at the bottom center. The sequence of the top strand is as follows: T, T, T (highlighted). The sequence of the bottom strand is as follows: A, A, A (highlighted). A caption reads, A missense mutation alters a single codon. An m R N A is produced below with the following sequence: U, U, U. A protein is formed below with Phenylalanine (Phe), at the center of the chain. A forward arrow from the D N A double strand with missense mutation represents Intragenic suppressor mutation and points to another D N A double strand, at the bottom right. The sequence of the top strand is as follows: C (highlighted), T, T. The sequence of the bottom strand is as follows: G (highlighted), A, A. An m R N A is produced below with the following sequence: C, U, U. A protein is formed below with Leucine (Leu), at the center of the chain. A caption reads, A second mutation at a different site in the same gene may restore the original amino acid. Intragenic suppressors may also work by suppressing a frameshi mutation. If the original mutation, for example, is a one-base deletion, then the addition of a single base elsewhere in the gene will restore the former reading frame. Consider the following nucleotide sequence on the template strand of DNA and the amino acids that it encodes: Description The D N A strand shows the following sequence: 3 prime-A A A, T C A, C T T, G G C, G T A, C A A-5 prime. The m R N A obtained below shows the following sequence: 5 prime-U U U, A G U, G A A, C C G, C A U, G U U-3 prime. The corresponding amino acids shown below are as follows: Phe, Ser, Glu, Pro, His, Val. The nucleotides are underlined in groups of three nucleotides. Suppose that a one-base deletion occurs in the ﬁrst nucleotide of the second codon. This deletion shi s the reading frame by one nucleotide and alters all the amino acids that follow the mutation: 1211 Description The D N A strand at top right shows the following sequence: 3 prime-A A A, T (crossed out) C A C, T T G, G C G, T A C, A A 5-prime. A caption from crossed out nucleotide reads, one-nucleotide deletion. the m R N A obtained below shows the following sequence: 5 prime-U U U, G U G, A A C, C G C, A U G, U U-3 prime. The corresponding amino acids shown below are as follows: Phe, Val, Asn, Arg, Met. The nucleotides are underlined in groups of three nucleotides. If a single nucleotide is added to the third codon (the suppressor mutation), the reading frame is restored, although two of the amino acids differ from those speciﬁed by the original sequence: Description The D N A strand at top right shows the following sequence: 3 prime-A A A, C A C, T T T (highlighted), G G C, G T A, C A A 5-prime. The highlighted nucleotide shows a caption that reads, one nucleotide insertion. The m R N A obtained below shows the following sequence: 5 prime-U U U, G U G, A A A, C C G, C A U, G U U-3 prime. The corresponding amino acids shown below are as follows: Phe, Val, Lys, Pro, His, Val. The nucleotides are underlined in groups of three nucleotides. Similarly, a mutation due to an insertion may be suppressed by a subsequent deletion in the same gene. A third way in which an intragenic suppressor mutation may work is by making compensatory changes in the protein. A ﬁrst missense mutation can alter the folding of a polypeptide chain by changing the way in which amino acids in the protein interact with one another. A second missense mutation at a different site (the suppressor mutation) can re-create the original folding pattern by restoring the interactions between the amino acids. INTERGENIC SUPPRESSOR MUTATIONS An intergenic suppressor mutation, in contrast, occurs in a gene other than the one bearing the original mutation that it suppresses. These mutations, also known as extragenic suppressor mutations, sometimes work by changing the way the mRNA is translated. In the example illustrated in Figure 18.9a, the original DNA sequence is AAC (UUG in the mRNA) and speciﬁes leucine. This sequence mutates to ATC (UAG in the mRNA), a stop codon (Figure 18.9b). The ATC nonsense mutation could be suppressed by a second mutation in a different gene that encodes a tRNA; this second mutation would result in a tRNA anticodon capable of pairing with the UAG stop codon (Figure 18.9c). For example, the gene that encodes the tRNA for tyrosine (tRNATyr), which has the anticodon AUA, might be mutated to have the anticodon AUC, which would then pair with the UAG stop codon. Instead of translation terminating at the UAG codon, tyrosine 1212 would be inserted into the protein, and a full-length protein would be produced, although tyrosine would now substitute for leucine. The effect of this change would depend on the role of this amino acid in the overall structure of the protein, but the effect of the suppressor mutation would probably be less detrimental than the effect of the nonsense mutation, which would halt translation prematurely. 18.9 An intergenic suppressor mutation occurs in a gene other than the one bearing the original mutation that it suppresses. (a) A wild-type sequence produces a full-length, functional protein. (b) A base substitution at a site in that gene produces a premature stop codon, resulting in a truncated, nonfunctional protein. (c) A base substitution at a site in another gene, which in this case encodes tRNA, alters the anticodon of tRNATyr; tRNATyr can then pair with the stop codon produced by the original mutation, allowing tyrosine to be incorporated into the protein and translation to continue. Description Part a is labeled wild type sequence. A double D N A strand is present at the top. The sequence of the top strand is as follows: T, T, G. The sequence of the bottom strand is as follows: A, A, C. Transcription occurs and an m R N A is obtained below with the following sequence: U, U, G. Translation occurs and a ribosome with complementary bases at its t R N A (A, A, C) attaches to the m R N A bases U, U, G. A leucine is obtained from the ribosome. Text reads, Leu is incorporated into a protein. A full-length, functional protein is obtained below. Part b is labeled wild base substitution. A double D N A strand is present at the top. The sequence of the top strand is as follows: T, A (highlighted), G. The sequence of the bottom strand is as follows: A, T (highlighted), C. Transcription occurs and an m R N A is obtained below with the following sequence: U, A, G. The sequence is labeled stop codon. Translation occurs and a ribosome attaches to the m R N A bases U, A, G. Translation is terminated. A caption reads, Protein synthesis is halted, resulting in a nonfunctional protein. A Shortened, nonfunctional protein is obtained below. 