DNA Mutations Flashcards
What is a mutation?
Heritable change in nucleotide sequence from a lesion (i.e. error) in DNA that is not repaired prior to replication round. May cause beneficial, detrimental or neutral changes.
What is the difference between spontaneous mutations and induced mutations?
Spontaneous: occur during normal growth due to replication errors (replication errors 10^-6/1000bp)
Induced: caused by external factors called mutagens that chemically modify DNA (10^-3/1000bp)
What is the difference between a wildtype and a mutant?
Wildtype: Strain of an organism isolated from nature with a certain observable phenotype
Mutant: Cell whose genome carries change in nucleotide sequence from that of the wildtype genotype, resulting into an observable mutant phenotype
What is a parental strain?
The strain from which a mutant is derived which may be either the wild-type strain or a previously mutated strain.
What is the difference between wildtype and mutant E.coli grown on MacConkey agar with a maltose carbon source?
Wildtype: pH indicator will turn red as maltose is fermented
Mutant strains (Mal-): pH indicator will remain yellow as maltose will not be fermented defectiveness in maltose metabolism
What are two ways mutants may be isolated?
Selection: favouring growth of mutants that have an advantage under specific conditions to differentiate from wild type. Conditions are severe enough that mutants are easily detectable.
Screening: examining all colonies for a specific trait (without providing selective advantage) to differentiate mutants from wildtype. Difficult and time consuming.
What are selectable mutations?
Mutation that provides growth advantage to mutant strain under specific environmental conditions, allowing its progeny to outcompete and replace the parent strain.
What are non-selectable mutaitons?
Mutations that neither provides an advantage or disadvantage over parent cells when grown in laboratory conditions. May be detected by screening morphologies for differences.
What is the difference between a nutritional auxotroph and prototroph?
auxotroph: A mutant strain that has an additional nutritional requirement compared to the parental strain from which it was derived. Written as (nutrient^-)
prototroph: A wild-type strain that can synthesize all required nutrients without needing additional growth factors beyond what is normally supplied in minimal medium. Written as (nutrient^+)
What are point mutations?
Mutations that change only one base pair, includes transitions (change within base category A-G, C-T) and transversions (change between base category, A/G-C/T)
What are silent mutations?
Point mutation in coding region of DNA results in codon that codes for the same amino acid as the wildtype. Results in normal protein.
What are missense mutations?
Point mutation in coding region of DNA result in codon that codes for a different amino acid than the wildtype. Impact on folding depends on location of amino acid and chemistry.
What is a nonsense mutation?
Point mutation in coding region of DNA that result in codon that codes for a stop rather instead of the wildtype amino acid. Unless occuring at the end of the gene, the product is incompletely made and truncated proteins are inactive or lack normal activity.
What is a frameshift mutation?
Mutation caused by an insertion or deletion that alters the reading frame by shifting to the right (+) or the left (-)
What is reversion?
The spontaneous/induced back mutation to reverse a point mutation and restore the original wild-type phenotype, producing a revertant.
What are two types of revertants?
- Same site revertant: mutation that restores activity is at the same site as the original mutation. aka true revertant if it restores the original sequence and phenotype.
- Second site revertant: mutation is at a different site in DNA that may compensate for the effect of the original mutation as a suppressor.
What are three classes of suppressor mutations?
- Mutation somewhere else in the same gene that restores enzyme function.
- A mutation in another gene that restores the function of the product of the original mutated gene.
- mutation in another gene that results in the production of an enzyme that can replace the nonfunctional one
What is a tRNA suppressor mutation and how does it rescue nonsense and missense mutations?
Type of second site suppressor mutation that happens in a tRNA anticodon.
Nonsense mutations: binds to the mutated stop codon and brings in its designated amino acid instead
Missense mutations: binds to mutated amino acid codon and brings in a functionally similar one.
Why are tRNA mutations possibly lethal? Why isn’t it?
If a tRNA mutated into a suppressor, it would no longer bind to its original codon, and the amino acid wouldn’t be incorporated where its needed. It isn’t lethal because most microorganisms have multiple tRNA genes for the same amino acid.
