Chapter 7 DNA Mutation, DNA Repair, and Transposable Elements

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台大農藝系遺傳學 601 20000 Chapter 19 slide 1

CHAPTER 7

DNA Mutation, DNA

Repair, and Transposable

Elements

Peter J. Russell

edited by Yue-Wen Wang Ph. D.

Dept. of Agronomy, NTU

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Adaptation Versus Mutation

1. A question that received much attention in the early 20

th

century was whether

mutation causes random variation leading to adaptation, or the environment induces

heritable adaptations.

a. Lamarckism is the doctrine of inheritance of acquired characteristics.

b. The random mutation doctrine says that sometimes chance changes happen to be adaptive, , thus altering phenotype by changing a protein (Figure 7.1).

c. The observation that phage T1-resistant E. coli arise could be interpreted to support either of these theories.

2. Luria and Delbrück (1943) demonstrated that the mutation mechanism is correct, by

doing a fluctuation test (Figure 7.2).

a. An E. coli population that started from one cell would show different patterns of T1 resistance depending on which model is correct.

i. The adaptive theory says that cells are induced to become resistant to T1 when it is added. Therefore, the proportion of resistant cells would be the same for all cultures with the same genetic background.

ii. The mutation theory says that random events confer resistance to T1, whether the phage is present or not. Cultures will therefore show different numbers of T1 resistant cells, depending on when the resistance mutation(s) occurred.

b. Luria and Delbrück observed fluctuating numbers of resistant bacteria from E. coli cultures, indicating that the random mutation model is correct.

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Fig. 7.2 Representation of a dividing population of T1 phage-sensitive wild-type

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Mutations Defined

1. A mutation is a change in a DNA base-pair or a

chromosome.

a. Somatic mutations affect only the individual in which they arise.

b. Germ-line mutations alter gametes, affecting the next generation.

2. Mutations are quantified in two different ways:

a. Mutation rate is the probability of a particular kind of mutation

as a function of time (e.g., number per gene per generation).

b. Mutation frequency is number of times a particular mutation

occurs in proportion to the number of cells or individuals in a

population (e.g., number per 100,000 organisms).

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Types of Point Mutations

Animation: Nonsense Mutation and Nonsense Suppressor Mutation

1. There are two general categories of point mutations: base-pair

substitutions and base-pair deletions or insertions.

2. A base-pair substitution replaces 1 base-pair with another. There are

two types (Figure 7.3):

a. Transitions convert a pyrimidine pair to the other

purine-pyrimidine pair (e.g., AT to GC or TA to CG).

b. Transversions convert a purine-pyrimidine pair to a pyrimidine-purine

pair (e.g., AT to TA, or AT to CG).

3. Base-pair substitutions in ORFs are also defined by their effect on the

protein sequence. Effects vary from none to severe.

a. Nonsense mutations change a codon in the ORF to a stop

(nonsense) codon, resulting in premature termination of translation,

and a truncated (often nonfunctional) protein (Figure 7.4).

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台大農藝系遺傳學 601 20000 Chapter 19 slide 9 b. Missense mutations have a base-pair change resulting in a different mRNA codon,

and therefore a different amino acid in the protein.

c. Phenotypic effects may or may not occur, depending on the specific amino acid change.

i. Neutral mutations change a codon in the ORF, but the resulting amino acid substitution produces no detectable change in the function of the protein (e.g., AAA to AGA substitutes arginine for lysine. The amino acids have similar properties, so the protein’s function may not be altered).

ii. Silent mutations occur when the mutant codon encodes the same amino acid as the wild-type gene, so that no change occurs in the protein produced (e.g.,

AAA and AAG both encode lysine, so this transition would be silent).

4. Deletions and insertions can change the reading frame of the mRNA

downstream of the mutation, resulting in a frameshift mutation.

a. When the reading frame is shifted, incorrect amino acids are usually incorporated. b. Frameshifts may bring stop codons into the reading frame, creating a shortened

protein.

c. Frameshifts may also result in read-through of stop codons, resulting in a longer protein.

d. Frameshift mutations result from insertions or deletions when the number of affected base pairs is not divisible by three.

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Reverse Mutations and Suppressor Mutations

1. Point mutations are divided into two classes based on their effect on phenotype:

a. Forward mutations change the genotype from wild type to mutant.

b. Reverse mutations (reversions or back mutations) change the genotype from mutant to wild-type or partially wild-type.

i. A reversion to the wild-type amino acid in the affected protein is a true reversion.

ii. A reversion to some other amino acid that fully or partly restores protein function is a partial reversion.