1213 Part c is labeled Base substitution at a second site. A double D N A strand is present at the top left and is labeled site 1 (first mutation). The sequence of the top strand is as follows: T, A (highlighted), G. The sequence of the bottom strand is as follows: A, T (highlighted), C. Another double D N A strand is present to the right and is labeled site 2. A caption reads, At site 2 is a gene encoding tyrosine t R N A. The sequence of the top strand is as follows: A, T, A. The sequence of the bottom strand is as follows: T, A, T. A t R N A with bases A, U, A is present to the right. A caption reads, Normal transcription produces a t R N A with an anticodon A U A (which would pair with the tyrosine codon U A U in translation). Second base substitution Mutation occurs at site 2 and a D N A double strand is obtained below. The sequence of the top strand is as follows: C, T, A. The sequence of the bottom strand is as follows: G (highlighted), A, T. Transcription occurs for site 1 and base substituted D N A obtained from site 2. For site 1, the m R N A produced below has the following sequence: U, A, G. A t R N A with bases A, U, C is obtained from the base substituted D N A double strand after transcription. T y r enters the process and translation occurs for m R N A obtained and t R N A obtained. The ribosome with t R N A (A, U, C) attaches on the m R N A (U, A, G). A t y r amino acid is obtained from the ribosome. A full-length, functional protein is obtained below. A caption reads, Introduction of an incorrect base (G) results in a mutant t R N A that has anticodon A U C (instead of A U A) which can pair with the stop codon U A G. A forward arrow indicates the ribosome will move. A caption reads, Translation continues past the stop codon, and T y r is incorporated into the protein. Because cells in many organisms have multiple copies of tRNA genes, other nonmutated copies of tRNATyr would remain available to recognize tyrosine codons in mRNA transcripts. We might expect that the tRNAs encoded by the gene with the suppressor mutation just described would suppress the normal stop codons at the ends of other coding sequences as well as the one in the transcript of the original mutant gene, resulting in the production of longer-than-normal proteins, but this event does not usually take place. Intergenic suppressor mutations can also work through gene interactions (see Chapter 5). For example, polypeptide chains that are produced by two different genes may interact to produce a functional protein. A mutation in one gene may alter the encoded polypeptide such that the interaction between the two polypeptides is destroyed, in which case a functional protein is not produced. A suppressor mutation in the second gene may produce a compensatory change in its polypeptide, therefore restoring the original interaction. Characteristics of some of the different types of mutations are summarized in Table 18.2. TABLE 18.2 Characteristics of diﬀerent types of mutations Type of Mutation Definition Base substitution Transition Base substitution in which a purine replaces a purine or a pyrimidine replaces a pyrimidine Transversion Base substitution in which a purine replaces a pyrimidine or a pyrimidine replaces a purine Insertions and deletions (indels) Insertion Addition of one or more nucleotides Deletion Deletion of one or more nucleotides Frameshi mutation Insertion or deletion that alters the reading frame of a gene In-frame deletion or Deletion or insertion of a multiple of three nucleotides that does not alter the reading frame insertion Expanding nucleotide Repeated sequence of a set of nucleotides in which the number of copies of the sequence increases 1214 repeats Direction Forward mutation Changes the wild-type phenotype to a mutant phenotype Reverse mutation Changes a mutant phenotype back to the wild-type phenotype Eﬀects Missense mutation Changes a sense codon into a diﬀerent sense codon, resulting in the incorporation of a diﬀerent amino acid in the protein Nonsense mutation Changes a sense codon into a nonsense (stop) codon, causing premature termination of translation Silent mutation Changes a sense codon into a synonymous codon, leaving unchanged the amino acid sequence of the protein Neutral mutation Changes the amino acid sequence of a protein without altering its ability to function Loss-of-function mutation Causes a complete or partial loss of function Gain-of-function mutation Causes the appearance of a new trait or function or causes the appearance of a trait in inappropriate tissue or at an inappropriate time Lethal mutation Causes premature death Suppression Suppressor mutation Suppresses the eﬀect of an earlier mutation at a diﬀerent site Intragenic suppressor Suppresses the eﬀect of an earlier mutation within the same gene mutation Intergenic suppressor Suppresses the eﬀect of an earlier mutation in another gene mutation CONCEPTS A suppressor mutation overrides the eﬀect of an earlier mutation at a diﬀerent site. An intragenic suppressor mutation occurs within the same gene that contains the original mutation; an intergenic suppressor mutation occurs in a diﬀerent gene. CONCEPT CHECK 2 How is a suppressor mutation diﬀerent from a reverse mutation? WORKED PROBLEM A gene encodes a protein with the following amino acid sequence: Met-Arg-Cys-Ile-Lys-Arg A mutation of a single nucleotide alters the amino acid sequence to Met-Asp-Ala-Leu-Lys-Gly-Glu-Ala-Pro-Val A second single-nucleotide mutation occurs in the same gene and suppresses the effects of the ﬁrst mutation (an intragenic suppressor). With the original mutation and the intragenic suppressor present, the protein has the following amino acid sequence: Met-Asp-Gly-Ile-Lys-Arg 1215 What is the nature and location of the ﬁrst mutation and the intragenic suppressor mutation? Solution Strategy What information is required in your answer to the problem? The nature and location of the ﬁrst mutation and the intragenic suppressor mutation. What information is provided to solve the problem? The amino acid sequence of the protein encoded by the original nonmutated gene. The amino acid sequence of the protein encoded by the mutated gene. The amino acid sequence of the protein encoded by the mutated gene and the intragenic suppressor. Solution Steps The ﬁrst mutation alters the reading frame, because all amino acids a er Met are changed, including the stop codon (which results in a longer protein). Insertions and deletions affect the reading frame; the original mutation consists of a single-nucleotide insertion or deletion in the second codon. The intragenic suppressor restores the reading frame; the intragenic suppressor is most likely a single-nucleotide insertion or deletion. If the ﬁrst mutation is an insertion, the suppressor must be a deletion; if the ﬁrst mutation is a deletion, then the suppressor must be an insertion. Notice that the protein produced by the suppressor still differs from the original protein at the second and third amino acids, but the second amino acid produced by the suppressor is the same as that in the protein produced by the original mutation. Thus, the suppressor mutation must have occurred in the third codon, because the suppressor does not alter the second amino acid. For more practice with analyzing mutations, try working Problem 24 at the end of the chapter. Mutation Rates The frequency with which a wild-type allele at a locus changes into a mutant allele is referred to as the mutation rate. The mutation rate is generally expressed as the number of