S.U.1: Gene mutations & DNA repair
 Gene responsible for heart problems is located on chrm 5, at a spot where the human tinman gene was mapped.
 All family members who inherited the heart defects also inherited a mutation in the tinman gene.
 People with congenital heart defects have a mutation in the tinman gene.
 Encodes a transcription factor that controls heart development.
 Analysis of mutants is often a source of key insights into NB biological processes.
19.1 Mutations are inherited alterations in the DNA sequence.
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DNA is a highly stable molecule that is replicated with accuracy.
 Changes in DNA structure & errors of replication occur.
Mutation= an inherited change in genetic info.
 Descendants may be cells or organisms.
Importance of mutations
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Mutations are the sustainers of life & cause suffering.
Mutations are the source of all genetic variation, raw material of evolution.
 Ability of organisms to adapt to environmental change depends critically on the presence of genetic variation
in natural populations & genetic variation is produced by mutation.
Many mutations have detrimental effects, & mutation is a source of many disease & disorders.
Genetic crosses are meaningless if all individual members of a species are identically homozygous for same alleles.
Mutations are useful for probing fundamental biological processes.
 Finding/creating mutations that affect biological system & studying their effects can lead to an understanding
of the system.
* Genetic dissection.
Categories of mutations – Fig 19.1
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In multicellular organisms, can distinguish between 2 categories of mutations.
1. Somatic mutations arise in somatic tissues, which do not produce gametes.
 When a somatic cell with a mutation divides, the mutation is passed on to the daughter cells.
 Leads to a population of genetically identical cells  Clone.
 Earlier in development that a somatic mutation occurs, the larger the clone of cells within that individual
organism that will contain the mutation.
 Huge number of cells present in a typical euk organism, somatic mutations are numerous
 Many somatic mutations have no obvious effect on the PT of the organism.
 Function of the mutant cell is replaced by that of the normal cells.
 However, cells with a somatic mutation that stimulates cell division can increase in number and
spread can give rise to cells with a selective advantage & is the basis for cancers
2. Germ-Line mutations arise in cells that ultimately produce gametes.
 Can be passed to future generations, producing individual organisms that carry the mutation in all their
somatic & germ-line cells.
 Use germ-line to speak of mutations in multicellular organisms.
 Mutations: Divided into those that affect a single gene (gene mutations), & those that affect the number
or structure of the chrm (chromosome mutations).
 Chromosome mutations could be seen with a microscope, whereas gene mutations could only be
detected by looking at the PT.
 Now: DNA sequencing allows direct observations of gene mutations.
* Mutations are distinguished on the basis of size of the DNA lesion.
 Use chromosome mutation for large-scale genetic alteration affecting chrm structure or number.
 Use gene mutation for a relatively small DNA lesion affecting a single gene.
GTS 261-Anya OberholzerPage 1
Types of gene mutations.
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Some classification schemes are based on the nature of the PT effect, others on the causative agent of the
mutation, & others focus on the molecular nature of the effect.
Base substitutions- Fig 19.2 & Fig 19.3
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Simplest type of gene mutation.
Two types:
1. Transition  A purine is replaced by a purine, or a pyrimidine is replaced by a pyrimidine.
 BASES ARE REPLACED BY THE SAME TYPE OF BASES
2. Transversion  A purine is replaced by a pyrimidine, or a pyrimidine is replaced by a purine.
BASES ARE REPLACED BY THE OPPOSITE TYPE OF BASES
The number of possible transversions is twice the number of possible transitions, but transitions arise more
frequently.
Insertions & Deletions- Fig 19.2
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Addition or removal of one or more nucleotide pairs.
More frequent than base substitutions.
Insertions & deletions within sequences that encode proteins may lead to Frameshift mutations.
 Frameshift mutations change the reading frame of the gene.
* Usually alter all A.A encoded by nucleotides following the mutation.
 Generally have drastic effects on the PT.
Insertions & deletions consisting of any multiple of 3 nucleotides will leave the reading frame intact, but insertion
or deletion of one or more A.A will affect the PT.
 Known as in-frame insertions & deletions.
Expanding nucleotide repeats- Fig 19.4, Fig 19.5 & Table 19.1
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Mutations in which the number of copies of a set of nucleotides increase in number.
First observed in 1991 in a gene FMR-1.
 FMR-1 causes fragile X-syndrome (most common hereditary cause of mental retardation).
* The tip of each long arm of the X chrm is attached only by a slender thread.