2. Suppressor mutations occur at sites different from the original mutation, and

mask or compensate for the initial mutation without actually reversing it.

Suppressor mutations have different mechanisms depending on the site at

which they occur.

a. Intragenic suppressors occur within the same gene as the original mutation, but at a different site. Two different types occur:

i. A different nucleotide is altered in the same codon as the original mutation. ii. A nucleotide in a different codon is altered (e.g., an insertion frameshift is

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台大農藝系遺傳學 601 20000 Chapter 19 slide 11 b. Intergenic suppressors occur in a different gene (the suppressor gene) from the

original mutation. Many work by changing mRNA translation.

i. Each suppressor gene works on only one type of nonsense, missense or frameshift mutation.

ii. A given suppressor gene suppresses all mutations for which it is specific.

iii. Suppressor genes often encode tRNAs that recognize stop codons and insert an amino acid, preventing premature termination of translation.

(1) Full or partial function of the polypeptide may be restored.

(2) The effect depends on how compatible the substituted amino acid is with protein function.

iv. Nonsense suppressors fall into three classes, one for each stop codon (UAG, UAA and UGA) (Figure 19.5).

v. Typical tRNA suppressor mutations are in redundant tRNA genes, so the wild-type tRNA activity is not lost.

vi. Nonsense suppression occurs by competition between release factors and suppressor tRNAs.

(1) UAG and UGA suppressor tRNAs do well in competition with release factors.

(2) UAA suppressor tRNAs are only 1–5% efficient.

vii. Suppression by a tRNA occurs at all of its specific stop codons (e.g., UGA or UAG), not just the mutant one. This may produce read-through proteins, but they are not as common as expected, possibly due to tandem stop codons (e.g., UAGUGA).

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Fig. 7.5 Mechanism of action of an intergenic nonsense suppressor mutation that

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Spontaneous and Induced Mutations

1. Most mutations are spontaneous, rather than

induced by a mutagen.

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Spontaneous Mutations

1. All types of point mutations can occur spontaneously, during S, G

1

and

G

2

phases of the cell cycle, or by the movement of transposons.

2. The spontaneous mutation rate in eukaryotes is between 10

-4

-to-10

-6

per

gene per generation, and in bacteria and phages 10

-5

-to-10

-7

/

gene/generation.

a. Genetic constitution of the organism affects its mutation rate.

i. In Drosophila, males and females of the same strain have similar

mutation rates.

ii. Flies of different strains, however, may have different mutation

rates.

b. Many spontaneous errors are corrected by the cellular repair systems,

and so do not become fixed in DNA.

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DNA Replication Errors

1. DNA replication errors can be either point mutations, or small insertions or

deletions.

2. Base-pair substitution mutations can result from “wobble” pairing. Bases are

normally in the keto form, but sometimes can undergo tautomeric shift to

form the abnormal (enol) form. The enol form can hydrogen bond with an

incorrect partner due to different spatial positioning of the atoms involved in

H–bonding (Figure 7.6). An example is a GC-to-AT transition (Figure 7.7):

a. During DNA replication, G could wobble pair with T, producing a GT pair.

b. In the next round of replication, G and A are likely to pair normally,

producing one progeny DNA with a GC pair, and another with an AT pair.

c. GT pairs are targets for correction by proofreading during replication, and

by other repair systems. Only mismatches uncorrected before the next round

of replication lead to mutations.

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Fig. 7.7 Production of a mutation as a result of a mismatch caused by wobble base

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3. Additions and deletions can occur spontaneously during

replication (Figure 7.8).

a. DNA loops out from the template strand, generally in a run of

the same base.

b. DNA polymerase skips the looped out bases, creating a deletion

mutation.

c. If DNA polymerase adds untemplated base(s), new DNA looping

occurs, resulting in additional mutation.

d. Insertions and deletions in structural genes generate frameshift

mutations (especially if they are not multiples of three).

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Fig. 7.8 Spontaneous generation of addition and deletion mutants by DNA looping-out errors during replication

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4. Spontaneous chemical changes include depurination and deamination of

particular bases, creating lesions in the DNA.

a. Depurination removes the purine (A or G) from DNA by breaking the

bond with its deoxyribose in the backbone.

i. Depurination is common.

ii. If not repaired before the next round of replication, it will result in

a random base at that site.

b. Deamination removes an amino group from a base (e.g., cytosine to

uradil) (Figure 7.9).

i. Uracil is an abnormal base in DNA, and it will usually be repaired.

ii. If uracil is not replaced, it will pair with an A during replication,

resulting in a CG-to-TA transition.

iii. Both prokaryotic and eukaryotic DNA have small amounts of

5-methylcytosine (5

m

C) in place of the normal C.