mutations per biological unit, which may be mutations per cell division, per gamete, or per round of replication. For example, achondroplasia is a type of hereditary dwarﬁsm in humans that results from a dominant mutation. On average, about four achondroplasia mutations arise in every 100,000 gametes, and so the mutation rate is 4 /100,000 , or 0.00004 mutations per gamete. The mutation rate provides information about how o en a mutation arises. FACTORS AFFECTING MUTATION RATES 1216 Calculations of mutation rates are affected by three factors. First, they depend on the frequency with which changes in DNA take place. Mutations can arise as spontaneous molecular changes in DNA, or they can be induced by chemical, biological, or physical agents in the environment. The second factor inﬂuencing the mutation rate is the probability that when an alteration in DNA takes place, it will be repaired. Most cells possess a number of mechanisms for repairing altered DNA (see Section 18.5), so most alterations are corrected before they are replicated. If these repair systems are effective, mutation rates will be low; if they are faulty, mutation rates will be elevated. Some mutations increase the overall rate of mutation at other genes; these mutations usually occur in genes that encode components of the replication machinery or DNA-repair enzymes. The third factor is the probability that a mutation will be detected. When DNA is sequenced, all mutations are potentially detectable. In practice, however, mutations are usually detected by their phenotypic effects. Some mutations may appear to arise at a higher rate simply because they are easier to detect. VARIATION IN MUTATION RATES We can draw several general conclusions about mutation rates, though they vary among genes and among species (Table 18.3). First, spontaneous mutation rates are low for all organisms studied. Typical mutation rates for bacterial genes range from about 1 to 100 mutations per 10 billion cells (from 1 × 10−8 to 1 × 10−10). The mutation rates for most eukaryotic genes are a bit higher, from about 1 to 10 mutations per million gametes (from 1 × 10−5 to 1 × 10−6). These higher values in eukaryotes may be due to the fact that the rates are calculated per gamete, and that several cell divisions are required to produce a gamete, whereas mutation rates in prokaryotic cells are calculated per cell division. TABLE 18.3 Mutation rates of diﬀerent genes in diﬀerent organisms Organism Mutation Rate Unit Influenza A Nonstructural genes 2.0 × 10−6 Per replication Bacteriophage T2 Lysis inhibition 1 × 10−8 Per replication Host range 3 × 10−9 Escherichia coli Lactose fermentation 2 × 10−7 Per cell division Histidine requirement 2 × 10−8 Neurospora crassa Inositol requirement 8 × 10−8 Per asexual spore Adenine requirement 4 × 10−8 Corn Kernel color 2.2 × 10−6 Per gamete Drosophila Eye color 4 × 10−5 Per gamete Allozymes 5.14 × 10−6 Mouse Albino coat color 4.5 × 10−5 Per gamete Dilution coat color 3 × 10−5 Human Huntington disease 1 × 10−6 Per gamete Achondroplasia 1 × 10−5 Neurofibromatosis (Michigan) 1 × 10−4 Hemophilia A (Finland) 1217 Duchenne muscular dystrophy (Wisconsin) 3.2 × 10−5 9.2 × 10−5 The differences in mutation rates among species may be due to differing abilities to repair mutations, unequal exposures to mutagens, or biological differences in rates of spontaneous mutations. Even within a single species, spontaneous mutation rates vary among genes. The reason for this variation is not entirely understood, but some regions of DNA are known hotspots for mutations. Recent research suggests that fewer mutations occur in DNA sequences that are associated with nucleosomes (see Chapter 11). Reduced mutation rates may occur in these sequences because DNA associated with nucleosomes is less exposed to mutagens, but they could also be explained by the effect of nucleosomes on DNA repair, recombination, or replication, all of which inﬂuence the rate of mutation. Several recent studies have measured mutation rates directly by sequencing genes of organisms over a number of generations. These new studies suggest that mutation rates are o en higher than those previously measured on the basis of changes in phenotype. In one study, geneticists sequenced randomly chosen stretches of DNA in the nematode Caenorhabditis elegans and found about 2.1 mutations per genome per generation, which was 10 times higher than previous estimates based on phenotypic changes. The researchers found that about half of the mutations were insertions and deletions. Recent genome sequencing has also provided more accurate information about mutation rates in humans. Several sequencing studies suggest that the overall rate of base substitutions in humans is about 1 × 10−8 mutations per base pair per generation. Other research suggests that each person carries approximately 100 new loss-of-function germ-line mutations. THINK-PAIR-SHARE Question 3 ADAPTIVE MUTATION As will be discussed in Chapters 24 through 26, genetic variation is critical for evolutionary change that brings about adaptation to new environments. New genetic variants arise primarily through mutation. For many years, genetic variation was assumed to arise randomly and at rates that are independent of the need for adaptation. However, some evidence suggests that stressful environments—where adaptation may be necessary to survive—can induce more mutations in bacteria, a process that has been termed adaptive mutation. The idea of adaptive mutation has been intensely debated; critics counter that most mutations are expected to be deleterious, and therefore increased mutagenesis would probably be harmful most of the time. CONCEPTS The mutation rate is the frequency with which a specific mutation arises. Rates of mutations are generally low and are aﬀected by environmental and genetic factors. 1218 CONCEPT CHECK 3 What three factors aﬀect mutation rates? 1219 18.2 Mutations May Be Caused by a Number of Diﬀerent Factors Mutations result from both internal and external factors. Those that occur under normal conditions are termed spontaneous mutations, whereas those that result from changes caused by environmental chemicals or radiation are induced mutations. Spontaneous Replication Errors Replication is amazingly accurate: less than one error in a billion nucleotides arises in the course of DNA synthesis (see Chapter 12). However, spontaneous replication errors do occasionally occur. THINK-PAIR-SHARE Question 4 TAUTOMERIC SHIFTS In their 1953 paper describing DNA structure, Watson and Crick (see Chapter 10) originally proposed that the primary cause of spontaneous replication errors is tautomeric shi s, in which the positions of protons (hydrogen atoms) in the DNA bases change. Each of the four DNA bases exists in different chemical forms, called tautomers (Figure 18.10a). The two tautomeric forms of each base are in dynamic equilibrium, although one form is much more common than the other. The standard Watson-and-Crick base pairings— adenine with thymine, and cytosine with guanine—occur between the common forms of the bases, but if the bases are in their rare tautomeric forms, other base pairings are possible (Figure 18.10b). For example, the common form of cytosine pairs with guanine, but the rare tautomer of cytosine pairs with adenine. 