 The normal FMR-1 allele has 60 copies of CGG, but in a person with fragile X-syndrome, the allele may contain
hundreds to thousands of copies.
Have been found in almost 30 human diseases.
 Most are caused by expansion of a set of 3 nucleotides (trinucleotide).
* Most often CNG N is any nucleotide.
* Some diseases are caused by more repeats.
* Number of copies of nucleotides correlates with severity & age of onset.
* Number of copies of the repeat also corresponds to instability of nucleotide repeats.
 When more repeats are present, the probability of expansion to even more repeats increases.
The association btwn the number of copies of nucleotide repeats, severity of disease, & probability of expansion
leads to anticipation, in which diseases caused by nucleotide-repeat expansions become more severe in each
generation. Decreasing in family is less common.
Also seen in microbes & plants.
Increase in the number of nucleotide repeats can produce disease symptoms in diff ways.
 In some diseases (Huntington), the nucleotide expands within the coding part of the gene, producing a toxic
protein that has extra Glu residues (Glu is encoded by CAG).
 In other diseases, the repeat is outside the coding region & affects expression.
 In fragile X-syndrome, additional copies of repeats cause the DNA to become methylated, & turn off
transcription of an essential gene.
May occur during DNA replication & appears to be related to the formation of hairpins & other DNA structures that
form in single-stranded DNA consisting if nucleotide repeats.
 May interfere with normal replication by causing strand slippage, misalignment of sequences or stalling of
replication.
Phenotypic effects of mutations- Fig 19.6
GTS 261-Anya OberholzerPage 2
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Mutations can be classified on the basis of their PT effect comparing the mutation to the wild-type.
Forward mutation alters the wild-type PT.
Reverse mutation changes the mutant PT back into the wild-type.
Use diff terms to describe the effects of mutations on protein structure.
 Missense mutation is a base substitution that results in a diff A.A in the protein.
 Nonsense mutation changes a sense codon (one that specifies an A.A) into a nonsense codon (one that
terminates translation).
* If a nonsense mutation occurs early in the mRNA sequence, the protein will be greatly shortened & is
usually non-functional.
 Silent mutation changes a codon to a synonymous codon that specifies the same A.A, altering the DNA without
changing the A.A sequence of the protein.
* Silent mutations may have PT effects when diff tRNAs are used for diff synonymous codons.
 Because some isoaccepting tRNAs are more abundant than others, which synonymous codon is used
may affect the rate of protein synthesis.
 Rate of synthesis can influence the PT by affecting the amount of protein present in the cell, &
sometimes the folding of the protein.
* Silent mutations may alter sequences near the exon- intron junctions that affect splicing.
* Silent mutations may influence the folding of mRNA, affecting its stability.
 Neutral mutation is a missense mutation that alters the A.A sequence of a protein without changing function.
* Occur when one A.A is replaced by another that is chemically similar or when the affected A.A acid has
little influence on protein function.
 May occur in genes that encode haemoglobin.
 Loss-of-function mutations cause the complete or partial absence of normal protein function.
* Alters the structure of the protein so that the protein no longer works correctly or the mutation can occur
in regulatory regions that affect the transcription, translation, or splicing of the protein.
* Frequently recessive & an individual diploid organism must be homozygous for loss- of-function mutation
before the loss of functional protein can be exhibited.
 E.g. Cystic fibrosis.
 Gain-of-function mutations produce an entirely new trait or it causes a trait to appear in an inappropriate
tissue or inappropriate stage of development.
* A mutation in a gene that encodes a receptor for a growth factor might cause the mutated receptor to
stimulate growth all the time, even in the absence of the growth factor.
* Frequently dominant in their expression.
 Conditional mutations are expressed only under certain conditions.
* E.g. PT affected only at certain temps.
 Lethal mutations cause premature death.
Suppressor Mutations- Fig 19.7
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A genetic change that hides or suppresses the effect of another mutation.
NOT A REVERSE MUTATION.
A suppressor mutation occurs at a site that is distinct from the site of the original mutation.
An individual with a suppressor mutation is a double mutant, possessing both original mutation & suppressor
mutation but exhibiting the wild-type PT.
Arise randomly.
Two classes
1. Intragenic
2. Intergenic
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Intragenic suppressor mutation- Fig 19.8
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Intragenic suppressor mutation is in the same gene as that containing the mutation being suppressed & may work
in several ways.
The suppressor may change the second nucleotide in the same codon altered by the original codon.
 Producing a codon that specifies the same A.A as that specified by the unmutated codon.
May also work by suppressing a Frameshift mutation.
