1Mutations

• 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.

2Mutations

• Our example:– information, 3 letters at a time, read consecutively

• Point mutations:

• Frameshift mutations: Insertion

3more Mutations

• Frameshift: deletion

• Transposon mutagensis: transposons are segments of DNA that can jump into another spot in the DNA; they have information.

4More 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?

5

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

6Loss of a purine, a natural process

saturn.roswellpark.org/.../ AP_site_generation.gif

Can lead to an incorrect base being added; a mutation.

7Pyrimidines and deamination

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 producesthymine, which is normal; results in a transition mutation.

8Frameshift mutations

• Cause misalignment during DNA replication; caused by intercalating agents such as ethidium bromide or acridine orange

http://www.photobiology.com/photoiupac2000/pierard/intintercal.jpg

9Radiation• UV light at 260 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.

http://academic.brooklyn.cuny.edu/biology/bio4fv/page/molecular%20biology/mutation-prym-dimers.jpeg

10Ionizing 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.

11Repair 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

http://earthobservatory.nasa.gov/Library/UVB/Images/dna_mutation.gif

12Repair 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 distortedDNA helix (such as caused by thymine dimers). The entire local section of DNA is removed and replaced.

http://www-personal.ksu.edu/~bethmont/excisio3.gif

In all prokaryotes & eukaryotes.

13Repair 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.

• Cytosines are methylated, keeps track of age of DNA strand– Deamination of these not always recognized as wrong

14Creation of an AP site

saturn.roswellpark.org/.../ AP_site_generation.gif

15AP 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.

16Bulky 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.

17Mismatch 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 DNAchain with the least amount of methyl cytosines.

cmgm.stanford.edu/.../DNA%20Repair%20-%20Doug/

18SOS 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.