1220 18.10 Purine and pyrimidine bases exist in diﬀerent forms called tautomers. (a) A tautomeric shi takes place when a proton changes its position, resulting in a rare tautomeric form. (b) Standard and anomalous base-pairing arrangements that arise if bases are in the rare tautomeric forms. Description Part a on the left shows common and rare forms of thymine, Guanine, Cytosine, and Adenine. For Thymine: A tautomeric shift in a common form of thymine results in a rare form of thymine. The common form of thymine has C 5 bonded to a methyl group. The hydrogen atom bonded to N 1 is highlighted. A proton shift takes place from N 1 to the oxygen atom at C 6. The rare form of thymine has a similar structure as that of common form, except that C 6 is now bonded to a hydroxyl group, in which the hydrogen atom is highlighted. For Guanine: A tautomeric shift in a common form of guanine results in a rare form of guanine. The common form of guanine has C 2 bonded to amino group. A proton shift takes place from N 1 to carbonyl group of C 6. The rare form of guanine has a similar structure as that of common form, except that C 6 is now bonded to a hydroxyl group, in which hydrogen atom is highlighted. For Cytosine: 1221 A tautomeric shift in a common form of cytosine results in a rare form of cytosine. The common form of cytosine has C 4 bonded to amino group. The hydrogen atom bonded to N 1 is highlighted. A proton shift takes place from the amino group at C 4 to N 3. The rare form of cytosine has a similar structure as that of common form, except that N 3 is now bonded to a hydrogen atom, in which the hydrogen atom is highlighted. For Adenine: An illustration shows a tautomeric shift in a common form of adenine, resulting in a rare form of adenine. The common form of adenine has C 6 bonded to an amino group, in which one of the two hydrogen atoms is highlighted. A proton shift takes place from the amino group at C 6 to N 1. The rare form of adenine has a similar structure as that of common form, except that N 1 is now bonded to a hydrogen atom, highlighted and C 6 is double bonded to an N H group. Part b on the right shows Standard base-pairing arrangements and anomalous base-pairing arrangements. Standard base-pairing arrangements show: Thymine (common form) and adenine (common form) show double bonds where hydrogen atom of N 1 of thymine is bonded to N 1 of adenine. The oxygen atom at C 6 is bonded to one of the hydrogen atoms of amino group at C 6 of adenine. Cytosine (common form) and Guanine (common form) show triple bonds. One of the H atoms from amino group at C 4 of cytosine is bonded to carbonyl O atom at C 6 of guanine. N 3 atom of cytosine is bonded to H atom at N 1 of guanine. One carbonyl O at C 2 of cytosine is bonded to one of the H atom of amino group at C 2 of guanine. Anomalous base-pairing arrangements show: Cytosine (rare form) and adenine (common form) show double bonds. N atom of the N H group at C 4 of cytosine is bonded to one of the H atom of amino group at C 6 of adenine. H atom bonded to N 3 of cytosine is bonded to N 1 of adenine. Thymine (common form) and Guanine (rare form) show triple bonds. One of the carbonyl O at C 4 of thymine is bonded to H atom of hydroxyl group at C 6 of guanine. One of the H atoms of N 3 of thymine is bonded to N 1 of guanine. One of the carbonyl O of thymine is bonded to one of the H atom of amino group at C 2 of guanine. Although Watson and Crick proposed that tautomeric shi s might produce mutations, tautomeric forms of the bases were difﬁcult to detect in DNA, and evidence of their role in mutation was lacking. Many researchers assumed that other structures, such as protonated bases (see the following discussion), were responsible for base mispairing during replication. Recently, however, researchers have detected base mispairings in DNA that involve rare base tautomers. MISPAIRING DUE TO OTHER STRUCTURES Mispairings between bases in DNA can arise through wobble (see Chapter 15), in which normal, protonated, and other forms of the bases are able to pair because of ﬂexibility in the DNA helical structure (Figure 18.11). These structures have been detected in DNA molecules and are responsible for some mispairings in replication. 1222 18.11 Nonstandard base pairings can occur as a result of the flexibility in DNA structure. Thymine and guanine in their normal forms can pair through wobble. Cytosine and adenine can pair through wobble when adenine is protonated (has an extra hydrogen atom). Description Thymine-guanine wobble shows a double bond between thymine and guanine. One of the H atom at N 3 of thymine is bonded to carbonyl O of guanine. The O bonded to C 2 of thymine is bonded to an H atom of N 1 of guanine. Cytosine-adenine protonated wobble shows a double bond. N 3 of Cytosine is bonded to one of the H atoms of amino group at C 6 of adenine. The O atom bonded to C 2 of cytosine is bonded to the H atom of N 1, which carries a positive change, of adenine. INCORPORATED ERRORS AND REPLICATED ERRORS When a base substitution causes a mispaired base to be incorporated into a newly synthesized nucleotide chain, an incorporated error is said to have occurred. Suppose that in replication, thymine (which normally pairs with adenine) mispairs with guanine through wobble (Figure 18.12). In the next round of replication, the two mismatched bases separate, and each serves as a template for the synthesis of a new nucleotide strand. This time, thymine pairs with adenine, producing another copy of the original DNA sequence. On the other strand, however, the incorrectly incorporated guanine serves as the template and pairs with cytosine, producing a new DNA molecule that has an error: a C G pair in place of the original T A pair (a T A → C G base substitution). The original incorporated error (the T–G mispairing) leads to a replicated error (the C G base pair instead of the original T A base pair), which creates a permanent mutation because all the base pairings are correct and there is no way for repair systems to detect the error. 