 If the original mutation is a one base deletion, then the addition of a single base elsewhere in the gene will
restore the former reading frame.
Consider:
DNA
AAA
TCA
CTT
GGC
GTA
CAA
A.A
Phe
Ser
Glu
Pro
His
Val
a
one-base
deletion
occurs
in
the
first
nucleotide
of the second codon.
Suppose
This deletion shifts the reading frame by one nucleotide & alters all A.A that follow the mutation.
AAA
TCAC TTG
GCG
TAC
AA
Phe
Val
Asn
Arg
Met
a
single
A.A
is
added
to
the
third
codon,
the
reading frame is restored, although 2 of the A.A differ from
If
those in the original sequence.
AAA
CAC
TTT
GGC
GTA
CAA
Phe
Val
Lys
Pro
His
Val
A mutation due to an insertion may be suppressed by a subsequent deletion in the same gene.
An intragenic suppressor may work by compensatory changes in the protein.
 A first missense mutation may alter the folding of a polypeptide chain by changing the way in which A.A in the
protein interact with one another.
 A second missense mutation at a diff site (suppressor) may recreate the original folding pattern by restoring
interactions btwn A.A.
Intergenic suppressor mutations- Fig 19.9 & Table 19.2
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An Intergenic suppressor mutation occurs in a gene other than the one bearing the original mutation.
May work by changing the way that the mRNA is translated.
The effect of this change would depend on the role of the A.A in the overall structure of the protein.
 The effect of the suppressor mutation is likely to be less detrimental than the effect of the nonsense mutation
which halts translation prematurely.
Because cells in many organisms have multiple copies of tRNA genes, other unmutated copies of tRNATyr would
remain available to recognise Tyr codons in the transcripts of the mutant gene & other genes would be expressed
concurrently.
May expect that tRNAs that have undergone suppressor mutation would also suppress the normal termination
codons at the ends of other coding sequences.
 Resulting in the production of longer-than-normal protein.
* This does not usually take place.
Can also work through genetic interactions.
 Polypeptide chains that are produced by 2 genes may interact to produce a functional protein.
* A mutation in one gene may alter the encoded polypeptide such that the interaction btwn the 2
polypeptides is destroyed functional protein is not produced.
* A suppressor mutation in the second gene may produce a compensatory change in its polypeptide.
Restoring the original interaction.
 TABLE 19.2 IS NB
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Mutation rates.
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Mutation rates are the frequency with which a wild-type allele at a locus changes into a mutant allele.
 Expressed as the number of mutations per biological unit.
* May be mutations per cell division, per gamete, or per round of replication.
 Achondroplasia is a hereditary dwarfism in humans that results from a dominant mutation.
 The mutation rate is 0.00004 per gamete, & provides info about how often a mutation arises.
Factors affecting mutation rates
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Calculations of mutation rates are affected by 3 factors.
1. The frequency with which a change takes place in DNA.
 A change in the DNA can arise from spontaneous molecular changes DNA or it can be induced by
chemical, biological, or physical agents in the environment.
2. The probability that, when changes take place, it will be repaired.
 Most cells possess a number of mechanisms for repairing altered DNA, & so most alterations are
corrected before they are replicated.
 If the repair systems are effective, mutation rates will be low
 If the repair systems are faulty, mutation rates will be elevated.
 Some mutations increase the overall rate of mutation at other genes.
 These mutations usually occur in genes that encode components of the replication machinery or
DNA-repair enzymes.
3. The probability that a mutation will be recognised & recorded.
 When DNA is sequenced, all mutations are potentially detectable.
 Mutations are usually detected by their PT effects.
 Some mutations may appear to arise at a higher rate simply because they are easier to detect.
Variation in mutation rates- Table 19.3
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Mutation rates vary among genes & species, but several conclusions about mutation rates can be made.
1. Spontaneous mutation rates are low for all organisms studied.
 Typical mutation rates for bacterial genes range from about 1 to 100 mutations per 10 billion cells.
 The mutation rates for most euk genes are a bit higher from about 1 to 10 million per million gametes.
 Higher values in euk may be due to the fact that the rates are calculated per gamete, &several cell
divisions are required to produce a gamete.
 Mutation rates in prok cells are calculated per cell division.
2. Diff among species may be due to differing abilities to repair mutations, unequal exposure to mutagens, or
biological differences in rates of spontaneously arising mutations.
 Even within a single species, spontaneous rates of mutation vary among genes.
Reasons for this variation are not entirely understood, but some regions of DNA are known to be more susceptible
to mutations than others.