(1) Deamination of 5

m

C produces T.

(2) T is a normal nucleotide in DNA, so it is not detected by repair

mechanisms.

(3) Deamination of 5

m

C results in CG-to-TA transitions.

(4) Locations of 5

m

C in the chromosome are often detected as

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Fig. 7.9 Deamination of cytosine to uracil (a); deamination of 5-methylcytosine to

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Fig. 7.x Distribution of spontaneous GC-to-AC transition mutations to stop codons

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Induced Mutations

1. Exposure to physical mutagens plays a role in genetic research, where they are used to increase mutation frequencies to provide mutant organisms for study.

2. Radiation (e.g., X rays and UV) induces mutations.

a. X rays are an example of ionizing radiation, which penetrates tissue and collides with molecules, knocking electrons out of orbits and creating ions.

i. Ions can break covalent bonds, including those in the DNA sugar-phosphate backbone. ii. Ionizing radiation is the leading cause of human gross chromosomal mutations.

iii. Ionizing radiation kills cells at high doses, and lower doses produce point mutations. iv. Ionizing radiation has a cumulative effect. A particular dose of radiation results in the

same number of mutations whether it is received over a short or a long period of time. b. Ultraviolet (UV) causes photochemical changes in the DNA.

i. UV is not energetic enough to induce ionization.

ii. UV has lower-energy wavelengths than X rays, and so has limited penetrating power. iii. However, UV in the 254–260 nm range is strongly absorbed by purines and pyrimidines,

forming abnormal chemical bonds.

(1)A common effect is dimer formation between adjacent pyrimidines, commonly thymines (designated T^T) (Figure 7.10).

(2)C^C, C^T and T^C dimers also occur, but at lower frequency. Any pyrimidine dimer can cause problems during DNA replication.

(3)Most pyrimidine dimers are repaired, because they produce a bulge in the DNA helix. If enough are unrepaired, cell death may result.

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Chemical Mutagens

Animation: Mutagenic Effects of 5BU

1. Chemical mutagens may be naturally occurring, or synthetic. They form

different groups based on their mechanism of action:

a. Base analogs depend upon replication, which incorpocates a base with

alternate states (tautomers) that allow it to base pair in alternate ways,

depending on its state.

i. Analogs are similar to normal nitrogen bases, and so are incorporated

into DNA readily.

ii. Once in the DNA, a shift in the analog’s form will cause incorrect

base pairing during replication, leading to mutation.

iii. 5-bromouradil (5BU) is an example. 5BU has a bromine residue

instead of the methyl group of thymine (Figure 7.11).

(1) Normally 5BU resembles thymine, pairs with adenine and is

incorporated into DNA during replication.

(2) In its rare state, 5BU pairs only with guanine, resulting in a

TA-to-CG transition mutation.

(3) If 5BU is incorporated in its rare form, the switch to its normal

state results in a CG-to-TA transition. Thus 5BU- induced

mutations may be reverted by another exposure to 5BU.

iv. Not all base analogs are mutagens, only those that cause base- pair

changes (e.g, AZT is a stable analog that does not shift).

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台大農藝系遺傳學 601 20000 Chapter 19 slide 28 b. Base-modifying agents can induce mutations at any stage of the cell cycle. They work

by modifying the chemical structure and properties of the bases. Three types are (Figure 7.12):

i. Deaminating agents remove amino groups. An example is nitrous acid (HNO2 ),

which deaminates G, C and A.

(1) HNO2 deaminates guanine to produce xanthine, which has the same base

pairing as G. No mutation results.

(2) HNO2 deaminates cytosine to produce uradil, which produces a CG-to-TA

transition.

(3) HNO2 deaminates adenine to produce hypoxanthine, which pairs with

cytosine, causing an AT-to-GC transition.

(4) Mutations induced by HNO2 can revert with a second treatment.

ii. Hydroxylating agents include hydroxylamine (NH2OH).

(1) NH2OH specifically modifies C with a hydroxyl group (OH), so that it pairs

only with A instead of with G.

(2) NH2OH produces only CG-to-TA transitions, and so revertants do not occur

with a second treatment.

(3) NH2OH mutants, however, can be reverted by agents that do cause

TA-to-CG transitions (e.g., 5BU and HNO2).

iii. Alkylating agents are a diverse group that add alkyl groups to bases. Usually alkylation occurs at the 6-oxygen of G, producing O6-allcylguanine.