1223 18.12 Wobble base pairing may lead to a replicated error. Description The illustration shows a D N A double strand. The sequence of the top strand is as follows: T, T, C, G. The sequence of the bottom strand is as follows: A, A, G, C. The strands separate and a caption reads, D N A strands separate for replication. A forward arrow divides into two on the right. The arrow on the top leads to a strand with the following sequence: T, T, C, G. It further leads to a double strand. The top strand reads the following: T, T, C, G. The complementary bottom strand is: A, G, G, C. The nucleotides in the center bulge outside. A caption reads, Thymine on the original template strand base pairs with guanine through wobble, leading to an incorporated error. Both the strands separate. A forward arrow from the strands divides into two. The top strand now (T, T, C, G) further changes to the wild type D N A double strand with T, T, C, G at the top strand and A, A, G, C at the bottom strand. The bottom strand (A, G, G, C) further changes to mutant type D N A double strand with T, C, C, G at the top strand and A, G, G, C at the bottom strand. A caption reads, At the next round of replication, the guanine nucleotide pairs with cytosine, leading to a transition mutation. The arrow at the bottom leads to strand A A G C, that further leads to a wild type D N A double strand with T T C G at top strand and A A G C at bottom strand. CAUSES OF DELETIONS AND INSERTIONS Small insertions and deletions can arise spontaneously in replication and crossing over. Strand slippage can occur when one nucleotide strand forms a small loop (Figure 18.13). If the looped-out nucleotides are on the newly synthesized strand, an insertion results. At the next round of replication, the insertion will be replicated, and both strands will contain the insertion. If the looped-out nucleotides are on the template strand, then the newly replicated strand will have a deletion, and this deletion will be perpetuated in subsequent rounds of replication. 1224 18.13 Insertions and deletions may result from strand slippage. Description The illustration shows a newly synthesized strand with the following sequence: 5 prime-T, A, C, G, G, A, C, T, G, A, A, A, A-3 prime. Template strand below has the following sequence: 3 prime-A, T, G, C, C, T, G, A, C, T, T, T, T, T, G, C, G, A, A ,G-5 prime. A downward arrow divides into two arrows below. The arrow on the left shows last second nucleotide of the newly synthesized strand loops out. The top strand now is as follows: A, C, G, G, A, C, T, G, A, A, A (looped out), A. The bottom strand is as follows: T, G, C, C, T, G, A, C, T, T, T, T, T, G, C, G, A, A. It then leads to a double strand below. The top strand now is as follows: A, C, G, G, A, C, T, G, A, A, A (looped out), A, A, A, C, G, C, T, T. The bottom strand is as follows: T, G, C, C, T, G, A, C, T, T, T, T, T, G, C, G, A, A. A caption reads, Newly synthesized strand loops out resulting in the addition of one nucleotide on the new strand. The arrow on the right shows a T nucleotide on the template strand loops out. The top strand now is as follows: A, C, G, G, A, C, T, G, A, A, A, A. The bottom strand is as follows: T, G, C, C, T, G, A, C, T, T, T (looped out), T, T, G, C, G, A, A. The top strand now is as follows: A, C, G, G, A, C, T, G, A, A, A, A, C, G, C, T, T. The bottom strand is as follows: T, G, C, C, T, G, A, C, T, T, T (looped out), T, T, G, C, G, A, A. A caption reads, Template strand loops out. This further leads to a D N A double strand below resulting in the omission of one nucleotide on the new strand. Another process that produces insertions and deletions is unequal crossing over (see Section 8.2). In normal crossing over, the homologous sequences of the two DNA molecules align, and crossing over produces no net change in the number of nucleotides in either molecule. Misaligned pairing, however, can cause unequal crossing over, which results in one DNA molecule with an insertion and the other with a deletion (Figure 18.14). 1225 18.14 Unequal crossing over produces insertions and deletions. Description The illustration shows two pairs of rod-shaped homologous chromosomes at the top. The first pair, orange- colored, at the top has the following sequence: Top chromosome: A, A, T, T, A, A, T, T; Bottom chromosome: T, T, A, A, T, T, A, A. The second pair, blue colored, below has the following sequence: Top chromosome: A, A, T, T, A, A, T, T; Bottom chromosome: T, T, A, A, T, T, A, A. Unequal crossing over takes place. The first longer, rod shaped chromosome pair obtained below has both orange and blue colored regions. The top chromosome obtained has the following sequence: Orange: A, A, T, T, A, A, T, T; blue: A, A, T, T. The bottom chromosome obtained has the following sequence: Orange: T, T, A, A, T, T, A, A; blue: T, T, A, A. The second short, rod shaped chromosome pair obtained below has both orange and blue colored regions. The top chromosome obtained has the following sequence: Blue: A, A, T, T; orange: no nucleotides present. The bottom chromosome obtained has the following sequence: Blue: T, T, A, A; orange: no nucleotides present. Captions throughout read, If homologous chromosomes misalign during crossing over, one crossover product contains an insertion and the other has a deletion. CONCEPTS Spontaneous replication errors arise from altered base structures and from wobble. Small insertions and deletions can occur through strand slippage during replication and through unequal crossing over. Spontaneous Chemical Changes 1226 In addition to spontaneous mutations that arise in replication, mutations also result from spontaneous chemical changes in DNA. One such change is depurination, the loss of a purine base from a nucleotide. Depurination results when the covalent bond connecting the purine to the 1′-carbon atom of the deoxyribose sugar breaks (Figure 18.15a), producing an apurinic site, a nucleotide that lacks its purine base. An apurinic site cannot act as a template for a complementary base in replication. In the absence of base-pairing constraints, an incorrect nucleotide (most o en adenine) is incorporated into the newly synthesized DNA strand opposite the apurinic site (Figure 18.15b), frequently leading to an incorporated error. The incorporated error is then transformed into a replicated error at the next round of replication. Depurination is a common cause of spontaneous mutation; a mammalian cell in culture loses approximately 10,000 purines every day. Loss of pyrimidine bases also occurs, but at a much lower rate than depurination. 18.15 Depurination (the loss of a purine base from a nucleotide) may lead to a base substitution. (a) Depurination occurs when the covalent bond connecting a purine to the 1′ carbon of the sugar is broken (indicated by dotted red line). (b) Replication of a template strand with an apurinic site may lead to an incorporated error. Description Part a on the left shows a D N A sugar phosphate backbone running from 5 prime at the top to 3 prime at the bottom. The pentose sugars have bases on the C 1 and phosphate molecules on the C 4. The first sugar molecule at the top has pyrimidine T attached to it. The sugar below it has purine G attached. The bond that joins purine and sugar is dissected by a red, vertical dotted line. The sugar below has purine G attached. Part b on the right shows a D N A double strand on the left side. The sequence of the top strand is as follows: T, G, G, C. The sequence of the bottom strand is as follows: A, C, C, G. One G nucleotide from the top strand gets separated, indicating depurination. A forward arrow leads to the D N A double strand obtained. The sequence of the top strand is as follows: T, apurinic site, G, C. The sequence of the bottom strand is as follows: A, C, C, G. Strand separation occurs and is represented by a forward arrow dividing into two arrows. The arrow at the top