Recent studies have measured mutation rates directly by sequencing genes of organisms before & after a number
of generations.
 Suggest that mutation rates are often higher than those previously measured on the basis of changes in PT.
 Geneticists sequenced randomly chosen stretches of DNA in the nematode worn Caenorhabditis elegans
& found  2.1 million mutations per genome per generation.
 Half of the mutations were insertions &deletions.
GTS 261-Anya OberholzerPage 5
Adaptive mutation
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Evolutionary change that brings about adaptation to new environments depends critically on the presence of
genetic variation.
New genetic variants arise primarily through mutation.
Adaptive mutation is a process in which more mutations in bacteria are induced in order to survive due to stressful
environments.
Critics counter that most mutations are expected to be deleterious, & so increased mutagenesis would likely be
harmful most of the time.
Mutation rates in bacteria collected from the wild do increase in stressful environment, such as those in which
nutrients are limited.
There is disagreement about whether:
1. The increased mutagenesis is a genetic strategy selected to increase the probability of evolving beneficial traits
in the stressful environment.
2. Mutagenesis is merely an accidental consequence of error –prone DNA-repair mechanisms that are more
active in stressful environments.
19.2 Mutations are potentially caused by a number of different natural & unnatural factors.
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Mutations result from internal & external factors.
Spontaneous mutations occur under normal conditions.
Induced mutations occur from changes caused by environmental chemicals or radiation.
Spontaneous replication errors.
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Less than one error in a billion nucleotides arises in the course of DNA synthesis, but errors do occur.
Tautomeric shifts- Fig19.10
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Thought to be primary cause of spontaneous replication errors.
In a Tautomeric shift, the positions of protons in the DNA bases change.
Purine & Pyrimidine bases exist in diff chemical formsTautomer.
 The two tautomeric forms of each base are in dynamic equilibrium, although one form is more common than
the other.
* Standard A with T, & C with G, is the common form of bases, but if the bases are in their rare Tautomeric
forms, other base pairings are possible.
Watson &Crick proposed that Tautomeric shifts might produce mutations
 Proposal was an accepted model for spontaneous replication errors for many years.
* Never been convincing evidence that the rare tautomers are the cause of spontaneous mutations.
* Research now shows little evidence of tautomers in DNA.
Mispairing due to other structures- Fig 19.11
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Mispairing can also occur through wobble.
 Normal, protonated, & other forms of the bases are able to pair due to the flexibility of the DNA helical
structure.
 These structures have been detected in DNA molecule &are now thought to be responsible for
mispairings in replication.
GTS 261-Anya OberholzerPage 6
Incorporation errors & replication errors – Fig 19.12
Example
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Incorporated error occurs when a mismatched base has been incorporated into a newly synthesised nucleotide
chain.
 Suppose that, in replication, T mispairs with G through wobble.
 In the next round of replication, the two mismatched bases separate, &each serves as a template for synthesis
of a new nucleotide strand.
 This time, T pairs with A, producing another copy of the original DNA sequence.
 On the other strand, the incorrectly paired G serves as a template & pairs with C, producing a new DNA
molecule that has an error  C·G pair in place of the original T·A pair.
 The original incorporated error leads to a replication error.
Replication error creates a permanent mutation.
 All base pairings are correct & there is no mechanism for repair systems to detect the error.
Causes of deletions &insertions- Fig 19.13 & Fig 19.14
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Mutations due to small insertions & deletions also arise spontaneously in replication & crossing over.
Strand slippage can occur when one nucleotide strand forms a small loop.
 If the looped out nucleotides are on the newly synthesised strand, insertion occurs.
 At the next round of replication, the insertion will be replicated &both strands will contain the insertion
 If the looped out nucleotides are on the template strand, the newly replicated strand has a deletion.
 This deletion will be replicated in subsequent rounds of replication.
Another process that produces insertions & deletions is unequal crossing over.
Unequal crossing over can be caused by misaligned pairing.

 One DNA molecule contains an insertion, & the other a deletion.
Some DNA sequences are more likely to undergo strand slippage or unequal crossing over.
 Stretches of repeated sequences (e.g. nucleotide repeats or homopolymeric repeats) Strand slippage.
 Duplicated or repetitive sequences may misalign during pairing Unequal crossing over.
 Both produce duplicated copies of sequences.
 Promotes further strand slippage & unequal crossing over.
 May explain anticipation often observed for expanding nucleotide repeats.