(1) An example is methylmethane sulfonate (MMS), which methylates G to produce O6-alkyl G.

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c. Intercalating agents insert themselves between adjacent bases in

dsDNA(Figure. 7.13). They are generally thin, plate-like hydrophobic

molecules.

i. At replication, a template that contains an intercalated agent will

cause insertion of a random extra base.

ii. The base-pair addition is complete after another round of

replication, during which the intercalating agent is lost.

iii. If an intercalating agent inserts into new DNA in place of a

normal base, the next round of replication will result in a deletion

mutation.

iv. Point deletions and insertions in ORFs result in frameshift

mutations. These mutations show reversion with a second

treatment.

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Site-Specific in vitro Mutagenesis of DNA

1. Recombinant DNA technology allows genes to be

mutated at specific positions, and then introduced

back into cells.

2. This allows study of genes of unknown function

and sequences used to regulate gene expression.

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Chemical Mutagens in the Environment

Animation: Ames Test Protocol

1. A wide variety of chemicals exist in our environment, and many can have mutagenic effects that can lead to genetic diseases and cancer. Examples include:

a. Drugs b. Cosmetics c. Food additives d. Pesticides

e. Industrial compounds

f. Chemical warfare agents such as mustard gas

2. The Ames test is a simple and inexpensive screen for potential carcinogens. It assays the reversion rate of mutant strains of Salmonella typhimurium back to wild-type (Figure 7.14).

a. Histidine (his) auxotrophs are tested for reversion in the presence of the chemical, by plating on media lacking the amino acid histidine.

i. Different his tester strains are available, to test for base-substitution and frameshift mutations.

ii. Liver enzymes (the S9 extract) are mixed with the test chemical to determine whether the liver’s detoxification pathways convert it to a mutagenic form.

iii. More revertants in the region of the test chemical than in the untreated control indicates that it may be a mutagen, and further tests are indicated.

b. The Ames test is used routinely to screen industrial and agricultural chemicals, and shows a good, but not perfect, correlation between mutagens and carcinogens.

c. The test can be made quantitative to produce a dose-response curve, allowing comparison of relative mutagenicity of different chemicals.

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Fig. 7.14 The Ames test for assaying the potential mutagenicity of chemicals

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Detecting Mutation

1. Mutants are often studied by geneticists.

Generally, mutation in haploid organisms is

readily detected, while recessive mutations in

diploid organisms are more difficult to

characterize. The problem is compounded in

humans, where controlled crosses cannot be done.

2. For some organisms, especially microorganisms,

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Visible Mutations

1. Some mutations affect the appearance of an

organism (e.g., Drosophila eyes or wing-shape,

coat color in animals, colony size in yeast, plaque

morphology of phages).

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Nutritional Mutations

1. Auxotrophic mutants are easily detected for

microorganisms that normally can grow on minimal

medium, using methods that have been developed for

selection and screening.

2. An example is replica plating. Cells are first grown on

supplemented medium, and then the colonies transferred

to minimal medium, as well as to a control plate of

supplemented medium. Colonies that grow on

supplemented, but not minimal, media are selected for

further study.

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Fig. 7.15 Replica-plating technique to screen for mutant strains of a colony-forming microorganism

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Conditional Mutations

1. Mutations in some genes (e.g., DNA and RNA

polymerases) are usually lethal, so these genes are

studied by isolating conditional mutations.

2. Heat sensitivity is a common conditional

mutation, in which a normal protein is produced

at permissive temperature, and a nonfunctional

protein results at the nonpermissive temperature.

Screening is generally by replica plating and

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Resistance Mutations

1. Microorganisms like E. coli and yeast are easily

screened for resistance to viruses, chemicals or

drugs, because resistant cells will grow when

wild-type cells will not.

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Repair of DNA Damage

1. Both prokaryotes and eukaryotes have

enzyme-based DNA repair systems that prevent mutations

and even death from DNA damage.

2. Repair systems are grouped by their repair

mechanisms. Some directly correct, while others

excise the damaged area and then repair the gap.

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Direct Reversal of DNA Damage

1. DNA polymerase proofreading corrects most of the incorrect nucleotide insertions that occur during DNA synthesis, which stalls until the wrong nucleotide is replaced with a correct one.

a. The role of 3’-to-5’ exonuclease activity is illustrated by mutator mutations in E. coli, which confer a much higher mutation rate on the cells that carry them.

b. The mutD gene, encoding the e subunit of DNA polymerase III, is an example. Cells mutant in mutD are defective in proofreading.

