Gene Mutation, DNA Repair, and Transposition (PDF)

14 Gene Mutation, DNA Repair, and Transposition CHAPTER CONCEPTS Mutations comprise any change in the nucleotide sequence of an organism’s genome....

14 Gene Mutation, DNA Repair, and Transposition CHAPTER CONCEPTS Mutations comprise any change in the nucleotide sequence of an organism’s genome. Mutations are a source of genetic varia- tion and provide the raw material for nat- ural selection. They are also the source Pigment mutations within an ear of corn, caused by transposition of the Ds element. of genetic damage that contributes to cell death, genetic diseases, and cancer. Mutations have a wide range of effects on organisms depending on the type of base-pair alteration, the location of the mutation within the chromosome, and T the function of the affected gene product. he ability of DNA molecules to store, replicate, transmit, and Mutations can occur spontaneously as decode information is the basis of genetic function. But equally a result of natural biological and chemi- important are the changes that occur to DNA sequences. Without cal processes, or they can be induced the variation that arises from changes in DNA sequences, there would by external factors, such as chemicals be no phenotypic variability, no adaptation to environmental changes, or radiation. and no evolution. Gene mutations are the source of new alleles and are Single-gene mutations cause a wide the origin of genetic variation within populations. On the downside, they variety of human diseases. are also the source of genetic changes that can lead to cell death, genetic Organisms rely on a number of DNA diseases, and cancer. repair mechanisms to counteract muta- Mutations also provide the basis for genetic analysis. The phenotypic tions. These mechanisms range from variations resulting from mutations allow geneticists to identify and study proofreading and correction of replica- the genes responsible for the modified trait. In genetic investigations, muta- tion errors to base excision and homolo- gous recombination repair. tions act as identifying “markers” for genes so that they can be followed during their transmission from parents to offspring. Without phenotypic Mutations in genes whose products variability, classical genetic analysis would be impossible. For example, if control DNA repair lead to genome hypermutability, human DNA repair all pea plants displayed a uniform phenotype, Mendel would have had no diseases, and cancers. foundation for his research. Transposable elements may move into We have examined mutations in large regions of chromosomes— and out of chromosomes, causing chro- chromosomal mutations! (see Chapter 6). In contrast, the mutations we mosome breaks and inducing muta- will now explore are those occurring primarily in the base-pair sequence tions both within coding regions and in of DNA within and surrounding individual genes—gene mutations. We gene-regulatory regions. will also describe how the cell defends itself from mutations using various mechanisms of DNA!repair. 261 M14_KLUG8414_10_SE_C14.indd 261 16/11/18 5:14 pm  262 14 GENE MUTATION, DNA REPAIR, AND TRANSPOSITION protein. This is known as a nonsense mutation. If the point 14.1 Gene Mutations Are Classified mutation alters a codon but does not result in a change in the in Various Ways amino acid at that position in the protein (due to degeneracy of the genetic code), it can be considered a silent mutation. A mutation can be defined as an alteration in the nucleotide Because eukaryotic genomes consist of so much more sequence of an organism’s genome. Any base-pair change in noncoding DNA than coding DNA!(see Chapter 11), the vast any part of a DNA molecule can be considered a mutation. majority of mutations are likely to occur in noncoding regions. A mutation may comprise a single base-pair substitution, a These mutations may be considered neutral mutations if deletion or insertion of one or more base pairs, or a major they do not affect gene products or gene expression. Most silent alteration in the structure of a chromosome. mutations, which do not change the amino acid sequence of Mutations may occur within regions of a gene that code the encoded protein, can also be considered neutral mutations. for protein or within noncoding regions of a gene such as You will often see two other terms used to describe introns and regulatory sequences, including promoters, base substitutions. If a pyrimidine replaces a pyrimidine or enhancers, and splicing signals. Mutations may or may not a purine replaces a purine, a transition has occurred. If a bring about a detectable change in phenotype. The extent to purine replaces a pyrimidine, or vice versa, a transversion which a mutation changes the characteristics of an organism has occurred. depends on which type of cell suffers the mutation and the Another type of change is the insertion or deletion of one degree to which the mutation alters the function of a gene or more nucleotides at any point within the gene. As illus- product or a gene-regulatory region. trated in Figure 14.1, the loss or addition of a single nucleo- Because of the wide range of types and effects of muta- tide causes all of the subsequent three-letter codons to be tions, geneticists classify mutations according to several changed. These are called frameshift mutations because different schemes. These organizational schemes are not the frame of triplet reading during translation is altered. A mutually exclusive. In this section, we outline some of the frameshift mutation will occur when any number of bases ways in which gene mutations are classified. are added or deleted, except multiples of three, which would reestablish the initial frame of reading!(see Figure 12.2). It is possible that one of the many altered triplets will be UAA, Classification Based on Type UAG, or UGA, the translation termination codons. When one of Molecular Change of these triplets is encountered during translation, polypep- Geneticists often classify gene mutations in terms of the tide synthesis is terminated at that point. Obviously, the nucleotide changes that constitute the mutation. A change results of frameshift mutations can be very severe, such as of one base pair to another in a DNA molecule is known as a producing a truncated protein or defective enzymes, espe- point mutation, or base substitution (see Figure 14.1). A cially if they occur early in the coding sequence. change of one nucleotide of a triplet within a protein-coding portion of a gene may result in the creation of a new triplet that codes for a different amino acid in the protein product. If Classification Based on Effect on Function this occurs, the mutation is known as a missense mutation. A As discussed earlier! (see Chapter 4), a