leads to the strand with the following sequence: T, blank, G, C. A caption reads, In replication, the apurinic site cannot provide a template for a complementary base on the newly synthesized strand. It further leads to a double strand. The sequence of the top strand is as follows: T, blank, G, C. The sequence of the bottom strand is as follows: A, A (highlighted), C, G. A caption reads, A nucleotide with the incorrect base (most often A) is incorporated into the newly synthesized strand. Strand separation occurs and is represented by a forward arrow that divides into two. The arrow at the top leads to template strands: T, blank, G, 1227 C. Replication occurs and a mutant D N A double strand is obtained. The top strand has the following sequence: T, blank, G, C. The bottom strand has the following sequence: A, A (highlighted), C, G. A caption reads, A nucleotide is incorporated into the newly synthesized strand opposite the apurinic site. The arrow at the bottom leads to the following strand: A, A (highlighted), C, G. Replication occurs and a D N A double strand is obtained. The top strand has the following sequence: T, T (highlighted), G, C. The bottom strand has the following sequence: A, A (highlighted), C, G. A caption reads, At the next round of replication, this incorrectly incorporated base will be used as a template, leading to a permanent mutation. The arrow at the bottom, after first strand separation, leads to the following strand: A, C, C, G. Replication occurs and a D N A double strand is obtained on the right. The top strand has the following sequence: T, G, G, C. The bottom strand has the following sequence: A, C, C, G. Text below reads, Normal D N A molecule (no mutation). Another spontaneously occurring chemical change that takes place in DNA is deamination, the loss of an amino group (NH2) from a base. Deamination may be spontaneous or may be induced by mutagenic chemicals. Deamination can alter the pairing properties of a base: the deamination of cytosine, for example, produces uracil (Figure 18.16a), which pairs with adenine in replication. A er another round of replication, the adenine will pair with thymine, creating a T A pair in place of the original C G pair (C G → U A → T A); this chemical change is a transition mutation. This type of mutation is usually prevented by enzymes that remove uracil whenever it is found in DNA. The ability of these enzymes to recognize the product of cytosine deamination may explain why thymine, not uracil, is found in DNA. In mammals, including humans, some cytosine bases in DNA are naturally methylated and exist in the form of 5-methylcytosine (5mC) (see Figure 10.20). When deaminated, 5mC becomes thymine (Figure 18.16b). Because thymine pairs with adenine in replication, the deamination of 5-methylcytosine changes an original C G pair to T A (C G → 5mC G → T G → T A). Consequently, C G → T A transitions are frequent in mammalian cells, and 5mC sites are mutation hotspots in humans. ❯ TRY PROBLEM 28 18.16 Deamination alters DNA bases. Description The part a on the left shows cytosine with an amino group on its C 4 (highlighted in green). A forward arrow represents deamination where an amino group is removed. Uracil is obtained on the right. The part b on the right shows 5 methyl cytosine (5 m C) which has a methyl group at C 5 (highlighted in red) and an amino group C 4 (highlighted in green). A forward arrow represents deamination where an amino group is removed. Thymine is obtained on the right. The methyl group at its C 5 is highlighted in red. 1228 CONCEPTS Some mutations arise from spontaneous alterations in DNA structure, such as depurination and deamination, which can alter the pairing properties of the bases and cause errors in subsequent rounds of replication. Chemically Induced Mutations Although many mutations arise spontaneously, a number of environmental agents are capable of damaging DNA, including certain chemicals and radiation. Any environmental agent that signiﬁcantly increases the rate of mutation above the spontaneous rate is called a mutagen. The ﬁrst discovery of a chemical mutagen was made by Charlotte Auerbach, who started her career in Berlin researching the development of mutants in Drosophila. Faced with increasing anti-Semitism in Nazi Germany, Auerbach immigrated to Britain in 1933. There she continued her research on Drosophila and, in 1940, began a collaboration with pharmacologist John Robson at the University of Edinburgh on the mutagenic effects of mustard gas, which had been used as a chemical weapon in World War I. The experimental conditions were crude: they heated liquid mustard gas over a Bunsen burner on the roof of the pharmacology building and exposed the ﬂies to the gas in a large chamber. A er developing serious burns on her hands from the gas, Auerbach let others carry out the exposures, and she analyzed the ﬂies. Auerbach and Robson showed that mustard gas is indeed a powerful mutagen, reducing the viability of gametes and increasing the numbers of mutations seen in the offspring of exposed ﬂies. Because the research was part of a secret project during World War II, publication of their ﬁndings was delayed until 1947. BASE ANALOGS One class of chemical mutagens consists of base analogs, chemicals with structures similar to those of any of the four standard nitrogenous bases of DNA. DNA polymerases cannot distinguish these analogs from the standard bases, so if base analogs are present during replication, they may be incorporated into newly synthesized DNA molecules. For example, 5-bromouracil (5BU) is an analog of thymine; it has the same structure as thymine except that it has a bromine (Br) atom on the 5-carbon atom instead of a methyl group (Figure 18.17a). Normally, 5BU pairs with adenine just as thymine does, but it occasionally mispairs with guanine (Figure 18.17b), leading to a transition (T A → 5BU A → 5BU G → C G), as shown in Figure 18.18. Through mispairing, 5BU can also be incorporated into a newly synthesized DNA strand opposite guanine. In the next round of replication, 5BU pairs with adenine, leading to another transition (G C → G 5BU → A 5BU → A T). 1229 18.17 5-Bromouracil (a base analog) resembles thymine, except that it has a bromine atom in place of a methyl group on the 5- carbon atom. Because of the similarity in their structures, 5-bromouracil may be incorporated into DNA in place of thymine. Like thymine, 5-bromouracil normally pairs with adenine, but when ionized, it may pair with guanine through wobble. Description The part a on the left shows normal base thymine with a methyl group at C 5 highlighted. 5 bromouracil which is a base analog shows B r group at C 5 highlighted. The part b on the right shows a normal pairing between 5 bromouracil and adenine. The carbonyl O at C 4 of 5 bromouracil is bonded to one of the H atom of amino group at C 6 of adenine. One of the H atoms of N 3 of 5 bromouracil is bonded to N 1 of adenine. Mispairing between 5-Bromouracil (ionized) and guanine is shown on the right. N 3 with a negative charge of 5 bromouracil is bonded to H atom of N 1 of guanine. Carbonyl O bonded to C 2 is bonded to H atom of amino group at C 2 of guanine. 