2. UV-induced pyrimidine dimers are repaired using photoreactivation (light repair).

a. Near UV light (320–370 nm) activates photolyase (product of the phr gene) to split the dimer.

b. Photolyases are found in prokaryotes and simple eukaryotes, but not in humans.

3. Damage by alkylation (usually methyl or ethyl groups) can be removed by specific DNA repair enzymes.

a. For example, O6-methylguanine methyltransferase (from the ada gene) recognizes O6

-methylguanine in DNA, and removes the methyl group, restoring the base to its original form.

b. A similar system repairs alkalated thymine.

4. Mutations of repair enzyme genes increase the organism’s rate of spontaneous mutations.

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Base Excision Repair

1. Base excision repair uses a repair glycosylase

enzyme to recognize and remove damaged bases.

a. Bond between base and deoxyribose is cleaved by the

glycosylase.

b. Other enzymes cleave sugar-phosphate from

backbone, leaving a gap in the DNA.

c. Repair DNA polymerase and DNA ligase use the

opposite strand as template to fill the gap.

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Repair Involving Excision of Nucleotides

1. A repair system, which does not require light, was discovered

in 1964. It is called dark repair, the excision repair system, or

the nucleotide excision repair (NER) system.

a. In E. coli, NER corrects pyrimidine dimers and other damage-induced distortions of the DNA helix.

b. The proteins required are UvrA, UvrB, UvrC and UvrD (encoded by genes of the same name) (Figure 7.16).

c. A complex of two UvrA and one UvrB proteins slides along the DNA. When it encounters a helix distortion, the UvrA subunits dissociate, and a UvrC binds the UvrB at the lesion.

d. When UvrBC forms, the UvrC cuts 4–5 nucleotides from the lesion on the 3’ side, and eight nucleotides away on the 5’ side. Then UvrB is released and UvrD binds the 5’ cut end.

e. UvrD is a helicase that unwinds the region between the cuts, releasing the short ssDNA, while DNA polymerase I fills the gap and DNA ligase seals the backbone.

f. In yeast and mammalian systems, about 12 genes encode proteins involved in excision repair.

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Fig. 7.16 Nucleotide excision repair (NER) of pyrimidine dimmer and other

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2. Methyl-directed mismatch repair recognizes mismatched base pairs, excises the

incorrect bases and then carries out repair synthesis.

a. In E. coli, initial stages involve products of the mutS, mutL and mutH genes (Figure 7.17).

i. MutS binds the mismatch, and determines which is the new strand by its lack of methylation.

ii. MutL and MutH bind unmethylated GATC sequences (site of methylation in E.

coli) and bring the GATC close to the mismatch by binding MutS.

iii. MutH then nicks the unmethylated GATC site, the mismatch is removed by an exonuclease and the gap is repaired by DNA polymerase III and ligase.

b. Eukaryotes also have mismatch repair, but it is not clear how old and new DNA strands are identified.

i. Four genes are involved in humans, hMSH2 (homologous to E. coli mutS), and

hMLH1, hPMS1 and hPMS2 (all homologous to mutL).

ii. All of these are mutator genes, and mutation in any 1 of them confers

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3. Translesion DNA synthesis is used to allow a cell to survive when specific

base-pairing cannot occur. Survival is usually at the cost of incurring new

mutations.

a. Bacteria use a system called SOS. In E. coli SOS is controlled by two genes, lexA and recA. Mutants in either of these genes have their SOS response permanently turned on.

i. When no DNA damage is present, LexA represses transcription of about 17 genes with products involved in various types of DNA repair.

ii. Sufficient DNA damage activates the RecA protein, which stimulates LexA to autocleave, removing repression of the DNA repair genes.

iii. After damage is repaired, RecA is inactivated, and newly synthesized LexA again represses the DNA repair genes.

b. The SOS system is a mutagenic bypass synthesis system.

i. DNA polymerase for translesion synthesis is made in SOS response. It replicates over and past the lesion.

ii. Nucleotides are incorporated at the lesion that may not match wild type, leading to mutations.

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Human Genetic Diseases Resulting from DNA

Replication and Repair Mutations

1. Many human genetic disorders result from gene

mutations, and are included in the OMIM website

(http://www3.ncbi.nlm.nih.gov/OMIM).

2. Some human genetic diseases resulting from defects in

DNA replication or repair are listed in Table 7.1.

Xeroderma pigmentosum is an example (Figure 7.18).

a. The disorder occurs in homozygotes for a mutation in a repair

gene.

b. Affected individuals are photosensitive, and portions of skin

exposed to light show intense pigmentation and warty growths

that may become malignant.

c. The defect is in excision repair, and the inability to repair

radiation damage to DNA often results in malignancies.