loss-of-function second possible outcome is that the triplet will be changed into mutation is one that reduces or eliminates the function of a stop codon, resulting in the termination of translation of the the gene product. Mutations that result in complete loss of THE CAT SAW THE DOG Change of Loss of Gain of one letter one letter one letter Substitution Deletion Insertion THE BAT SAW THE DOG THE ATS AWT HED OG THE CMA TSA WTH EDO G THE CAT SAW THE HOG THE CAT SAT THE DOG Loss of C Insertion of M Point mutation Frameshift mutation Frameshift mutation F I G UR E 1 4.1 Analogy showing the effects of substitution, deletion, and insertion of one letter in a sentence composed of three-letter words to demonstrate point and frameshift mutations. M14_KLUG8414_10_SE_C14.indd 262 16/11/18 5:14 pm  14.1 GENE MUTATIONS ARE CLASSIFIED IN VARIOUS WAYS 263 function are known as null mutations. Any type of muta- A suppressor mutation can occur within the same gene that tion, from a point mutation to deletion of the entire gene, suffered the first mutation (intragenic mutation) or else- may lead to a loss of function. where in the genome (intergenic mutation). Most loss-of-function mutations are recessive. A Depending on their type and location, mutations can recessive mutation results in a wild-type phenotype when have a wide range of phenotypic effects, from none to severe. present in a diploid organism and the other allele is wild type. Some examples of mutation types based on their phenotypic In this case, the presence of less than 100 percent of the gene outcomes are listed in Table 14.1. product is sufficient to bring about the wild-type phenotype. Some loss-of-function mutations can be dominant. A dominant mutation results in a mutant phenotype in Classification Based on Location of Mutation a diploid organism, even when the wild-type allele is also Mutations may be classified according to the cell type or present. Dominant mutations in diploid organisms can have chromosomal locations in which they occur. Somatic muta- several different types of effects. A dominant negative tions are those occurring in any cell in the body except germ mutation in one allele may encode a gene product that cells, whereas germ-line mutations occur only in germ is inactive and directly interferes with the function of the cells. Autosomal mutations are mutations within genes product of the wild-type allele. For example, this can occur located on the autosomes, whereas X-linked and Y-linked when the nonfunctional gene product binds to the wild-type mutations are those within genes located on the X or Y gene product in a homodimer, inactivating or reducing the chromosome, respectively. activity of the homodimer. Mutations arising in somatic cells are not transmitted A dominant negative mutation can also result from to future generations. When a recessive autosomal muta- haploinsufficiency, which occurs when one allele is inacti- tion occurs in a somatic cell of an adult multicellular diploid vated by mutation, leaving the individual with only one func- organism, it is unlikely to result in a detectable phenotype. tional copy of a gene. The active allele may be a wild-type The expression of most such mutations is likely to be masked copy of the gene but does not produce enough wild-type gene by expression of the wild-type allele within that cell and the product to bring about a wild-type phenotype. In humans, presence of nonmutant cells in the remainder of the organ- Marfan syndrome is an example of a disorder caused by ism. Somatic mutations will have a greater impact if they are haploinsufficiency—in this case as a result of a loss-of- dominant or, in males, if they are X-linked, since such muta- function mutation in one copy of the fibrillin-1 (FBN1) gene. tions are most likely to be immediately expressed. In addi- In contrast, a gain-of-function mutation codes for a tion, the impact of dominant or X-linked somatic mutations gene product with enhanced, negative, or new functions. This will be more noticeable if they occur early in development, may be due to a change in the amino acid sequence of the pro- when a small number of undifferentiated cells replicate to tein that confers a new activity, or it may result from a muta- give rise to several differentiated tissues or organs. tion in a regulatory region of the gene, leading to expression Mutations in germ cells have the potential of being of the gene at higher levels or at abnormal times or places. expressed in all cells of an offspring. Inherited dominant Typically, gain-of-function mutations are dominant. autosomal mutations will be expressed phenotypically in A suppressor mutation is a second mutation that the first generation. X-linked recessive mutations arising either reverts or relieves the effects of a previous mutation. in the gametes of a female (the homogametic sex; having TABLE 14.1 Classifications of Mutations by Phenotypic Effects Classification Phenotype Example Visible Visible morphological trait Mendel’s pea characteristics Nutritional Altered nutritional characteristics Loss of ability to synthesize an essential amino acid in bacteria Biochemical Changes in protein function Defective hemoglobin leading to sickle-cell anemia in humans Behavioral Behavior pattern changes Brain mutations affecting Drosophila mating behaviors Regulatory Altered gene expression Regulatory gene mutations affecting expression of the lac operon in E. coli Lethal Altered organism survival Tay-Sachs and Huntington disease in humans Conditional Phenotype expressed only under certain environ- Temperature-sensitive mutations affecting coat mental conditions color in Siamese cats M14_KLUG8414_10_SE_C14.indd 263 16/11/18 5:14 pm  264 14 GENE MUTATION, DNA REPAIR, AND TRANSPOSITION two X chromosomes) may be expressed in male offspring, most organisms are exposed and, as such, may be considered who are by definition hemizygous for the gene mutation natural agents that cause induced mutations. because they have one X and one Y chromosome. This will We will next describe several aspects of spontaneous occur provided that the male offspring receives the affected mutations, including mutation rates. X chromosome. Because of heterozygosity, the occurrence of an autosomal recessive mutation in the gametes of either males or females (even one resulting in a lethal allele) may Spontaneous Mutation Rates in Nonhuman go unnoticed for many generations, until the resultant allele Organisms has become widespread in the population. Usually, the new Several generalizations can be made regarding spontane- allele will become evident only when a chance mating brings ous mutation rates. The mutation rate is defined as the two copies of it together into the homozygous condition. likelihood that a gene will