Spontaneous chemical changes- Fig 19.15 & Fig 19.16
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Mutations can also result from spontaneous chemical changes in DNA.
Depurination is the loss of a purine base from a nucleotide.
 Results when the covalent bond connecting the purine to the 1’-C atom of the deoxyribose sugar breaks.
 This produces an apurinic site (a nucleotide that lacks its purine base).
 An apurinic site cannot act as a template for complementary base in replication.
 In the absence of base-pairing constraints, an incorrect nucleotide (mostly A) is incorporated into the
newly synthesised DNA strand opposite the apurinic site.
 This leads to an incorporated error.
 The incorporated error is transformed into a replication error at the next round of replication.
 Common cause of spontaneous mutation.
Deamination is the loss of a NH2 group from a base.
 Can be spontaneous or induced by mutagenic chemicals.
 Can alter pairing properties of a base (e.g. deamination of C produces U, which pairs with A in replication).
 A pairs with T, creating a T·A pair in place of the original C·G pair this is a transition mutation.
 Transition mutation is usually repaired by enzymes that remove U when it is found in DNA.
 The ability to recognise the product of C deamination may explain why T, not U is found in DNA.
 In mammals, some C bases in DNA are naturally methylated & exist in the form of 5-methylcytosine (5mC).
 When deaminated, 5mC becomes T.
 Because T pairs with A in replication, the original C·G changes to T·A.
 C·GT·A transitions in mammalian cells, & 5mC sites are mutation “hotspots” in humans.
GTS 261-Anya OberholzerPage 7
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Environmental agents are capable of damaging DNA, including certain chemicals & radiation.
Mutagen is any environmental agent that significantly increases the rate of mutation above the spontaneous rate.
 Discovered by Charlotte Auerbach, who was researching the development of mutants in Drosphilia.
 Auerbach &Robson showed that mustard gas was a powerful mutagen, reducing the viability of gametes
&increasing the number of mutations seen in offspring of exposed flies.
Base analogs- Fig 19.17 & Fig 19.18
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Example
Insert boring history here.
Chemically induced mutations.
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Base analogs are chemicals with structures similar to that of any of the 4 standard DNA bases.
 DNA poly can’t distinguish these analogs from the standard bases.
If base analogs are present during replication, they may be incorporated into the newly synthesised DNA.
 5-bromouracil (5BU) is an analog of T, & has the same structure of T, except it has Br on the 5-C atom, and
not –CH3.
 Normally, 5BU pairs with A, but it can mispairs with G, leading to a transition.
 Through mispairing, 5BU can be incorporated into the new DNA strand opposite G, leading to another
round of transition.
 2-aminopurine (2AP) is the analog of A.
 5BU & 2AP can produce transition mutations.
Mutations made by base analogs can be reversed by treatment with the same analog or treatment of a diff analog.
Alkylating agents – Fig 19.19
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Chemicals that donate alkyl groups (-CH3 & CH3-CH2) to nucleotide bases.
 Ethylmethyl sulfonate (EMS) adds an ethyl group to G, producing O6-ethylguanine, which pairs with T.
EMS produces C·GT·A transitions.
 Mutations produced by EMS can be reversed by additional treatment with EMS.
 Mustard gas is another alkylating agent.
Deamination –Fig 19.19
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Can be induced by chemicals.
 Nitrous acid deaminates C, creating U, which will later pair with A, producing C·GT·A transition mutation.
 Nitrous acid changes A into hypoxanthine, which pairs with C, leading to T·AC·G transition.
 Nitrous acid can change G, producing xanthine, which pairs with C, but also with T.
 Nitrous acid produces exclusively transition mutations, & can be reversed with nitrous acid.
Hydroxylamine – Fig 19.19
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Very specific base modifying mutagen that adds –OH to C, converting it to hydroxylaminocytosine.
 Conversion increases the frequency of a rare tautomer that pairs with A instead of G.
 Leads to C·GT·A transitions.
Only acts on C, & will not generate T·AC·A transitions.
Hydroxylamine will not reverse the mutations that it produces.
Oxidative reaction –Fig 19.20
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Reactive forms of oxygen are produced during normal aerobic metabolism, radiation, ozone, peroxides, & some
drugs.
 Reactive forms of oxygen damage DNA & induce mutations by bringing about chemical changes in DNA.
Oxidation converts G into 8-oxy-7,8-dihydrodeoxyguanine, which mispairs with A instead of C.
 Causes G·CT·A transversion mutation.