What are macrolesions and examples?
Macrolesions are changes in large segments of DNA that can span 1 or more genes. Includes:
- Large insertions or deletions
- Duplications: segment of DNA is repeated at different site
- Translocations: segment of DNA is moved to a different location on the same strand
- Inversions: segment is removed and inserted in the opposite direction (opposite strand)
What are types of spontaneous mutations?
- Apurinic/Apyrimidinic sites
- Deamination
- Tautomeric shift
- Insertions and deletions
What are Apurinic/Apyrimidinic sites? How do they occur?
Loss of nitrogenous bases as a result of the spontaneous hydrolysis of glycosyl bond between base and deoxyribose. This is typically corrected in DNA repair, but if not, A MUTATION IS FORMED by the addition of ANY base via DNA polymerase.
What is deamination?
The loss of amino group from cytosine, adenine and guanine (NOT thymine/uracil)
What are the results of deamination?
- Adenine becomes hypoxanthine, and pairs with cytosine instead of thymine
- Guanine becomes xanthine, pairs with cytosine as normal
- Cytosine becomes uracil, pairs with Adenine instead of guanine.
What is the tautomeric shift?
Low frequency proton shift in DNA creating a temporary change in base structure (isomer) called a tautomer. This results keto form turning to enol form (Guanine, thymine) and an amino form turning to imino form (Adenine, Cytosine)
What is the mechanism of the tautomeric proton shifts from keto to enol, and amino to imino?
Keto to enol (G + T): Hydrogen from adjacent nitrogen moves to carbonyl oxygen
Amino to imino (A + C): Hydrogen from NH2 group moves to adjacent nitrogen.
What is the change in base pairing due to tautomeric shifts?
- Thymine (enol) binds to Guanine (keto)
- Guanine (enol) binds to Thymine (keto)
- Adenine (imino) binds to Cytosine (amino)
- Cytosine (imino) binds to Adenine (amino)
What are insertions and deletion slips?
Insertion slips: Slippage in new strand forms a loop at short stretch of same nucleotide type, adding an extra nucleotide in the new strand to compensate for whats hidden in the loop.
Deletion slips: slippage in parent strand forms a loop at short stretch of same nucleotide type, resulting in new strand skipping base-pairing with the bases what is hidden in the loop.
What are agents that induce mutagenesis?
- 5 bromouracil
- Nitrous acid (HNO2)
- Ethyl methanesulfonate
- Acrinidines, ethidium bromide.
How do nucleotide base analogs induce mutagenesis?
Molecules that resemble purine/pyrimidine bases in structure, but display faulty base pairing proteins. Despite the DNA may replicate normally, replication errors (mutations) occur at higher frequencies due to incorrect base pairing.
How does bromouracil work?
- 5BU in keto form replaces thymine and normally binds with adenine.
- 5BU switches to enol form when the strands separate to act as templates
- Daughter strand may have an adenine or guanine base paired to 5BU.
How does nitrous acid induce mutagenesis?
- sodium nitrate (NaNO3) is converted to Nitrous acid (HNO2) in the stomach
- Nitrous acid deaminates DNA bases resulting in adenine to hypoxanthine, guanine to xanthine, and cytosine to uracil.
- hypoxanthine base pairs with cytosine instead of thymine and uracil binds to adenine instead of guanine.
How does ethyl methanesulfonate (EMS) induce mutagenesis?
Methylation or ethylation of any oxygen group in the four bases will cause a transition mutation. (G/C:T/A)
What are intercalating agents?
Planar molecules that insert between adjacent basepairs and push them apart to distort the helical structure. Promotes single base insertions or deletions, causing frameshift mutations.
How does ethidium bromide induce metagenesis?
- Slips between adjacent base pairs in the double helix, physically distorting the structure
- When DNA polymerase tries to replicate the distorted DNA, it can insert or delete nucleotides at the site of intercalation.