undergo a mutation in a single generation or in forming a single gamete. First, the rate of ES S ENTIAL P OINT spontaneous mutation is exceedingly low for all organisms. Mutations can have many different effects on gene function, depend- Second, the rate varies between different organisms. Third, ing on the type of nucleotide changes that comprise the mutation even within the same species, the spontaneous mutation rate and their locations. Phenotypic effects can range from neutral or varies from gene to gene. silent to loss of function, gain of function, or lethality. Viral and bacterial genes undergo spontaneous muta- tion at an average of about 1 in 100 million (10-8) replica- tions or cell divisions. Maize and Drosophila demonstrate N O W S O LV E T H I S rates several orders of magnitude higher. The genes stud- 14.1 If a point mutation occurs within a human egg cell ied in these groups average between 1 in 1,000,000 (10-6) genome that changes an A to a T, what is the most likely and 1 in 100,000 (10-5) mutations per gamete formed. effect of this mutation on the phenotype of an offspring Some mouse genes are another order of magnitude higher that develops from this mutated egg? in their spontaneous mutation rate:1 in 100,000 to 1 in HINT: This problem asks you to predict the effects of a single 10,000 (10-5 to 10-4). It is not clear why such large varia- base-pair mutation on phenotype. The key to its solution involves tions occur in mutation rates. an understanding of the organization of the human genome as The variation in rates between organisms may, in part, well as the effects of mutations on coding and noncoding regions reflect the relative efficiencies of their DNA proofreading of genes and the effects of mutations on development. and repair systems. We will discuss these systems later in the chapter. Variation between genes in a given organism may be For more practice, see Problems 4–7. due to inherent differences in mutability in different regions of the genome. Some DNA sequences appear to be highly sus- ceptible to mutation and are known as mutation hot spots. 14.2 Mutations Can Be Spontaneous Mutation Rates in Humans Spontaneous or Induced Now that whole-genome sequencing is becoming both rapid and economical, it is possible to examine entire genomes, Mutations can be classified as either spontaneous or induced, both coding and noncoding regions, and to compare genomes although these two categories overlap to some degree. Spon- from parents and offspring and estimate spontaneous germ- taneous mutations are changes in the nucleotide sequence line mutation rates. of genes that appear to occur naturally. No specific agents are In 2012, a research group in Iceland sequenced the associated with their occurrence. Many of these mutations genomes of 78 parent/offspring sets, comprising 219 arise as a result of normal biological or chemical processes individuals, and compared the single-nucleotide in the organism that alter the structure of nitrogenous bases. polymorphisms (SNPs)!(see Chapter 7) throughout their Often, spontaneous mutations occur during the enzymatic genomes.1 Their data revealed that a newborn baby’s process of DNA replication, as we discuss later in this chapter. genome contains an average of 60 new mutations, com- In contrast to spontaneous mutations, the mutations pared with those of his or her parents. Their research also that result from the influence of extraneous factors are con- revealed that the number of new mutations depends sig- sidered to be induced mutations. Induced mutations may nificantly on the age of the father at the time of conception. be the result of either natural or artificial agents. For exam- ple, radiation from cosmic and mineral sources and ultra- 1 Kong, A., et al. (2012). Rate of de novo mutations and the impor- violet radiation from the sun are energy sources to which tance of father’s age to disease risk. Nature 488:471–475. M14_KLUG8414_10_SE_C14.indd 264 16/11/18 5:14 pm  14.3 SPONTANEOUS MUTATIONS ARISE FROM REPLICATION ERRORS AND BASE MODIFICATIONS 265 For example, when the father is 20 years old, he contributes mutations. Replication errors due to mispairing predomi- approximately 25 new mutations to the child. When he is 40 nantly lead to point mutations. The fact that bases can take years old, he contributes approximately 65 new mutations. several forms, known as tautomers, increases the chance In contrast, the mother contributes about 15 new mutations, of mispairing during DNA replication, as we will explain at any age. The researchers estimated that the father con- shortly. tributes approximately 2 mutations per year of his age, with In addition to mispairing and point mutations, DNA repli- the mutation rate doubling every 16.5 years. The large pro- cation can lead to the introduction of small insertions or dele- portion of mutations contributed by fathers is likely due to tions. These mutations can occur when one strand of the DNA the fact that male germ cells go through more cell divisions template loops out and becomes displaced during replication, during a lifetime than do female germ cells. or when DNA polymerase slips or stutters during replication— Of the 4933 new SNP mutations that were identified in events termed replication slippage. If a loop occurs in the this study, only 73 occurred within gene exons. Other stud- template strand during replication, DNA polymerase may ies have suggested that about 10 percent of single-nucleotide miss the looped-out nucleotides, and a small deletion in the mutations lead to negative phenotypic changes. If so, then new strand will be introduced. If DNA polymerase repeatedly an average spontaneous mutation rate of 60 new mutations introduces nucleotides that are not present in the template might yield about six deleterious phenotypic effects per strand, an insertion of one or more nucleotides will occur. generation. Insertions and deletions may lead to frameshift mutations or It is estimated that somatic cell mutation rates are amino acid insertions or deletions in the gene product. between 4 and 25 times higher than those in germ-line cells. Replication slippage can occur anywhere in the DNA but It is well accepted that somatic mutations are responsible for seems more common in regions containing tandemly repeated the development of most cancers. We will discuss the effects sequences. Repeat sequences are hot spots for DNA mutation of somatic mutations on the development of cancer in more and in some cases contribute to hereditary diseases, such as detail later!