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General Features of Transposable Elements

1. Transposable elements are divided into two classes on the basis of their

mechanism for movement:

a. Some encode proteins that move the DNA directly to a new position or replicate the DNA to produce a new element that integrates elsewhere. This type is found in both prokaryotes and eukaryotes.

b. Others are related to retroviruses, and encode reverse transcriptase for making DNA copies of their RNA transcripts, which then integrate at new sites. This type is found only in eukaryotes.

2. Transposition is nonhomologous recombination, with insertion into DNA

that has no sequence homology with the transposon.

a. In prokaryotes, transposition can be into the cell’s chromosome, a plasmid or a phage chromosome.

b. In eukaryotes, insertion can be into the same or a different chromosome.

3. Transposable elements can cause genetic changes, and have been involved in

the evolution of both prokaryotic and eukaryotic genomes. Transposons may:

a. Insert into genes.

b. Increase or decrease gene expression by insertion into regulatory sequences.

c. Produce chromosomal mutations through the mechanics of transposition.

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Transposable Elements in Prokaryotes

1. Prokaryotic examples include:

a. Insertion sequence (IS) elements.

b. Transposons (Tn).

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Insertion Sequences

Animation: Insertion Sequences in Prokaryotes

1. IS elements are the simplest transposable elements found in

prokaryotes, encoding only genes for mobilization and insertion of its

DNA. IS elements are commonly found in bacterial chromosomes and

plasmids.

2. IS elements were first identified in E. coli’s galactose operon,

wheresome mutations’ were shown to result from insertion of a DNA

sequence now called IS1 (Figure 7.19)

3. Prokaryotic IS elements range in size from 768bp to over 5kb. Known

E. coli IS elements include IS1 which is 768bp long and present in 4–

19 copies on the E. coli chromosome, IS2, and IS10R.

4. The ends of all sequenced IS elements show inverted terminal repeats

(IRs) of 9–41bp. IS1 has 23bp of nearly identical sequence.

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5. Random integration of IS elements may:

a. Disrupt coding sequences or regulatory regions.

b. Alter expression of nearby genes by the action of IS element promoters.

c. Cause deletions and inversions in adjacent DNA.

d. Serve as a site for crossing-over between duplicated IS elements.

6. When an IS element transposes:

a. Transposition requires transposase, an enzyme encoded by the IS element.

b. Transposase recognizes the IR sequences to initiate transposition at a

frequency characteristic for each IS element (range is 105 to 107 per



generation).

c. IS elements insert into the chromosome without sequence homology

(illegitimate recombination) at target sites (Figure 7.20).

i. A staggered cut is made in the target site, and the IS element inserted.

ii. DNA polymerase and ligase fill the gaps, producing small direct

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Fig. 7.20 Schematic of the integration of an IS element into chromosomal DNA

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Transposons

1. Transposons are similar to IS elements, but carry additional genes, and

have a more complex structure. There are two types of prokaryotic

transposons:

a. Composite transposons carry genes (e.g., antibiotic resistance) flanked

on both sides by IS elements (IS modules).

i. The IS elements are of the same type, and called ISL (left) and ISR

(right).

ii. ISL and ISR may be in direct or inverted orientation to each other.

iii.Transposition of composite transposons results from the IS

elements, which supply transposase and its recognition signals, the

IRs (Figure 7.21).

(1) Tn10’s transposition is rare (

＜ 10

-7

/molecule/generation).

(2) Transposons, like IS elements, produce target site

duplications (e.g., a 9-bp duplication for Tn10).

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b. Noncomposite transposons also carry

genes (e.g., drug resistance) but do not

terminate with IS elements.

i. Transposition proteins are encoded in

the central region.

ii. The ends are repeated sequences (but

not IS elements).

iii. Noncomposite transposons cause

target site duplications (like composite

transposons).

iv. An example is Tn3, which produces a

5-bp duplication upon insertion.

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2. Models have been generated for transposition:

a. Cointegration is an example of the replicative transposition that

occurs with Tn3 and its relatives (Figure 7.22).

i. Donor DNA containing the Tn fuses with recipient DNA.

ii. The Tn is duplicated, with one copy at each donor-recipient DNA

junction, producing a cointegrate.

iii. The cointegrate is resolved into two products, each with one copy

of the Tn.

b. Conservative (nonreplicative) transposition is used by Tn10, for

example. The Tn is lost from its original position when it transposes.

3. Transposons cause the same sorts of mutations caused by IS elements:

a. Insertion into a gene disrupts it.

b. Gene expression is changed by adjacent Tn promoters.

c. Deletions and insertions occur.

