1. Mutations Mutations are inheritable changes in the DNA
“Failure to faithfully store genetic information”
Changes can be to chromosomes or genes
Current focus: changes to DNA sequences.
This means an alteration in a basepair or
in the order of the basepairs.

2. Types of mutations-1 Mutations can be classified in many, many ways
Some ways mutually exclusive, some not.
Spontaneous vs. Induced
Spontaneous happens naturally
Enzymatic errors, especially in copying
Various chemical reactions
Induced mutations: specifically caused, as by researcher
Treatment with various chemicals, radiation
Gametic (germ line) vs. somatic
Gametic mutations can be passed on to next generation
Somatic only affects individual (in metazoans)

3. Types of mutations-2 Morphological Nutritional/biochemical
Change in physical structure, readily observed
Nutritional/biochemical
Mutated enzyme results in phenotypic change
Bacterial auxotrophs; sickle cell anemia
Behavioral mutations
Regulatory mutations
Affect control of gene expression rather than protein

4. Types of mutations-3 Lethal mutations: not easily studied unless:
Conditional mutations: expressed depending on environmental conditions, especially temperature.
a way to study lethal mutations: permissive and restrictive temperatures; esp. useful with bacteria
temp sensitive mutations occur
naturally, continued in offspring:
Siamese cats, Himalayan rabbits
farpoint/saavik1.jpg

5. Types of mutations-4 Classification of mutations by FUNCTION
Loss of function: knockout or null.
Hypomorphic: lowered expression, “leaky”
Hypermorphic: greater activity or more visible trait
typically regulatory mutation, results in increased expression
Gain of function: e.g. new enzymatic activity
a factor in evolution;
Dominant negative: bad apple spoils the bunch e.g. bad protein in multicomponent enzyme

6. Detection of mutations
Bacteria and fungi
Prototrophs and auxotrophs: microbe no longer able to synthesize or breakdown particular nutrient.
Change in behavior, e.g. motility
Various methods in plants and animals
Humans (“not suitable experimental organisms”)
Reliance on pedigrees
Possible to determine sex linkage, dominance

7. Mutations are rare (but not equally so)
Mutation rate depends on species and on gene
Hot spot: a location in DNA where mutations occur significantly more often than the usual 1/ 106.
Monotonous run of single nucleotide or tandem repeats: GGGGGGGGG or ATGGATGGATGG
Methylated cytosines
methylation is added a CH3 group to something
Cytosines are methylated to help indicate which DNA strand is older (helps with DNA repair).
Problem occurs when a cytosine is chemically damaged by deamination. (more later)

8. Mutations Our example: Point mutations:
information, 3 letters at a time, read consecutively
Point mutations:
Frameshift mutations: Insertion

9. more Mutations Frameshift: deletion
Transposon mutagensis: transposons are segments of DNA that can jump into another spot in the DNA; they have information.

10. More types of mutations
Switch between A & G, or C & T: transition
Switch between purine and pyrimidine: transversion
Silent: 3rd position of codon usually means same amino acid, so change here has no effect.
Missense: typically a single nucleotide change, causes change in amino acid and noticeable effect.
Nonsense: change amino acid codon to STOP codon
Additions, deletions, and “stuttering”
Stuttering: repeated sequences sometimes copied incorrectly; enzyme gets confused?

11. Mutagens: that which causes mutations
Base analogs: e.g. 5-bromouracil. In equilibrium between keto and enol forms
In keto form, looks like T
In enol form looks like C
Used one way, but when copied, mispairing can occur.
Modifying agents: chemically change bases
HNO2 nitrous acid: deaminates (amino to keto)
See upcoming slide: deamination
Alkylating agents (ethylmethane sulfonate): add methyl or ethyl group to bases (-CH3 or CH2CH3), cause mispairing during synthesis

12. Loss of a purine, a natural process
Can lead to an incorrect base being added; a mutation.
saturn.roswellpark.org/.../ AP_site_generation.gif

13. Pyrimidines and deamination
Loss of an amine group, replacement w/ a keto group.
Deamination of cytosine makes uracil; recognized as wrong
and repaired. Deamination of 5-methyl cytosine produces
thymine, which is normal; results in a transition mutation.

14. Frameshift mutations
Cause misalignment during DNA replication; caused by intercalating agents such as ethidium bromide or acridine orange

15. Radiation UV light at 265 nm
causes thymine dimers; covalent connections between adjacent thymines. Hurried repair makes mistakes.
Ionizing radiation
short wavelength, high energy radiation, e.g. x-rays, gamma radiation.
Causes ss, ds breaks in DNA.
biology/bio4fv/page/molecular%
20biology/mutation-prym-dimers.jpeg

16. Ionizing Radiation Major damage is from free radicals
Most abundant substance in cell is water; radiation produces radicals that attack DNA, causing breaks.
The effects of radiation are a matter of considerable scientific and political controversy.
Effects of high levels of radiation are well understood, but effects of low levels are very difficult to study.
Brief soapbox: after Chernobyl tragedy, people vacated many square miles around damaged reactor. Now, endangered animals making a comeback despite radiation.

17. Repair of DNA damage
Despite the constant bombardment of DNA with radiation and chemicals, cells possess repair mechanisms.
Repair systems exist for
UV light damage
Chemical changes to bases
Loss of bases
Incorrect copying
Ss and ds breaks in DNA

18. Repair of Thymine dimers
Photoreactivation: Enzyme
uses a photon of blue light to
separate thymines from each
other. (When using UV as a
mutagen, put cells in dark
afterwards!) (in E. coli)
Excision Repair: DNA repair
enzymes recognize a distorted
DNA helix (such as caused by
thymine dimers). The entire
local section of DNA is
removed and replaced.
In all prokaryotes & eukaryotes.

19. Repair of chemical changes
Deamination of cytosine
as shown previously, converts cytosine to uracil
the enzyme uracil glycosylase cuts off uracil, leaving deoxyribose as part of backbone, creating an “AP” site
AP = apurinic or apyrmidinic, meaning purine etc. NOT there.
AP repair, mechanism that specifically fixes such places.

20. Creation of an AP site
saturn.roswellpark.org/.../ AP_site_generation.gif

21. AP repair- continued Activity of uracil glycosylase or
spontaneous loss of base from DNA
can create an AP site.
An endonuclease cuts out the
remaining sugar-phosphate and
replaces it with
a complete nucleotide.

22. Bulky excision repair
Like in repair of UV-induced damage, cells sense bulges, kinks, or similar damage to DNA
Chunk of DNA containing the damaged area is excised, replaced by DNA polymerase I enzyme (or equivalent)
13 bases removed in bacteria
Eukaryotes (always more elaborate) take out 28 nucleotides
In humans, failure in this repair system causes disease xeroderma pigmentosum with increased risk of skin cancer.

23. Mismatch repair
If Proofreading misses: Other enzymes recognize that the wrong base pair is in place, cuts out incorrect one and replaces it. Which one is incorrect?
Presumably the newest one = the one on the DNA
chain with the least amount of methyl cytosines.
cmgm.stanford.edu/.../DNA%20Repair%20-%20Doug/

24. SOS Repair
Especially in bacteria, when damage to DNA is severe, an emergency system goes into effect where damage is repaired rapidly, but sloppily. Introduces many mutations, some possibly fatal, but DNA damage would surely be fatal otherwise.