(see Chapter 19). fragile-X syndrome and Huntington disease. The hypermuta- bility of repeat sequences in noncoding regions of the genome is the basis for several current methods of forensic DNA analysis. ES SENT IA L P OINT Spontaneous mutations can occur naturally without the action of extraneous agents. Induced mutations occur as a result of Tautomeric Shifts extraneous agents which can be either natural or human-made. Spontaneous mutation rates vary between organisms and between Purines and pyrimidines can exist in tautomeric forms—that different regions of genome. is, in alternate chemical forms that differ by the shift of a single proton in the molecule. The biologically important tautomers are the keto–enol forms of thymine and gua- nine and the amino–imino forms of cytosine and adenine. 14.3 Spontaneous Mutations Arise Tautomeric shifts change the covalent structure of the molecule, allowing hydrogen bonding with noncomplemen- from Replication Errors and Base tary bases, and hence, may lead to permanent base-pair Modifications changes and mutations. Figure 14.2 compares normal base-pairing arrangements with rare unorthodox pairings. In this section, we will outline some of the processes that Anomalous T-G and C-A pairs, among others, may be formed. lead to spontaneous mutations. Many of the DNA changes A mutation occurs during DNA replication when a tran- that occur during spontaneous mutagenesis also occur, at a siently formed tautomer in the template strand pairs with a higher rate, during induced mutagenesis. noncomplementary base. In the next round of replication, the “mismatched” members of the base pair are separated, and each becomes the template for its normal complemen- DNA Replication Errors and Slippage tary base. The end result is a point mutation (Figure 14.3). As we learned earlier!(see Chapter 10), the process of DNA replication is imperfect. Occasionally, DNA polymerases insert incorrect nucleotides during replication of a strand of Depurination and Deamination DNA. Although DNA polymerases can correct most of these Some of the most common causes of spontaneous mutations replication errors using their inherent 3′ to 5′ exonuclease are two forms of DNA base damage: depurination and proofreading capacity, misincorporated nucleotides may deamination. Depurination is the loss of one of the persist after replication. If these errors are not detected nitrogenous bases in an intact double-helical DNA molecule. and corrected by DNA repair mechanisms, they may lead to Most frequently, the base is either guanine or adenine—in M14_KLUG8414_10_SE_C14.indd 265 16/11/18 5:14 pm  266 14 GENE MUTATION, DNA REPAIR, AND TRANSPOSITION (a) Standard base-pairing arrangements H H H N H O H N H C CH3 O H N N C C C C C N C C C C H C N H N C H C N H N C N N C C N N C C N O H N O H H Thymine (keto) Adenine (amino) Cytosine (amino) Guanine (keto) (b) Anomalous base-pairing arrangements CH3 O H O H H H N F IG U R E 14.2 Examples C H C C C C H N H N of standard base-pairing N N C arrangements (a) com- H C N H N C C C C C pared with examples of the N C C N H C N H N C N anomalous base pairing that occurs as a result of tautomeric O H N N C C N shifts (b). The long triangles indicate the point at which H O H each base bonds to a backbone Thymine (enol) Guanine (keto) Cytosine (imino) Adenine (amino) sugar. other words, a purine. These bases may be lost if the glycosidic A T bond linking the 1′@C of the deoxyribose and the number 9 T A position of the purine ring is broken, leaving an apurinic site G C on one strand of the DNA. Geneticists estimate that thousands of such spontaneous lesions are formed daily in the DNA of C G mammalian cells in culture. If apurinic sites are not repaired, there will be no base at that position to act as a template A T during DNA replication. As a result, DNA polymerase may introduce a nucleotide at random at that site. No tautomeric T A Tautomeric shift shift to imino form In deamination, an amino group in cytosine or ade- G C nine is converted to a keto group. In these cases, cytosine is C G converted to uracil, and adenine is changed to the guanine- Replication resembling compound hypoxanthine (Figure 14.4). The (round 1) major effect of these changes is an alteration in the base-pair- A T A T ing specificities of these two bases during DNA replication. T A Anomalous C A Tautomer For example, cytosine normally pairs with guanine. Follow- C-A base ing its conversion to uracil, which pairs with adenine, the G C pair formed G C original G-C pair is converted to an A-U pair and then, in the C G C G next replication, is converted to an A-T pair. When adenine is No mutation deaminated, the original A-T pair is ultimately converted to a A T G-C pair because hypoxanthine pairs naturally with cytosine, which then pairs with guanine in the next replication. C A Tautomeric shift back to G C amino form Oxidative Damage C G DNA may also suffer damage from the by-products of Replication (round 2) normal cellular processes. These by-products include reac- tive oxygen species (electrophilic oxidants) that are gen- A T A T erated during normal aerobic respiration. For example, C G Transition T A mutation G C G C C G C G FI G U R E 1 4.3 Formation of an A-T to G-C transition muta- tion as a result of a transient tautomeric shift in adenine. M14_KLUG8414_10_SE_C14.indd 266 16/11/18 5:14 pm  14.4 INDUCED MUTATIONS ARISE FROM DNA DAMAGE CAUSED BY CHEMICALS AND RADIATION 267 H H H 14.4 Induced Mutations Arise H N H H O H N N C from DNA Damage Caused by C C C C C C N Chemicals and Radiation H C N H C N H N C N C N C C N All cells on Earth are exposed to a plethora of agents called mutagens, which have the O O H Cytosine Uracil Adenine potential to damage DNA and cause induced mutations. Some of these agents, such as some H H fungal toxins, cosmic rays, and UV light, are H N H H O H N H natural components of our environment. Oth- N N C C ers, including some industrial pollutants, medi- C C C C C C N N cal X rays, and chemicals within tobacco smoke, C N C N H N C can be considered as unnatural or human-made N C N C C N additions to our modern world. On the positive H H O side, geneticists have harnessed some mutagens Adenine Hypoxanthine Cytosine for use in analyzing genes and gene functions. The mechanisms by which some of these natural and F I G U R E 1 4.4 Deamination of cytosine and adenine, leading to new base pairing and mutation. Cytosine is converted to uracil, which unnatural agents lead to mutations are outlined base-pairs with adenine. Adenine is converted to hypoxanthine, which in this section. base-pairs with cytosine. Base Analogs One category of mutagenic chemicals is base analogs, com- superoxides (O2-), hydroxyl radicals ( # OH), and hydrogen pounds that can substitute for purines or pyrimidines during peroxide (H2O2) are created during cellular metabolism and nucleic acid biosynthesis. For example, the synthetic chemi- are constant threats to the integrity of DNA. Such reactive cal 5-bromouracil (5-BU), a derivative of uracil, behaves oxidants, also generated by exposure to high-energy radia- as a thymine analog but with a bromine atom substituted tion, can produce more than 100 different types of chemi- at the number 5 position of the pyrimidine ring. If 5-BU cal modifications in DNA, including modifications to bases, is chemically linked to deoxyribose, the nucleoside ana- loss of bases, and single-stranded breaks. log bromodeoxyuridine (BrdU) is formed. Figure 14.5 compares the structure of 5-BU with that of thymine. The presence of the bromine atom in place of the methyl group ES SENT IA L P OINT increases the probability that a tautomeric shift will occur. Spontaneous mutations result from many different causes includ- If BrdU is incorporated into DNA in place of thymidine and a ing errors during DNA replication and changes in DNA base pairing tautomeric shift to the enol form of 5-BU occurs, 5-BU base- related to tautomeric shifts, depurinations, deaminations, and reac- tive oxidant damage. pairs with guanine. After one round of replication, an A-T to G-C transition results. Furthermore, the presence of 5-BU within DNA increases the sensitivity of the molecule to UV N O W S O LV E T H I S light, which itself is mutagenic. 14.2 One of the most famous cases of an X-linked reces- Alkylating, Intercalating, and Adduct-Forming sive mutation in humans is that of hemophilia found in the Agents descendants of Britain’s Queen Victoria. The pedigree of the royal family indicates that Victoria was heterozygous A number of naturally occurring and human-made chemi- for the trait; however, her father was not affected, and no cals alter the structure of DNA and cause mutations. The other member of her maternal line appeared to carry the sulfur-containing mustard gases, used during World War mutation. What are some possible explanations of how I, were some of the first chemical mutagens identified in the mutation arose? What types of mutations could lead chemical warfare studies. Mustard gases are alkylating to the disease? agents—that is, they donate an alkyl group, such as CH3 HINT: This problem asks you to determine the sources of new or CH2CH3, to amino or keto groups in nucleotides. Ethyl- mutations. The key to its solution is to consider the ways in which methane sulfonate (EMS), for example, alkylates the keto mutations occur, the types of cells in which they can occur, and groups in the number 6 position of guanine and in the num- how they are inherited. ber 4 position of thymine. As with base analogs, base-pairing affinities are altered, and transition mutations result. M14_KLUG8414_10_SE_C14.indd 267 16/11/18 5:14 pm  268 14 GENE MUTATION, DNA REPAIR, AND TRANSPOSITION H during the cooking of meats such as beef, chicken, and fish. HCAs are formed at high temperatures CH3 O Br O H N H N from amino acids and creatine. Many HCAs cova- C lently bind to guanine bases. At least 17 different C C C C C C 5 4 N HCAs have been linked to the development of can- H C6 3N H H C N H N C 1 2 cers, such as those of the stomach, colon, and breast. N C N C C N O O Ultraviolet Light H Thymine 5-BU (keto form) All electromagnetic radiation consists of energetic Adenine waves that we define by their different wavelengths Br O H O H (Figure 14.7). The full range of wavelengths is N C referred to as the electromagnetic spectrum, and C C C C the energy of any radiation in the spectrum varies H C N H N C N inversely with its wavelength. Waves in the range of N C C N visible light and longer are benign when they inter- act with most organic molecules. However, waves O H N of shorter length than visible light, being inher- H ently more energetic, have the potential to disrupt 5-BU (enol form) Guanine organic molecules. Purines and pyrimidines absorb ultraviolet F I G U R E 1 4. 5 Similarity of the chemical structure of 5-bromouracil (UV) radiation most intensely at a wavelength (5-BU) and thymine. In the common keto form, 5-BU base-pairs nor- mally with adenine, behaving as a thymine analog. In the rare enol of about 260 nanometers (nm). Although Earth’s form, it pairs anomalously with guanine. ozone layer absorbs the most dangerous types of UV radiation, sufficient UV radiation can induce thousands of For!example, 6-ethylguanine acts as an analog of adenine DNA lesions per hour in any cell exposed to this radiation. and pairs with thymine (Figure 14.6). One major effect of UV radiation on DNA is the creation Intercalating agents are chemicals that have dimen- of pyrimidine dimers—chemical species consisting of sions and shapes that allow them to wedge between the base two identical pyrimidines—particularly ones consisting pairs of DNA. Wedged intercalating agents cause base pairs of two thymidine residues (Figure 14.8). The dimers dis- to distort and DNA strands to unwind. These changes in tort the DNA conformation and inhibit normal replication. DNA structure affect many functions including transcrip- As a result, errors can be introduced in the base sequence tion, replication, and repair. Deletions and insertions occur of DNA during replication through the actions of error- during DNA replication and repair, leading to frame-shift prone DNA polymerases. When UV-induced dimerization mutations. is extensive, it is responsible (at least in part) for the killing Another group of chemicals that cause mutations are effects of UV radiation on cells. known as adduct-forming agents. A DNA adduct is a substance that covalently binds to DNA, altering its con- formation and interfering with replication and repair. Two Ionizing Radiation examples of adduct-forming substances are acetaldehyde As noted above, the energy of radiation varies inversely with (a component of cigarette smoke) and heterocyclic amines wavelength. Therefore, X rays, gamma rays, and cosmic (HCAs). HCAs are cancer-causing chemicals that are created rays are more energetic than UV radiation (Figure 14.7). CH2CH3 H O H O O CH3 N N C C C C C C C C N N C N H EMS C N H N C H C N FI G U R E 14.6 Conversion of N C N C guanine to 6-ethylguanine by the alkylating agent ethylmethane NH2 H N H O sulfonate (EMS). The 6-ethylgua- Guanine 6-Ethylguanine Thymine nine base-pairs with thymine. M14_KLUG8414_10_SE_C14.indd 268 16/11/18 5:14 pm  14.4 INDUCED MUTATIONS ARISE FROM DNA DAMAGE CAUSED BY CHEMICALS AND RADIATION 269 Visible spectrum (wavelength) 750 nm 700 nm 650 nm 600 nm 550 nm 500 nm 450 nm 380 nm Gamma Cosmic Radio waves Microwaves Infrared UV X rays rays rays 103 m 109 nm 106 nm 103 nm 1 nm 10–3 nm 10–5 nm (1 m) Decreasing wavelength F IG U R E 14.7 The regions of the electromagnetic Increasing energy spectrum and their associated wavelengths. As a result, they penetrate deeply into tissues, causing ion- chromosomes, and producing a variety of chromosomal