18.18 Incorporation of 5-bromouracil into a DNA strand can lead to a replicated error. Description The illustration shows a D N A double strand on the left. The sequence of the top strand is as follows: 3 prime G, A, C 5 prime. The sequence of the bottom strand is as follows: 5 prime C, T, G 3 prime. Two curved forward arrows represent strand separation. The arrow at the top leads to the following strand with sequence: G, A, C. It further leads to a D N A double strand. The sequence of the top strand is as follows: G, A, C. The sequence of the bottom strand is as follows: C, B (highlighted), G. B is labeled, incorporated error. A caption reads, In replication, 5-bromouracil may become incorporated into D N A in place of thymine, producing an incorporation error. A forward arrow dividing into two parts indicates strand separation. The top arrow leads to the following strand: G, A, C. This further leads to a D N 1230 A double strand. The sequence of the top strand is as follows: G, A, C. The sequence of the bottom strand is as follows: C, T, G. The bottom arrow leads to the following strand: C, B (highlighted), G. Replication occurs and a D N A double strand is obtained. The sequence of the top strand is as follows: G, G, C. The sequence of the bottom strand is as follows: C, B (highlighted), G. A caption reads, 5-Bromouracil may mispair with guanine in the next round of replication. A forward arrow dividing into two parts indicates strand separation. The top arrow leads to the following strand: G, G, C that further leads to a mutant D N A double strand with replicated errors. The sequence of the top strand is as follows: G, G (highlighted), C. The sequence of the bottom strand is as follows: C, C (highlighted), G. A caption reads, In the next replication, this guanine nucleotide pairs with cytosine, leading to a permanent mutation. The bottom arrow leads to the following strand: C, B (highlighted), G. Replication occurs and a D N A double strand is obtained. The sequence of the top strand is as follows: G, A, C. The sequence of the bottom strand is as follows: C, B (highlighted), G. A caption reads, If 5-bromouracil pairs with adenine, no replicated error occurs. The arrow at the bottom, after first strand separation, leads to the following strand: C, T, G. Replication occurs and a D N A double strand is obtained. The sequence of the top strand is as follows: G, A, C. The sequence of the bottom strand is as follows: C, T, G. A caption below reads, Conclusion: Incorporation of bromouracil followed by mispairing leads to a T A to C G transition mutation. Another mutagenic chemical is 2-aminopurine (2AP), which is a base analog of adenine. Normally, 2AP pairs with thymine, but it may mispair with cytosine, causing a transition (T A → T 2AP → C 2AP → C G). Alternatively, 2AP may be incorporated through mispairing into the newly synthesized DNA opposite cytosine and then later pair with thymine, leading to a C G → C 2AP → T 2AP → T A transition. In the laboratory, mutations caused by base analogs can be reversed by treatment with the same analog or by treatment with a different analog. ALKYLATING AGENTS Alkylating agents are chemicals that donate alkyl groups, such as methyl (CH3) and ethyl (CH3—CH2) groups, to nucleotide bases. For example, ethylmethylsulfonate (EMS) adds an ethyl group to guanine, producing O6-ethylguanine, which pairs with thymine (Figure 18.19a). Thus, EMS produces C G → T A transitions. EMS is also capable of adding an ethyl group to thymine, producing 4-ethylthymine, which then pairs with guanine, leading to a T A → C G transition. Because EMS produces both C G → T A and T A → C G transitions, mutations produced by EMS can be reversed by additional treatment with EMS. Mustard gas is another alkylating agent. 1231 18.19 Chemicals may alter DNA bases. Shown here are some examples of mutations produced by chemical agents. Description The table has 3 rows and 5 columns. The column headers are as follows: Original base, Mutagen, Modified base, pairing partner, and type of mutation. The row entries are as follows: Row 1: Original base: Guanine; Mutagen: E M S alkylation; Modified base and pairing partner: ethyl group bonded to O atom at C 6 is highlighted in O superscript 6 ethyl guanine, N 3 of O superscript 6 ethyl guanine is bonded to H atom of N 1 of thymine, one of the H atom of amino group at C 2 of O superscript 6 ethyl guanine is bonded to carbonyl O at C 2 of thymine; type of mutation: C G converts to T A and T A converts to C G. Row 2: Original base: Cytosine with amino group at C 5 highlighted; Mutagen: Nitrous acid (H N O superscript 2) Deamination; Modified base and pairing partner: Carbonyl O at C 4 of uracil is bonded to one of the H atom of amino group at C 6 of adenine, H atom of N 3 of uracil is bonded to N 1 of adenine; type of mutation: C G converts to T A and T A converts to C G. Row 3: Original base: Cytosine with amino group at C 5 highlighted; Mutagen: Hydroxylamine (N H subscript 2 O H) Hydroxylation; Modified base and pairing partner: N atom at C 4 of Hydroxyl aminocytosine has a hydroxyl group attached (highlighted) and is bonded to one of the H atom of amino group at C 6 of adenine, H atom of N 3 of Hydroxyl aminocytosine is bonded to N 1 of adenine; type of mutation: C G converts to T A. DEAMINATING CHEMICALS In addition to its spontaneous occurrence (see Figure 18.16), deamination can be induced by some chemicals. For instance, nitrous acid deaminates cytosine, creating uracil, which in the next round of replication pairs with adenine (Figure 18.19b), producing a C G → T A transition mutation. Nitrous acid also changes adenine into hypoxanthine, which pairs with cytosine, leading to a T A → C G transition. 1232 And nitrous acid also deaminates guanine, producing xanthine, which pairs with cytosine just as guanine does; however, xanthine can also pair with thymine, leading to a C G → T A transition. Nitrous acid produces exclusively transition mutations, and because both C G → T A and T A → C G transitions are produced, these mutations can be reversed with nitrous acid. HYDROXYLAMINE Hydroxylamine is a very speciﬁc base- modifying mutagen that adds a hydroxyl group to cytosine, converting it into hydroxylaminocytosine (Figure 18.19c). This conversion increases the frequency of a rare tautomer that pairs with adenine instead of guanine and leads to C G → T A transitions. Because hydroxylamine acts only on cytosine, it does not generate T A → C G transitions; thus, hydroxylamine will not reverse the mutations that it produces. ❯ TRY PROBLEM 26 OXIDATIVE RADICALS Reactive forms of oxygen (including superoxide radicals, hydrogen peroxide, and hydroxyl radicals) are produced in the course of normal aerobic metabolism as well as by radiation, ozone, peroxides, and certain drugs. These reactive forms of oxygen damage DNA and induce mutations by bringing about chemical changes in DNA. For example, oxidation converts guanine into 8-oxy-7,8-dihydrodeoxyguanine (Figure 18.20), which frequently mispairs with adenine instead of cytosine, causing a G C → T A transversion. 