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Transcript
Peter J. Russell
CHAPTER 19
DNA Mutation and Repair
edited by Yue-Wen Wang Ph. D.
Dept. of Agronomy,台大農藝系
NTU
遺傳學 601 20000
Chapter 19 slide 1
Adaptation Versus Mutation
1. A question that received much attention in the early 20th 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.
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 19.2).
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
Chapter 19 slide 2
cultures, indicating that the random mutation台大農藝系
model is 遺傳學
correct.601 20000
Fig. 19.2 Representation of a dividing population of T1 phage-sensitive wild-type
E. coli
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 3
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).
台大農藝系 遺傳學 601 20000
Chapter 19 slide 4
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 19.3):
a. Transitions convert a purine-pyrimidine pair to the other purinepyrimidine 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 19.4).
台大農藝系 遺傳學 601 20000
Chapter 19 slide 5
Fig. 19.3a-d Types of base-pair substitution mutations
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 6
Fig. 19.3e-g Types of base-pair substitution mutations
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 7
Fig. 19.4 A nonsense mutation and its effect on translation
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 8
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.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 9
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
suppressed by a nearby deletion event).
台大農藝系 遺傳學 601 20000
Chapter 19 slide 10
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 wildtype 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).
台大農藝系 遺傳學 601 20000
Chapter 19 slide 11
Fig. 19.5 Mechanism of action of an intergenic nonsense suppressor mutation that
results from mutation of a tRNA gene
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 12
Spontaneous and Induced Mutations
1. Most mutations are spontaneous, rather than
induced by a mutagen.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 13
Spontaneous Mutations
1. All types of point mutations can occur spontaneously, during S, G1 and
G2 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.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 14
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. A
normal form of the base-pairs with an incorrect partner due to different
spatial positioning of the atoms involved in H-bonding (Figure 19.6).
An example is a GC-to-AT transition (Figure 19.7):
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.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 15
Fig. 19.6 Normal and wobble base pairing in DNA
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 16
Fig. 19.7 Production of a mutation as a result of a mismatch caused by wobble base
pairing
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 17
3. Additions and deletions can occur spontaneously during
replication (Figure 19.8).
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).
台大農藝系 遺傳學 601 20000
Chapter 19 slide 18
Fig. 19.8 Spontaneous generation of addition and deletion mutants by DNA
looping-out errors during replication
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 19
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 19.9).
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 5methylcytosine (5mC) in place of the normal C.
(1) Deamination of 5mC produces T.
(2) T is a normal nucleotide in DNA, so it is not detected by repair
mechanisms.
(3) Deamination of 5mC results in CG-to-TA transitions.
(4) Locations of 5mC in the chromosome are often detected as
mutational hot spots (Figure 19.10).
台大農藝系 遺傳學 601 20000
Chapter 19 slide 20
Fig. 19.9 Deamination of cytosine to uracil (a); deamination of 5-methylcytosine to
thymine
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 21
Fig. 19.10 Distribution of spontaneous GC-to-AC transition mutations to stop codons
in the lac repressor gene of E. coli
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 22
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 19.11).
(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.
台大農藝系
遺傳學 601 20000
Chapter 19 slide 23
Fig. 19.11 Production of thymine dimers by ultraviolet light irradiation
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 24
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 19.12).
(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 TAto-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). Chapter 19 slide 25
台大農藝系 遺傳學 601 20000
Fig. 19.12a, b Mutagenic effects of the base analog 5-bromouracil (5BU)
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 26
Fig. 19.12c Mutagenic effects of the base analog 5-bromouracil (5BU)
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 27
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 19.13):
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-toCG transitions (e.g., 5BU and HNO2).
iii. Allcylating agents are a diverse group that add ailcyl 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.
(2) O6-aIkylG pairs with T rather than C, causing GC-to-AT transitions.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 28
Fig. 19.13a Action of three base-modifying agents
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 29
Fig. 19.13b, c Action of three base-modifying agents
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 30
c. Intercalating agents insert themselves between adjacent bases in
dsDNA. 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.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 31
Fig. 19.14 Intercalating mutations
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 32
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. Many procedures have been developed for site-specific mutagenesis, often
using PCR (polymerase chain reaction). An example is shown in Figure 19.15.
a. This method uses four primers:
i. Primers are needed for each end of the sequence to be amplified, as in normal
PCR (primers 1 and 2 in the figure).
ii. A pair of complementary primers, 1M and 2M, that create the mutation by
matching the target sequence except for where the mutation is desired.
iii. Two PCR reactions are done, one with primers 1 and 1M, the other with
primers 2 and 2M.