ization of the molecules encountered along the way. Hence, aberrations, such as deletions, translocations, and chro- this type of radiation is called ionizing radiation. mosomal fragmentation. As ionizing radiation penetrates cells, stable molecules Although it is often assumed that radiation from artifi- and atoms are transformed into free radicals—chemi- cial sources such as nuclear power plant waste and medical cal species containing one or more unpaired electrons. X rays are the most significant sources of radiation exposure Free radicals can directly or indirectly affect the genetic for humans, scientific data indicate otherwise. Scientists esti- material, altering purines and pyrimidines in DNA, break- mate that less than 20 percent of human radiation exposure ing phosphodiester bonds, disrupting the integrity of arises from human-made sources. The greatest radiation exposure comes from radon gas, cosmic rays, and natural soil radioactivity. More than half of human-made radia- Single strand tion exposure comes from medical X rays and radioactive of DNA pharmaceuticals. P O H O C N E SS ENT IAL P OINT N Thymine Sugar C O Mutations can be induced by many types of chemicals and radia- base tions. These agents can damage both DNA bases and the sugar- C C phosphate backbones of DNA molecules. H CH3 P O H O N O W S O LV E T H I S C N Sugar N C O 14.3 The cancer drug melphalan is an alkylating agent C C of the mustard gas family. It acts in two ways: by caus- ing alkylation of guanine bases and by cross linking DNA H CH3 Thymine strands together. Describe two ways in which melphalan O H dimer P might kill cancer cells. What are two ways in which cancer O C N cells could repair the DNA-damaging effects of melphalan? N C O HI NT: This problem asks you to consider the effect of the alkyla- Sugar C C tion of guanine on base pairing during DNA replication. The key H CH3 to its solution is to consider the effects of mutations on cellular processes that allow cells to grow and divide. In Section 14.6, you will learn about the ways in which cells repair the types of muta- F I G U R E 1 4.8 Depiction of a thymine dimer induced by UV radiation. The covalent crosslinks (shown in red) occur tions introduced by alkylating agents. between carbon atoms of the pyrimidine rings. M14_KLUG8414_10_SE_C14.indd 269 16/11/18 5:14 pm  270 14 GENE MUTATION, DNA REPAIR, AND TRANSPOSITION degradation of the abnormal mRNA or creation of abnor- 14.5 Single-Gene Mutations Cause a mal protein products. Wide Range of Human Diseases Although most human genetic diseases are polygenic— Single-Gene Mutations and B Thalassemia that is, caused by variations in several genes—even a single Although some single-gene diseases, such as sickle-cell base-pair change in one of the approximately 20,000 human anemia!(discussed in Chapter 13), are caused by one spe- genes can lead to a serious inherited disorder. These mono- cific base-pair change within a gene, most are caused by genic diseases can be caused by many different types of any of a large number of different mutations. The muta- single-gene mutations. Table 14.2 lists some examples of tion profile associated with b@thalassemia provides an the types of single-gene mutations that can lead to serious example of the latter, more common, type of monogenic genetic diseases. A comprehensive database of human disease. genes, mutations, and disorders is available in the Online b@thalassemia is an inherited autosomal recessive Mendelian Inheritance in Man (OMIM) database!(described blood disorder resulting from a reduction or absence of in the Exploring Genomics feature in Chapter 3). As of 2018, hemoglobin. It is the most common single-gene disease in the OMIM database has cataloged approximately 5000 the world, affecting people worldwide, but especially popu- human phenotypes for which the molecular basis is known. lations in Mediterranean, North African, Middle Eastern, Geneticists estimate that approximately 30 percent Central Asian, and Southeast Asian countries. of mutations that cause human diseases are single base- People with b@thalassemia have varying degrees of pair changes that create nonsense mutations. These muta- anemia—from severe to mild—with symptoms including tions not only code for a prematurely terminated protein weakness, delayed development, jaundice, enlarged organs, product, but also trigger rapid decay of the mRNA. Many and often a need for frequent blood transfusions. more mutations are missense mutations that alter the Mutations in the b@globin gene (HBB gene) cause amino acid sequence of a protein and frameshift muta- b@thalassemia. The HBB gene encodes the 146-amino-acid tions that alter the protein sequence and create inter- b@globin polypeptide. Two b@globin polypeptides associate nal nonsense codons. Other common disease-associated with two a@globin polypeptides to form the adult hemoglobin mutations affect the sequences of gene promoters, mRNA tetramer. The HBB gene spans 1.6 kilobases of DNA on the splicing signals, and other noncoding sequences that affect short arm of chromosome 11. It is made up of three exons transcription, processing, and stability of mRNA or pro- and two introns. tein. One recent study showed that about 15 percent of Scientists have discovered approximately 400 differ- all point mutations that cause human genetic diseases ent mutations in the HBB gene that cause b@thalassemia, result in abnormal mRNA splicing. Approximately 85 although most cases worldwide are associated with only percent of these splicing mutations alter the sequence of about 20 of these mutations. Table 14.3 provides a sum- 5′ and 3′ splice signals. The remainder create new splice mary of the types of single-gene mutations that cause sites within the gene. Splicing defects often result in b@thalassemia. TABLE 14.2 Examples of Human Disorders Caused by Single-Gene Mutations Type of Mutation Disorder Molecular Change Missense Achondroplasia Glycine to arginine at position 380 of FGFR3 gene Nonsense Marfan syndrome Tyrosine to STOP codon at position 2113 of fibrillin-1 gene Insertion Familial hypercholesterolemia Various short insertions throughout the LDLR gene Deletion Cystic fibrosis Three-base-pair deletion of phenylalanine codon at position 508 of CFTR gene Trinucleotide repeat expansions Huntington disease 740 repeats of (CAG) sequence in coding region of Huntingtin gene M14_KLUG8414_10_SE_C14.indd 270 16/11/18 5:14 pm  14.6 ORGANISMS USE DNA REPAIR SYSTEMS TO COUNTERACT MUTATIONS 271 TABLE 14.3 Types of Mutations in the HBB Gene That Cause B@Thalassemia Gene Region Affected Number of Mutations Known Description 5′ upstream region 22 Single base-pair mutations occur between - 101 and - 25 upstream from transcription start site. For exam- ple, a T S A transition in the