18.20 Oxidative radicals convert guanine into 8-oxy-7,8-dihydrodeoxyguanine. Description The illustration shows oxidative radicals convert guanine into 8-oxy-7,8-dihydrodeoxyguanine. The illustration shows guanine on the left. A forward arrow shows text that reads, oxidative radicals. The product, 8-Oxy-7,8- dihydrodeoxyguanine is shown on the right. It has a highlighted oxygen atom which is double bonded to C 8. Text below reads, may mispair with adenine. INTERCALATING AGENTS 1233 Proﬂavin, acridine orange, ethidium bromide, and dioxin are intercalating agents (Figure 18.21a), which produce mutations by sandwiching themselves (intercalating) between adjacent bases in DNA, distorting the three-dimensional structure of the helix and causing single-nucleotide insertions and deletions in replication (Figure 18.21b). These insertions and deletions frequently produce frameshi mutations, so the mutagenic effects of intercalating agents are o en severe. Because intercalating agents generate both insertions and deletions, they can reverse mutations they produce. 18.21 Intercalating agents are mutagens. Intercalating agents, such as proflavin and acridine orange (a), insert themselves between adjacent bases in DNA, distorting the three-dimensional structure of the DNA double helix (b). Description Part a on the left shows a proflavin at the top. An amino group is present at its C 3 and another amino group is present at C 6. Both the amino groups are highlighted. An Acridine orange is present below. The structure of Acridine orange is similar to that of proflavin except the N atoms at C 3 and C 6, have two methyl groups each instead of H atoms. All four methyl groups are highlighted. Part b on the right shows a double helical D N A strand with nitrogenous bases at the center. There is a thick Intercalated molecule after every two bases. The intercalated molecules distort the shape of the D N A double helix. CONCEPTS Mutagenic chemicals can produce mutations by a number of mechanisms. Base analogs are incorporated into DNA and frequently pair with the wrong base. Alkylating agents, deaminating chemicals, hydroxylamine, and oxidative radicals change the structure of DNA bases, thereby altering their pairing properties. Intercalating agents wedge between the bases and cause single-base insertions and deletions in replication. CONCEPT CHECK 4 Base analogs are mutagenic because of which characteristic? a. They produce changes in DNA polymerase that cause it to malfunction. b. They distort the structure of DNA. c. They are similar in structure to the normal bases. 1234 d. They chemically modify the normal bases. Radiation In 1927, Hermann Muller demonstrated that mutations in fruit ﬂies could be induced by X-rays. The results of subsequent studies showed that X-rays greatly increase mutation rates in all organisms. Because of their high energies, X-rays, gamma rays, and cosmic rays are all capable of penetrating tissues and damaging DNA. These forms of radiation, called ionizing radiation, dislodge electrons from the atoms they encounter, changing stable molecules into free radicals and reactive ions, which then alter the structures of bases and break phosphodiester bonds in DNA. Ionizing radiation also frequently results in double-strand breaks in DNA. Attempts to repair these breaks can produce chromosome mutations (discussed in Chapter 8). Ultraviolet (UV) light has less energy than ionizing radiation and does not dislodge electrons but is nevertheless highly mutagenic. Pyrimidine bases readily absorb UV light, which causes chemical bonds to form between adjacent pyrimidine molecules on the same strand of DNA, creating pyrimidine dimers (Figure 18.22a). Pyrimidine dimers consisting of two thymine bases (called thymine dimers) are most frequent, but cytosine dimers and thymine–cytosine dimers can also form. These dimers are bulky lesions that distort the conﬁguration of DNA (Figure 18.22b) and o en block replication. Most pyrimidine dimers are immediately repaired by mechanisms discussed in Section 18.5, but some escape repair and inhibit replication and transcription. 18.22 Pyrimidine dimers result from ultraviolet light. (a) Formation of a thymine dimer. (b) A thymine dimer distorts the DNA molecule. Description Part a on the left shows a sugar–phosphate backbone running from 3 prime at the top to 5 prime at the bottom. The two pentose sugars have thymine bases (T) on the C 1 and a phosphate molecule is present between C 4 of both the sugars. A forward arrow labeled covalent bonds is present. When the sugar phosphate backbone is exposed to U V light, two covalent bonds are also formed between the thymine bases. The covalent bonds are shown by thick red vertical lines between two corresponding carbon atoms of the thymine bases. C 2 of first 1235 thymine base is covalently bonded to C 2 of the second thymine base. C 3 of the first thymine base is covalently bonded to C 3 of second thymine base. Part b on the right shows a thymine dimer. The first thymine molecule has the following sequence from top to bottom: T, C, C, A, A, C, G, T, A, G. The second thymine molecule has the corresponding sequence: A, G, G, T double bonded to T, G, C, A, T, C. The double bond is represented by red, vertical parallel lines. When pyrimidine dimers block replication, cell division is inhibited and the cell usually dies; for this reason, UV light kills bacteria and is an effective sterilizing agent. For a mutation—a hereditary error in the genetic instructions—to occur, the replication block must be overcome. Bacteria can sometimes circumvent replication blocks produced by pyrimidine dimers and other types of DNA damage by means of the SOS system. This system can overcome these blocks and allow replication to proceed, but in the process, it makes numerous mistakes and greatly increases the rate of mutation. Indeed, the very reason that replication can proceed in the presence of a block is that the enzymes in the SOS system do not strictly adhere to the base-pairing rules. The trade-off is that replication continues and the cell survives, but only by sacriﬁcing the normal accuracy of DNA synthesis. CONCEPTS Ionizing radiation such as X-rays and gamma rays damages DNA by dislodging electrons from atoms; these electrons then break phosphodiester bonds and alter the structure of bases. Ultraviolet light causes mutations primarily by producing pyrimidine dimers that disrupt replication and transcription. The SOS system enables bacteria to overcome replication blocks but introduces mistakes in replication. 1236 18.3 Mutations Are the Focus of Intense Study by Geneticists Because mutations o en have detrimental effects, they are frequently studied by geneticists. These studies have included the development of tests to determine the mutagenic properties of chemical compounds and the investigation of human populations tragically exposed to high levels of radiation. Detecting Mutagens with the

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