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Fig. 7.22 Cointegration model for transposition of a transposable element by

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IS Elements in the E. coli F factor

1. The E. coli F factor is an episome, able to

integrate into the bacterial chromosome and

capable of self-replication.

a. The F plasmid contains four IS elements: two copies

of IS3, one of IS2, and one of γδ(gamma-delta).

b. The E. coli chromosome also has copies of these

insertion sequences in different sites and orientations.

c. Homologous recombination moves the F factor into

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Transposable Elements in Eukaryotes

1. Rhoades (1930s) working with sweet corn, observed

interactions between two genes:

a. A gene for purple seed color, the Al locus. Homozygous mutants

(a/a) have colorless seeds.

b. A gene on a different chromosome, Dt (dotted) that causes seeds

with genotype a/a Dt/-- to have purple dots.

i. Dt appears to mutate the a allele back to the Al wild-type in

regions of the seed, producing a dotted phenotype.

ii. The effect of the Dt allele is dose dependent.

(1) One dose gave an average of 7.2 dots per seed.

(2) Two doses gave an average of 22.2 dots/seed.

(3) Three doses gave an average of 121.9 dots/seed.

c. Rhoades interpreted Dt as a mutator gene.

2. McClintock (1940s-50s), working with corn (Zea mays)

proposed the existence of “controlling elements” that

regulate other genes and are mobile in the genome.

(66)

3. The genes studied by both Rhoades and McClintock have turned out to

be transposable elements, and many others have been identified in

various eukaryotes.

a. Most studied are transposons of yeast, Drosophila, corn and humans.

b. Their structure is very similar to that of prokaryotic transposable

elements.

c. Eukaryotic transposable elements have genes for transposition and

integration at a number of sites, as well as a variety of other genes.

d. Random insertion results from non-homologous recombination, and

means that any chromosomal gene may be regulated by a transposon.

4. The result of transposon insertion into a chromosome will depend on the

properties of the transposon, with possible effects including:

a. Chromosome mutations such as duplications, deletions, inversions,

translocations, or breakage.

b. Disruption of genes to produce a null mutation (gene is nonfunctional).

c. Activation or repression of adjacent genes by disrupting a cellular

(67)

Transposons in Plants

Animation: Transposable Elements in Plants

1. Plant transposons also have IR sequences, and generate short direct

target site repeats.

2. Several families of transposons have been identified in corn, each with

characteristic numbers, types and locations.

a. Each family has two forms of transposon. Either can insert into a gene

and produce a mutant allele.

i. Autonomous elements, which can transpose by themselves. Alleles

produced by an autonomous element are mutable alleles, creating

mutations that revert when the transposon is excised from the gene.

ii. Nonautonomous elements, which lack a transposition gene and

rely on the presence of another transposon to supply the missing

function. Mutation by these elements is stable (except when an

autonomous element from the family is also present).

(68)

3. Multiple genes control corn color, and classical genetics indicates that a

mutation in any of these genes leads to a colorless kernel. McClintock

studied the unstable mutation that produces spots of purple pigment on

white kernels (Figure 7.23).

a. She concluded that spots do not result from a conventional mutation,

but from a controlling element (now Tn).

b. A corn plant with genotype c/c will have white kernels, while C/-- will

result in purple ones.

i. If a reversion of c to C occurs in a cell, that cell will produce purple

pigment, and hence a spot.

ii. The earlier in development the reversion occurs, the larger the

spot.

(69)

iii. McClintock concluded that the c allele resulted from insertion of a

“mobile controlling element” into the C allele. (Figure 7.24)

(1) The element is Ds (dissociation), now known to be a

nonautonomous transposon.

(2) Its transposition is controlled by Ac (activator), an

autonomous transposon.

c. McClintock’s evidence of transposable elements did not fit the

prevailing model of a static genome. More recent studies have

confirmed and characterized the elements involved, the Ac-Ds system.

i. The autonomous Ac element is 4,563bp, with short inverted terminal

repeats and one gene (transposase). Insertion generates an 8-bp target

site duplication.

ii. The nonautonomous Ds elements vary in length and sequence, but

all have the same terminal IRs as Ac, and most are deleted or

(70)

(71)

iii. Ac transposes only during chromosome replication, by a

conservative transposition mechanism (Figure 7.25). There

are two possible results of Ac transposition, depending on

whether the target DNA has replicated or not.