TATA sequence at - 30 results in decreased gene transcription and severe disease. mRNA CAP site 1 Single base-pair mutation (A S C transversion) at + 1 posi- tion leads to decreased levels of mRNA. 5′ untranslated region 3 Single base-pair mutations at + 20, + 22, and + 33 cause decreases in transcription and translation and mild disease. ATG translation initiation codon 7 Single base-pair mutations alter the mRNA AUG sequence, resulting in no translation and severe disease. Exons 1, 2, and 3 coding regions 36 Single base-pair missense and nonsense mutations, and mutations that create abnormal mRNA splice sites. Disease severity varies from mild to extreme. Introns 1 and 2 38 Single base-pair transitions and transversions that reduce or abolish mRNA splicing and create abnormal splice sites that affect mRNA stability. Most cause severe disease. Polyadenylation site 6 Single base-pair changes in the AATAAA sequence reduce the efficiency of mRNA cleavage and polyadenylation, yield- ing long mRNAs or unstable mRNAs. Disease is mild. Throughout and surrounding the 7100 Short insertions, deletions, and duplications that alter HBB gene coding sequences, create frameshift stop codons, and alter mRNA splicing. Proofreading and Mismatch Repair 14.6 Organisms Use DNA Repair Some of the most common types of mutations arise during Systems to Counteract Mutations DNA replication when an incorrect nucleotide is inserted by DNA polymerase. The major DNA synthesizing enzyme Living systems have evolved a variety of elaborate repair in bacteria (DNA polymerase III) makes an error approxi- systems that counteract both spontaneous and induced mately once every 100,000 insertions, leading to an error rate DNA damage. These DNA repair systems are absolutely of 10-5. Fortunately, DNA polymerase proofreads each step, essential to the maintenance of the genetic integrity of catching 99 percent of those errors. If an incorrect nucleotide organisms and, as such, to the survival of organisms on is inserted during polymerization, the enzyme can recognize Earth. The balance between mutation and repair results in the error and “reverse” its direction. It then behaves as a 3′ the observed mutation rates of individual genes and organ- to 5′ exonuclease, cutting out the incorrect nucleotide and isms. Of foremost interest in humans is the ability of these replacing it with the correct one. This improves the efficiency systems to counteract genetic damage that would otherwise of replication 100-fold, creating only 1 mismatch in every 107 result in genetic diseases and cancer. The link between insertions, for a final error rate of 10-7. defective DNA repair and cancer susceptibility is described To cope with errors such as base–base mismatches, later!(see Chapter 19). small insertions, and deletions that remain after proof- We now embark on a review of these and other DNA reading, another mechanism, called mismatch repair repair mechanisms, with the emphasis on the major (MMR), may be activated. During MMR, the mismatches approaches that organisms use to counteract genetic are detected, the incorrect nucleotide is removed, and the damage. correct nucleotide is inserted in its place. M14_KLUG8414_10_SE_C14.indd 271 16/11/18 5:14 pm  272 14 GENE MUTATION, DNA REPAIR, AND TRANSPOSITION Following replication, the repair enzymes mentioned Postreplication repair below are able to recognize any mismatch that is introduced on the newly synthesized DNA strand and bind to the strand. T T Lesion An endonuclease enzyme creates a nick in the backbone of the newly synthesized DNA strand, either 5′ or 3′ to the mis- match. An exonuclease unwinds and degrades the nicked DNA strand, until the region of the mismatch is reached. AA Complementary region Finally, DNA polymerase fills in the gap created by the exo- DNA unwound prior nuclease, using the correct DNA strand as a template. DNA to replication ligase then seals the gap. A series of E. coli gene products, MutH, MutL, and T T MutS, as well as exonucleases, DNA polymerase III, and DNA ligase, are involved in MMR. Mutations in the mutH, mutL, and mutS genes result in bacterial strains deficient in MMR. In humans, mutations in genes that code for DNA MMR AA Replication skips over lesion and continues proteins (such as hMSH2 and hMLH1, which are the human equivalents of the mutS and mutL genes of E. coli) are associ- T T ated with the hereditary nonpolyposis colon cancer. MMR defects are commonly found in other cancers, such as leu- Recombined AA kemias, lymphomas, and tumors of the ovary, prostate, and complement endometrium. Cells from these cancers show genome-wide increases in the rate of spontaneous mutation. The link Undamaged complementary region of parental strand between defective MMR and cancer is supported by experi- is recombined New gap formed ments with mice. Mice that are engineered to have deficien- cies in MMR genes accumulate large numbers of mutations T T and are cancer-prone. AA Postreplication Repair and the SOS Repair System AA New gap is filled by DNA Another type of DNA repair system, called postreplication polymerase and DNA ligase repair, responds after damaged DNA has escaped repair F IG U RE 14. 9 Postreplication repair occurs if DNA replica- and has failed to be completely replicated. As illustrated in tion has skipped over a lesion such as a thymine dimer. Figure 14.9, when DNA bearing a lesion of some sort (such as Through the process of recombination, the correct com- a pyrimidine dimer) is being replicated, DNA polymerase may plementary sequence is recruited from the parental strand and inserted into the gap opposite the lesion. The new gap stall at the lesion and then skip over it, leaving an unreplicated is filled by DNA polymerase and DNA ligase. gap on the newly synthesized strand. To correct the gap, RecA protein directs a recombinational exchange with the corre- sponding region on the undamaged parental strand of the same minimize DNA damage, hence its name. During SOS repair, polarity (the “donor” strand). When the undamaged segment DNA synthesis becomes error-prone, inserting random and of the donor strand DNA replaces the gapped segment, a gap possibly incorrect nucleotides in places that would normally is created on the donor strand. The gap can be filled by repair stall DNA replication. As a result, SOS repair itself becomes synthesis as replication proceeds. Because a recombinational mutagenic—although it may allow the cell to survive DNA event is involved in this type of DNA repair, it is considered to damage that would otherwise kill it. be a form of homologous recombination repair. Another postreplication repair pathway, the E. coli SOS repair system, also responds to damaged DNA, but in a dif- Photoreactivation Repair: Reversal of UV ferent way. In