Intercalating agents- Fig 19.21
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Intercalating agents produce mutations by sandwiching themselves btwn adjacent bases in DNA, distorting the 3D
structure of the helix & causing single- nucleotide insertions &deletions in replication.
 In-dels frequently produce Frameshift mutations.
 Mutagenic effects of intercalating agents are often severe.
Intercalating agents produce in-dels, & can reverse the effects of their own mutations.
Examples of intercalating agents: Proflavin, acridine orange, ethidium bromide, & dioxin.
GTS 261-Anya OberholzerPage 8
Radiation- Fig 19.22
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1927, Hermann Muller.
Demonstrated that mutations in fruit flies could be induced by X-rays.
X-rays greatly increase mutation rates in all organisms.
Due to high energies, X-ray, gamma rays, & cosmic rays are able to penetrate tissue & damage DNA.
 These forms of radiation are called ionizing radiation, & dislodge e- from the atoms that they encounter.
 Change stable molecules into free radicals & reactive ions.
 These alter the structure of bases & break phoshodiester bonds in DNA.
 Ionization can result in double-strand breaks in DNA.
 Attempts to repair the DNA may result in chrm mutations.
UV light has less energy than ionizing radiation & does not eject e-, but is highly mutagenic.
 Purine & pyrimidine bases readily absorb UV light.
 Results in the formation of chemical bonds btwn adjacent pyrimidine molecules on the same strand of
DNA, creating pyrimidine dimers.
 T dimers are more frequent, but T-C dimers may form.
 Dimers distort DNA configuration, & often block replication.
 When dimers block replication, cell division is inhibited & the cell usually dies.
UV light kills bacteria & is an effective sterilizing agent.
 For a mutation to occur, the replication block must be overcome.
 Bacteria can bypass replication blocks produced by pyrimidine dimers & other DNA damage by means of
the SOS system.
* SOS system allows replication blocks to be overcome but, in the process, makes numerous mistakes &
increases the rate of mutation.
 SOS system does not strictly adhere to base-pairing rules.
o Replication may continue & the cell survives, but only by sacrificing the normal accuracy of
DNA synthesis.
* A protein, RecA binds to the damaged DNA at the blocked replication fork & becomes activated.
 Activation promotes the binding of a protein LexA, a repressor of the SOS system.
 Activated RecA complex induces LexA to undergo self-cleavage, destroying its repressive activity.
o Inactivation enables other SOS genes to be expressed, & products of these genes allow
replication of damaged DNA to proceed.
o SOS system allows bases to be inserted into new DNA in the absence of bases on the
template strand, but these result in errors in the base sequence.
19.3 Mutations are the focus of intense study by Geneticists.
Detecting mutations with the Ames test- Fig 19.23
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Based on the principle that both cancer &mutations result from damage to DNA.
 90% of known carcinogens are mutagens.
Mutagenesis in bacteria could serve as an indicator of carcinogens in humans.
Uses diff auxotrophic strains of Salmonella typhimurium that have defects in the lipopolysaccharide coat, which
protects it from the environment.
 DNA-repair system in these strains has been inactivated by enhancing susceptibility to mutagens.
Ames II uses several auxotrophic strains that detect diff types of base-pair substitutions.
 Other strains detect diff types of Frameshift mutations.
 Each strain carries a his- mutation, which makes it unable to synthesise His, & the bacteria are plated onto a
medium that lacks His.
 Only bacteria that have undergone a reverse mutation of the His gene (his-his+) can synthesise His & grow
on the medium.
 Diff dilutions of a chemical to be tested are added to plates inoculated with the bacteria, & the no. of mutant
bacterial colonies that appear on each plate is compared to the no. that appears on control plates with no
chemical.
GTS 261-Anya OberholzerPage 9
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Any chemical that significantly increases the no. of colonies appearing on a treated plate is mutagenic &
probably carcinogenic.
Some compounds are not active carcinogens but can be converted into cancer causing compounds.
 To make the Ames test sensitive for potential carcinogens, a sample to be tested is incubated in mammalian
liver extract that contains metabolic enzymes.
Radiation exposure in humans- Fig 19.24
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Study of biological effects of radiation exposure from Hiroshima is being made.
Somatic mutations examined by studying radiation sickness &cancer among survivors.
Germ-line mutations were assessed by looking at birth defects, chrm abnormalities, & gene mutations in children
born to people who were exposed.
 A mutation rate of 3.4 x 10-6 was estimated for children whose parents were exposed
 In range of spontaneous mutation rates observed for other euk.
 Neel &colleagues also examined the frequency of chrm mutation, sex ratios of children born to exposed parents, &
frequencies of chrm aneuploidy.