(1) If Ac transposes during replication into a replicated

target site, its chromatid’s donor site will be empty since

that copy of Ac has inserted elsewhere. In the

homologous donor site on the other chromatid, a copy

will remain. There is no net increase in copies of Ac.

(2) Transposition to an unreplicated chromosome site also

leaves one donor site empty (and the other with a copy of

Ac). The DNA into which Ac inserts will then be

replicated, resulting in a net gain of one copy of Ac.

iv. Replication of Ds is the same, except that the

(72)

(73)

Ty Elements in Yeast

1. Ty element is diagrammed in Figure 7.26:

a. It is 5.9 kb including 2 terminal direct repeats of 334 bp, the long terminal repeats (LTR) or deltas (δ).

b. Each delta contains a promoter and transposase recognition sequences.

c. Ty elements encode one 5.7 kb mRNA beginning at the delta 5’ promoter (Figure 20.14).

d. There are two ORFs in the mRNA, designated TyA and TyB, encoding two different proteins.

e. Ty copy number varies between yeast strains, with an average of about 35.

2. Ty elements also share similarities with retroviruses, ssRNA viruses that replicate

via dsDNA intermediates.

a. Ty elements transpose by making an RNA copy of the integrated DNA sequence, then making DNA using reverse transcriptase. This DNA can integrate at a new

chromosomal site. Evidence for this includes:

i. An experimentally introduced intron in the Ty element (which normally lacks

introns) was monitored through transposition. The intron was removed, indicating an RNA intermediate.

ii. Ty elements encode a reverse transcriptase.

iii. Virus-like particles containing Ty RNA and reverse transcriptase activity occur. b. Ty elements are referred to as retrotransposons.

(74)

(75)

Drosophila transposons

1. It is estimated that 15% of the Drosophila genome is mobile!

The P element is an example. P elements vary from full-length

autonomous elements through shorter nonautonomous versions that

lack the transposase gene (Figure 7.27).

2. P elements are used experimentally to transfer genes into the germ

line of Drosophila embryos. For example (Figure 7.28):

a. The wild-type rosy

+

(ry

+

) gene was inserted into a P element,

cloned in a plasmid, and microinjected into a mutant ry/ry

strain.

b.Insertion of the recombinant P element into the recipient

chromosome introduced the ry

+

allele, and produced wild-type

(76)

Fig. 7.27 Structure of the autonomous P transposable element found in

Drosophila

(77)

Fig. 7.28 Illustration of the use of P elements to introduce genes into the

Drosophila

(78)

Human Retrotansposons

1. Retrotransposons in humans include:

a. LINEs (long interspersed sequences), repeated sequences >5kb interspersed with unique-sequence DNA up to 35kb. LINEs occur as:

i. Autonomous elements with functional transposition genes. ii. Nonautonomous derivatives with internal deletions.

b. SINEs (short interspersed sequences), 100–400bp repeats interspersed with unique-sequence DNA 1–2kb in length.

2. LINEs make up about 20% of the human genome. L1 is the best studied type.

a. L1 elements are about 5% of the human genome. The full-length element (6.5kb) is not abundant, and most L1 elements are deleted versions.

b. The full-length L1 element contains a large ORF with homology to known reverse transcriptases. Experimentally, the L1 ORF can substitute for the yeast Ty reverse transcriptase gene.

c. L1 elements transpose via RNA, but unlike other retrotransposons, do not have LTRs, so use a different mechanism for transposition.

d. Clinically, cases of hemophilia have been shown to result from newly transposed L1 insertions into the gene for factor VIII (required for normal blood clotting). This

insertional mutagenesis has been found in families where a child has L1 in the factor VIII gene, but neither parent does.

(79)

台大農藝系遺傳學 601 20000 Chapter 19 slide 79 3. SINEs lack genes for transposition, and so are nonautonomous, requiring enzymes

encoded by LINEs in order to move. The Alu family is a very abundant type of SINE.

a. Alu sequences (named for the restriction site contained in their sequences) are about 300bp, repeated 300,000–500,000 times in the genome. Alu sequences make up to 3% of total human DNA.

b. A human case of a genetic disease, neurofibromatosis, provides some evidence. i. Neurofibromas (tumorlike growths on the body) result from an

autosomal dominant mutation.

ii. In a patient’s DNA, an unusual Alu sequence was detected in one of the introns of the neurofibromatosis gene.

iii. The resulting longer transcript is incorrectly processed, removing an exon from the mRNA and producing a nonfunctional protein.

iv.Neither parent had this Alu sequence in the neurofibromatosis gene. v. Divergent Alu sequences made it possible to track this particular version to an insertion event in the germ line of the patient’s father.