the presence of a large number of unrepaired Damage DNA mismatches and gaps, the bacteria can induce expres- As was illustrated in Figure 14.8, UV light introduces mutations sion of about 20 genes (including lexA, recA, and uvr) whose by the creation of pyrimidine dimers. UV-induced damage to products allow DNA replication to occur even in the pres- E. coli DNA can be partially reversed if, following irradiation, ence of DNA lesions. This type of repair is a last resort to the cells are exposed briefly to visible light, especially in the M14_KLUG8414_10_SE_C14.indd 272 16/11/18 5:14 pm  14.6 ORGANISMS USE DNA REPAIR SYSTEMS TO COUNTERACT MUTATIONS 273 blue range of the visible spectrum. The process is dependent Base excision repair on the activity of a protein called photoreactivation enzyme (PRE) or photolyase. The enzyme’s mode of action is to cleave ACUAGT 5' the cross-linking bonds between thymine dimers. Although Duplex with the enzyme will associate with a thymine dimer in the dark, U–G mismatch 3' it must absorb a photon of blue light to cleave the dimer. The T GGT C A enzyme is also detectable in many organisms, including other Uracil DNA glycosylase U recognizes and excises bacteria, fungi, plants, and some vertebrates—though not in incorrect base humans. Humans and other organisms that lack photoreacti- AC AGT 5' vation repair must rely on other repair mechanisms to reverse the effects of UV radiation. 3' T GGT C A AP endonuclease recognizes lesion and nicks DNA strand Base and Nucleotide Excision Repair AC AGT 5' A number of light-independent DNA repair systems exist in all bacteria and eukaryotes. The basic mechanisms involved 3' T GGT C A in these types of repair—collectively referred to as excision DNA polymerase and repair or cut-and-paste mechanisms—consist of the follow- DNA ligase fill gap ing three steps. ACCAGT 5' 1. The damage, distortion, or error present on one of the two Mismatch repaired strands of the DNA helix is recognized and enzymatically 3' T GGT C A clipped out by an endonuclease. Excisions in the phospho- diester backbone usually include a number of nucleotides FI G U R E 14.10 Base excision repair (BER) accomplished by adjacent to the error as well, leaving a gap on one strand uracil DNA glycosylase, AP endonuclease, DNA polymerase, and DNA ligase. Uracil is recognized as a noncomplemen- of the helix. tary base, excised, and replaced with the complementary base (C). 2. A DNA polymerase fills in the gap by inserting nucleo- tides complementary to those on the intact strand, which it uses as a replicative template. The enzyme adds these Although much has been learned about the mechanisms nucleotides to the free 3′@OH end of the clipped DNA. In E. of BER in E. coli, BER systems have also been detected in coli, this step is usually performed by DNA polymerase I. eukaryotes from yeast to humans. Experimental evidence shows that both mouse and human cells that are defective 3. DNA ligase seals the final “nick” that remains at the in BER activity are hypersensitive to the killing effects of 3′@OH end of the last nucleotide inserted, closing the gap. gamma rays and oxidizing agents. There are two types of excision repair: base excision Nucleotide excision repair (NER) pathways repair repair and nucleotide excision repair. Base excision repair “bulky” lesions in DNA that alter or distort the double helix. (BER) corrects DNA that contains incorrect base pairings These lesions include the UV-induced pyrimidine dimers due to the presence of chemically modified bases or uridine and DNA adducts discussed previously. nucleosides that are inappropriately incorporated into DNA The NER pathway (Figure 14.11) was first discovered or created by deamination of cytosine. The first step in the in 1964 by Paul Howard-Flanders and coworkers, who iso- BER pathway involves the recognition of an inappropriately lated several independent E. coli mutants that are sensitive paired base by enzymes called DNA glycosylases. There are to UV radiation. One group of genes was designated uvr a number of DNA glycosylases, each of which recognizes a (ultraviolet repair) and included the uvrA, uvrB, and uvrC specific base. For example, the enzyme uracil DNA glycosyl- mutations. In the NER pathway, the uvr gene products are ase recognizes the presence of uracil in DNA (Figure 14.10). involved in recognizing and clipping out lesions in the DNA. DNA glycosylases first cut the glycosidic bond between the Usually, a specific number of nucleotides are clipped out target base and its sugar, creating an apyrimidinic (or around both sides of the lesion. In E. coli, usually a total of apurinic) site. The sugar with the missing base is then rec- 13 nucleotides are removed, including the lesion. The repair ognized by an enzyme called AP endonuclease. The AP is then completed by DNA polymerase I and DNA ligase, in endonuclease makes cuts in the phosphodiester backbone a manner similar to that occurring in BER. The undamaged at the apyrimidinic or apurinic site. The gap is filled by DNA strand opposite the lesion is used as a template for the repli- polymerase and DNA ligase. cation, resulting in repair. M14_KLUG8414_10_SE_C14.indd 273 16/11/18 5:14 pm  274 14 GENE MUTATION, DNA REPAIR, AND TRANSPOSITION from normal individuals and those with XP. (Fibroblasts are Nucleotide excision repair undifferentiated connective tissue cells.) 5' The involvement of multiple genes in NER and XP has been investigated using somatic cell hybridization. 3' Fibroblast cells from any two unrelated XP patients, when DNA is grown together in tissue culture, can fuse together, forming damaged heterokaryons. A heterokaryon is a single cell with two Lesion nuclei from different organisms but a common cytoplasm. If the mutation in each of the two XP cells occurs in the same gene, the heterokaryon, like the cells that fused to form it, will still be unable to undergo NER. This is because there is no Nuclease uvr gene normal copy of the relevant gene present in the heterokaryon. excises lesion products However, if NER does occur in the heterokaryon, the mutations in the two XP cells must have been present in two different genes. Hence, the two mutants are said to demonstrate complementation, a concept discussed ear- lier!(see Chapter 4). Complementation occurs because the DNA heterokaryon has at least one normal copy of each gene Gap is filled polymerase I in the fused cell. By fusing XP cells from a large number of XP patients, researchers were able to determine how 5' many genes contribute to the XP phenotype. Based on 3' these and other studies, XP patients were divided into Gap is sealed; seven complementation groups, indicating that at least normal pairing DNA ligase seven different genes code for proteins that are involved is restored