 Germ-line mutations were not elevated.
 Animal studies clearly show that germ-line mutations increase due to radiation, so why are the same results not
seen in the Hiroshima example?
 Could be that people who received most of the radiation died soon after the blast.
Read p513 (Last paragraph) - p514 for another example
19.4 A number of pathways repair changes in DNA- Table 19.4
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Rate of mutations remains low due to efficiency with which DNA is repaired.
Several general statements about DNA repair.
1. Most DNA-repair mechanisms require 2 nucleotide strands of DNA
 Most replace whole nucleotides, & a template strand is needed to specify base sequence.
2. Redundancy.
 Many types of DNA damage can be corrected by more than 1 pathway of repair.
 Testifies to extreme importance of DNA repair to the survival of the cell.
 Ensures that almost all mistakes are corrected, if a mistake escapes 1 repair system, it is likely to be
repaired by another.
Mismatch repair- Fig19.25
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Replication= very accurate.
In replication, mismatched bases are incorporated into new DNA at a frequency of about 10-4 to 10-10.
 Most errors that initially arise are corrected & never become permanent mutations.
 Some corrections are made in proofreading by DNA poly.
Many incorrectly inserted nucleotides that escape detection by proofreading are corrected by mismatch repair.
 Incorrectly inserted nucleotides are detected & corrected by mismatch-repair enzymes
 Mismatch repair system corrects small unpaired loops in the DNA that may be caused by strand slippage.
 Some nucleotide repeats may form secondary structures on the unpaired strand, allowing them to escape
detection.
After the error has been recognised, mismatch enzymes cut out a section of the newly synthesised strand, & fill the
gap with new nucleotides, using original DNA as a template.
 Mismatch repair must have a way of distinguishing btwn old & new strand of DNA, so that the error & not the
original strand are removed.
Proteins that carry out mismatch repair in E.coli differentiate btwn old & new strands by the presence of –CH3 on
special sequences of the old strand.
 After replication, A nucleotides in GATC are methylated.
* Methylation is delayed & so, immediately after replication, the old strand is methylated & the new strand is
not.
* Mismatch repair complex brings an unmethylated GATC sequence in close range to the mismatched bases.
GTS 261-Anya OberholzerPage 10
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 It nicks the unmethylated strand at the GATC site, & degrades the strand btwn the nick & mismatched
bases.
 DNA poly & DNA ligase fill in the gap on the unmethylated strand with correctly paired nucleotides.
Mismatch repair in euk cells is similar to that in E.coli, but how old & new strands are recognised is unknown.
 In some euk, like yeast & fruit flies, there is no detectable methylation of DNA, but mismatch still takes place.
 Humans who possess mutations in mismatch repair genes often exhibit elevated somatic mutations, & are
susceptible to colon cancer.
Direct repair- Fig 19.26
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Does not replace altered nucleotides, but changes them back to their original structures.
Most well understood direct repair is photoreactivation of UV-induced pyrimidine dimers
 E.coli & some Euk cells possess an enzyme, photolyase, which uses energy from light to break the covalent
bonds that link the pyrimidine dimer.
 O6-methylguanine DNA methyltransferase removes the methyl from O6-methylguanine, restoring the base to
G.
Base-excision repair- Fig 19.27
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Repair is maintained
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A modified base is first excised & then the entire nucleotide is replaced.
 Excision of modified bases is catalysed by a set of enzymes DNA glycosylase.
* Each recognises & removes a specific type of modified base by cleaving the bond that links the base to the
1’-C atom of the deoxyribose sugar.
After the base has been removed, an enzyme, AP endonuclease cuts the phosphodiester bond, & other enzymes
remove the deoxyribose sugar.
 DNA poly then adds one or more new nucleotides to the exposed 3’-OH.
* Replaces a section of nucleotides on the damaged strand.
 DNA ligase seals the nick in the phosphodiester backbone, & the original sequence is restored.
Bacteria use DNA poly I, where euk use DNA poly.
 DNA poly  has no proofreading ability & tends to make mistakes.
* It may introduce as many as 10 million mutations per day.
 How are these errors corrected?
 AP endonuclease has the ability to proofread.
 When DNA poly  inserts the wrong base into the DNA, DNA ligase can’t seal the nick in the
backbone.
o 3’-OH & 5’-P groups of adjacent nucleotides are not in the right orientation for ligase to
connect to them.
 AP endonuclease 1 detects a mispairing & uses its 3’5’ exonuclease activity to excise the
incorrectly paired bases.
