Wednesday

Genetics Lecture 27, 11/5; Chapter 15, mutations, DNA repair

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How do you determine if a compound is mutagenic? - AMES Test



  • Use 4 strains of the bacteria strain salmonella typherium (all oxytrophic which means they can't grow on minimal media)



  • One strain is especially susceptable to base pair substitution.  The other 3 are susceptible to frameshift mutation 


  • (3:10) Can this compound cause mutation and result in a significant appearence of prototrophs.



    • Auxotrophic strain + liver enzymes.  Many mutagenic compounds only become mutagenic after exposure to liver enzymes.


    • Plate the strain on two minimal media plates


    • Mix your potential mutagen with liver enzyes - add to a piece of filter paper


    • filter paper is placed on one of two minimal media plates


    • Incubate overnight and determin number of prototrophs on both minimal media plates.



  • if experimental plate has a substantially larger number of prototrophs then the control then compound is a mutagen


  • If your experimental and control plates have similar numbers of prototrophs then it is most likely not a mutagen.  (test was developed in 1970's and was used on carcinogens, 80% resulted in mutation)


(13:15) DNA Repair
  • Photoreactivation repair (involves prokaryotes) - requires activation from UV light
  • How it was discovered:
    • What they knew: If you exposed DNA to light from the blue spectrum shortly after exposing DNA o UV light you can reverse some damage done to the DNA.  Relied on light-dependent enzyme which was also temperature sensitive.
    • What they discovered: Found a protein called photoreactivation enzyme (originally isolated from E. coli) and discovered that this eznyme absorbed 1 photon of light and thus became active.  As it became active the enzyme gains the ability to cleave the bonds in a thymine dimer.  
  • Nucleotide excision repair (found in the vast majority of organisms)  3 step process.
    •  - Begin with damaged piece of DNA -
    • Nucleases search for and identify these lesions.  When they find it they cut out the damaged DNA along with a number of bases on either side.  This results in a gap in the DNA.  
    • (23:40) Form of DNA polymerase which fills in the gap with new nucleotides and in the process corrects the mutation.
    • (25:05) DNA ligase functions to seal gaps in the DNA strand.
  • (26:45) Base excision repair: Corrects minor alteration to DNA molecules (a bit more focused then nucleotide excision repair)
    • DNA glycosylase (an enzyme) functions to cleave a base from the sugar phosphate backbone
    • AP endonuclease recognizes a sugar without a base (whatever glycosylase leaves behind).  When it does this is leaves a nick in the DNA strand upsteram of the sugar where there is no base.
    • DNA polymerase comes in and removes the sugar and replaces it with a new nucleotide.
    • DNA ligase comes and seals the gap in the DNA strand.
  • (32:20) Proofreading during DNA replication
    • During DNA replication, DNA polymerase functions to add nucleotides to a growing DNA chain.
    • After DNA polymerase adds a base it pauses to check that base looking specifically for "correctness".  Did the polymerase add the right base.  TWO things can happen: if the base is correct DNA polymerase moves on.  If the base is not correct DNA polymerase (with its 3' to 5' exonuclease activity) will function to cleave and remove an incorrect base.  After removal DNA polymerase replaces the base and moves on.
  • (37:10) SOS repair system: responds to gaps in DNA strands
    • Use an alternative DNA polymerase (DNA POL V).
    • Typically gaps cause DNA replication to stall out.  DNA POL V comes to a gap like this and can fill, it solves the problem.  However, in filling the gap, DNA POL V uses less stringent base pairing rules.  By-product is increased mismatched base pairs.


Monday

Genetics Lecture 26, 11/3; Chapter 15, Mutations, Tautomeric Shift, Spontaneous DNA changes






Observations on the fact that the rate of mutation varies from organism to organism.
  • The rate of spontaneous mutation is exceedingly low
  • Great deal of variety from organism to organism.
  • Within a single organism different genes mutate different rates

Deleterious mutations.
  • 5-10% of the DNA in an organism actually codes for protein. Majority of mutations are in non-coding regions.
  • Deleterious mutation: Any mutation that causes a change in phenotype.
  • On average you will see 1.6 deleterious mutations/person/generation

(6:00) Specific changes a mutation can create on a DNA sequence
  • Missense mutation: a single nucleotide change in the sequence of a gene. EFFECT: Typically is that there is a single amino change (sickle-cell anemia). Also known as Point Mutation and Base substitution.
    • Two types
    • Transition: exchange purine for purine or pyrimidine for pyrimidine
    • Transversion: exchange purine for pyrimidine.
    • Frameshift mutation:
      • deletion or insertion: change the reading frame (way a gene is read to be converted to protein) for the gene. Change both the sequence and the length of the protein.
    • Nonsense mutation
      • create a mutation which creates a stop codon where a stop codon was not present previously. Results in prematrue termination of translation.

(15:15) Chemical agents that can damage DNA
  • Tautomeric Shift - alternative versions of a compound. Nitrogenous bases have tautomeres.
  • If a nitrogenous base undergoes a tautomeric shift. The tautomere of the nitrogenous base typically experiences non-watson&crick base pairing.
  • Compounds that function as base analogs: Mutagenic chemicals with the ability to substitute for one of the bases.
    • (19:45) EX. 5-Bromouracil: a derivative of uracil. Functions as an analog for thymine. Normal version of 5-Bromouracil base pairs with adenine. 5- Bromouracil can undergo tautomeric shift. When this happens base pairs with guanine. The presnce of 5-Bromouracil makes DNA more susceptible to mutagenesis with UV light.
    • (23:45) EX. 2-aminopurine: analog for adenine. Base pairs with cytosine.
  • Alkylating agents --> EMS (Ethylmethyl sulfonate). Add an alkyl group to a base. Creates bases that act like base analogs. EMS alkylates guanine and creates 6-ethyl guanine which functions as an analog for adenine and thus binds to thymine.
  • (29:45) Arsidine Dyes - cause frameshift mutations. They tend to remove 1 to 2 bases from a sequence of DNA in their role as an intercalating agent. Intercalating agent - a compound that is similar in size to a nitrogenous base pair. Because of this it is able to slip in between spaces in a DNA sequence. This creates torsion on the DNA and the DNA loses 1 to 2 BP in order to relieve the torsion.
    • EX. of intercalating agent: Proflavin, Arsidine orange, Ethidium bromide.

(33:40)Environmental/ Spontaneous changes to DNA
  • Depurination: spontaneous loss of a purine base from its sugar-phosphate backbone.
  • Apurinic site - gap in the sequenc of bases. This is a problem in DNA replication
  • EFFECTS:
    • DNA replication STOPS.
    • DNA polymerase gets to the opening and inserts any base (most likely to be wrong)
  • Deamination: nitrogenous base has an amino group converted to a keto group. Cytosine is converted to uracil. The uracil then base pairs with adenine. Adenine can also undergo a deamination. Adenine will become hypoxanthine and then base pairs with cytosine.
  • Thymine Dimers: occur when you have two bases of thymine next to each other on a single strand of DNA. UV (260nm wavelength) hits the DNA and the two bases of thymine break their bonds with the base that they are paired to. Instead they form a double bond with each other. This can cause large scale mutations during DNA replication to the point that you can see the death of an organism. This is why UV light can be used to sterilize things.