iv. Primers are removed, the two PCR reactions are mixed, and the DNA
denatured and allowed to reanneal.
v. Some reannealing will result in strands capable of producing the full-length
product in a PCR reaction.
vi. Amplification using primers 1 and 2 produces mutant full-length molecules
that can be transformed into a cell to replace the wild-type sequence.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 33
Fig. 19.15 Site-specific mutagenesis using PCR
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 34
3. Mutant mice have been created with this technique, providing mammalian models
for genetic disease.
a. If a human gene is cloned, its mouse equivalent gene can be cloned as well, due to
strong homology.
b. The cloned mouse gene can be mutagenized in vitro (e.g., a deletion) so that its
product is completely nonfunctional.
c. The mutant DNA is microinjected into mouse embryos, and may replace one of
their wild-type alleles.
d. Progeny mice are screened for the mutation (e.g., PCR, restriction analysis or
DNA probing) to identify heterozygotes.
e. Interbreeding of the heterozygotes produces some knockout progeny that are
homozygous for the mutation (if it is not lethal) and have a null phenotype.
f. Examples of knockout mice include:
i. Tumor suppressor gene p53, producing knockout mice that rapidly develop
tumors and other mutant phenotypes.
ii. Cystic fibrosis (CF) gene, producing mucous membrane defects throughout
the body due to defective chloride ion transport.
4. Any organism that can be transformed by cloned DNA and undergo homologous
recombination can be mutated using the knockout approach.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 35
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.
a. Mutagens typically produce base-pair substitutions or insertions or deletions.
b. Most cancer development results from accumulated mutations in a number of genes
(oncogenes, tumor suppressor and perhaps mutator genes).
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
19.16).
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.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 36
Fig. 19.16 The Ames test for assaying the potential mutagenicity of chemicals
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 37
DNA Repair Mechanisms
1. Both prokaryotes and eukaryotes have enzymebased 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.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 38
Direct Correction of Mutational Lesions
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.
b. Demethylation restores the base to its original form.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 39
Repair Involving Excision of Base Pairs
1. Another 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 19.17).
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.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 40
Fig. 19.17 Nucleotide excision repair (NER) of pyrimidine dimmer and other damageinduced distortions of DNA
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 19 slide 41
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
19.18).
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
hereditary predisposition to hereditary nonpolyposis colon cancer (HNPCC).
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Fig. 19.18 Mechanism of mismatch correction repair
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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3. The SOS response in bacteria results when specific base pairing cannot occur.
It allows the cell to survive otherwise lethal events, but usually at the cost of
incurring new mutations.
a. 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 an error-prone bypass synthesis system.
i. Some lesions, like T^T dimers, are easily copied to give AA in the new DNA
strand.
ii. Others, like C^C dimers, stall the SOS repair system, creating a delay during
which C can be deaminated to a U (forming a C^U), creating a transition
mutation.
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Human Genetic Diseases Resulting from DNA
Replication and Repair Mutations
1. Many human genetic disorders result from gene mutations,. and a number are
discussed elsewhere in the text. Another example is familial
hypercholesterolemia (FH).
2. FH is an autosomal dominant trait that includes high blood cholesterol levels,
artheroscierosis, heart attacks and usually early death.
a. Individuals with LH have defective or absent cell surface receptors for low-density
lipoproteins (LDLs).
b. LH individuals are defective in LDL uptake, and so LDL accumulates on artery walls
as plaque, resulting in heart disease.
c. Different alleles for LH may result in either defective or completely absent LDL
receptors.
3. Some human genetic diseases resulting from defects in DNA replication or
repair are listed in Table 19.1. Xeroderma pigmentosum is an example (Figure
19.19).
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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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,
selection and screening procedures exist.
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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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Auxotrophic 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. 19.20 Replica-plating technique to screen for mutant strains of a colony-forming
microorganism
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
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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
incubation at different temperatures.
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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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Molecular Screens
1. Targeted mutations require screening to detect individuals with the
desired mutation. Techniques are similar to screens for human disease
and DNA typing, and use PCR, restriction enzyme analysis and DNA
probing.
2. An example is yeast genome analysis, which attempts to systematically
identify the approximately 6,000 ORFs in that organism by knocking
out each one in turn and determining the phenotype (if any).
a. Knocked-out gene is replaced with a kanamycin resistance (kanR) gene.
The knockout is confirmed by PCR (Figure 19.21).
b. Size of PCR product with defined primers indicates whether
substitution of kanR for the target DNA has been successful.
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Fig. 19.21 Example of molecular screening: Confirmation of gene deletions in yeast
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
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