 DNA poly  then uses its polymerase activity to fill the missing nucleotide.
Nucleotide excision repair- Fig 19.28
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Remove bulky lesions that distort the double helix.
Versatile & can repair many diff types of DNA damage.
Found in all organisms.
In humans, a large number of genes take part.
1. A complex of enzymes scans the DNA, looking for distortions of its 3D structure.
 When a distortion is detected, more enzymes separate the nucleotide strands at damaged region.
 Single-strand-stabilizing- proteins stabilize the strand.
2. The backbone of the damaged strand is cleaved on both sides of the damage.
 Part of the damaged strand is peeled away by helicase enzymes, DNA poly fills the gaps, & DNA ligase
seals the nick.
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Connecting concepts- The basic pathway of DNA repair.
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Most methods of DNA repair depend on the presence of 2 strands.
 Nucleotides in damaged areas are removed & replaced.
 Nucleotides are replaced in mismatch repair, base excision repair, & nucleotide excision repair, but not in
direct repair.
 Repair mechanisms that include nucleotide removal utilize a common 4- step pathway
1. Detection: The damaged section of the DNA is recognised.
2. Excision: DNA-repair endonucleases nick the backbone on one or both sides, & one or more nucleotides are
removed.
3. Polymerization: DNA pol adds nucleotides to the newly exposed 3’-OH by using the other strand as a
template to replace nucleotides.
4. Ligation: DNA ligase seals the nick in the backbone.
Primary diff are in details of detection & excision.
Detection
Excision
Base-excision
1 nick is made in the backbone on
DNA poly displaces old nucleotides
the damaged side
as it adds new ones to 3’ of nick.
Mismatch
Old nucleotides are degraded
Nucleotide-excision
Nicks made on both sides of DNA
Nucleotides displaced by helicase.
lesion.
All 3 mechanisms use DNA poly & DNA ligase to fill gaps produced by excision & removal of damaged nucleotides.
Repair of double-strand breaks.

Double strand break is a common type of DNA damage.
 Both strands of double helix are broken
 Caused by ionizing radiation, oxidative free radicals, & other DNA damaging agents.
 Detrimental to the cell because they stall DNA replication & may lead to chrm rearrangements, such as
deletions, duplications, inversions, & translocations.
 2 major pathways for repairing double strand breaks.
Homologous recombination
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Repairs a broken DNA molecule by using identical or nearly identical genetic info contained in another DNA
molecule (usually sister chromatid).
DNA repair uses the same mechanism used in the process of homologous recombination responsible for crossing
over.
 Begins with removal of some nucleotides at the broken ends, followed by strand invasion, displacement &
replication.
 Many of the same enzymes used in crossing over are used in the repair of double-strand breaks by
homologous recombination.
* NB enzymes= BRCA1 & BRCA2.
 Genes for these proteins are frequently mutated in breast cancer cells.
Nonhomologous end joining
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Repair double-strand breaks without using a homologous template.
Used when the cell is in G1 & sister chromatid is not available for repair through homologous recombination.
Uses proteins that recognise the broken ends of the DNA, binding to the ends & joining them together.
More error prone than homologous recombination & leads to deletions, insertions, & translocations.
GTS 261-Anya OberholzerPage 12
Translesion DNA polymerases
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Some mutations, such as pyrimidine dimers, produce distortions in the 3D structure of DNA, blocking replication.
Translesion poly are able to bypass bulky lesions, but often make errors.
 Allow replication to proceed past the bulky lesion, but introduce mutations into the sequence.
* Some mutations are corrected by DNA repair systems, but others escape detection.
* Example: poly .
Genetic diseases & faulty DNA repair- Fig 19.29 & table 19.5
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Read through only!
Several human diseases are linked to defects in DNA repair.
 Associated with high incidences of cancer, because defects in DNA repair lead to increase rate of mutations.
 Xeroderma pigmentosum is a rare autosomal recessive condition that includes abnormal skin pigmentation & acute
sensitivity to sunlight.
 People with this have an increased chance in getting skin cancer.
 Sunlight includes a strong UV- component.
* Exposure to sunlight produces pyrimidine dimers in the DNA of skin cells.
* Most pyrimidine dimers in humans can be corrected by nucleotide excision repair.
 Cells of people with this condition are defective in nucleotide excision repair, & many of their
pyrimidine dimers go uncorrected, which may lead to cancer.
 Can result from defects in several diff genes.
See page 519 for more on genetic diseases
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