- Insertions and deletions cause frameshift mutations.
What is the ames test?
- Salmonella typhimurium is his- due to a point mutation. This means it will not grow in minimal media without histidine
- S. typhimurium is spread onto two plates. One is minimal agar with a small amount of histidine and a mutagen (rat liver extract), the other is minimal agar with a little histidine and no mutagen.
- Mutagen induces mutations that may reverse the original mutation, making the strain his+ again, creating many revertants. Without the mutagen, there are few spontaneous revertants.
- This tests for the safety of the mutagen, as if many revertants are produced, the chemical may be deemed carcinogenic and unsafe.
How does non-ionizing radiation induce mutagenesis?
Bases in DNA absorb UV light, cross-linking pyrimidines on the same DNA strand to create dimers that distort the helical structure of DNA and prevents DNA polymerase from using the strand with dimer as a template. This causes damage that increases the likelihood of mutation.
How does ionizing radiation induce metagenesis?
Xrays, cosmic rays, gamma rays cause water and other substances ionize, forming free radicals (OH.) that causes double stranded and single stranded breaks that may lead to rearrangements or largre deletions.
What is the dose-dependent effect?
DNA damage increases with the amount of free radicals present. The higher the damage or the more prolonged the repairs, the more likely the cell dies.
What are the mechanisms to repair damaged DNA?
- Error-free repair system: restores original bp sequence
- Error-prone repair system: can repair incorrectly and introduce mutations
What are types of error-free DNA repair?
- Alkyltransferase
- photoreactivation repair system
- base excision repair
- nucleotide exision repair
- methyl directed mismatch repair
How does the alkyltransferase work?
Recognizes alkylated DNA and catalyze transfer of methyl or ethyl group from base to cysteine side (one time use)
What is the photoreactivation repair system?
photoreactivating enzyme (photolyase) has reduced FADH2 that absorbs blue light and uses the energy to SEPARATE fused pyrimidines. When there is no light, it cooperates with the nucleotide excision repair system.
What is base excision repair?
Mechanism that ELIMINATE bases and pyrimidine dimers. Includes general purpose AP lyase and specific DNA glycolases.
How does an general purpose AP lyase work
The enzyme scans DNA for apurinic/apyrimidinic sites where a base has been lost. It then cuts DNA backbone at AP site and leaves a nick to make DNA accessible for repair enzymes to fill the missing base and seal the strand.
How do DNA glycosylases work?
The enzyme recognizes specific damaged bases and breaks the glycosyl bond between the damaged base and sugar, leaving a sugar within the backbone. AP endonucleases cut out small section to create a nick for DNA pol and ligase to fill in the correct base and ligate the backbone.
How does nucleotide excision repair work? What types does E. coli have?
- UvrA-UvrB complex scans DNA for distortions by bulky lesions (thymine dimers). Once found by UvrA, it dissociates, leaving UvrB bound to the lesion.
- UvrC binds to UvrB and acts as an endonuclease, making one cut 4-5 nucleotides downstream of the lesion (3’), and another 8 nucleotides upstream of the lesion (5’)
- UvrD acts as a helicase to unwind the damaged DNA fragment, removing the oligonucleotide containing the lesion.
- DNA polymerase fills the gap and DNA ligase joins the fragments.
How does methyl-directed mismatch repair work?
- MutS protein dimer binds to alteration in DNA causing minor distortion in helix
- Two MutL proteins bind to MutS, and one MutH binds to the nearest GATC/CTAG
- MutH nuclease cuts unmethylated DNA at GATC/CTAG sequence
- UvrD helicase removes small segment of DNA containing the mismatch, and is enlarged by exonucleases
- New DNA region is synthesized by DNA polymerase III and ligated to ends by ligase
8.GATC sites are methylated by deoxyadenosine methylase (DAM)
What is the SOS response?
Broad emergency DNA repair system activated when DNA suffers extensive damage. Includes error-prone repair due to prioritization of survival over accuracy.
What are DNA pol IV and DNA pol V?
DNA pol IV (dinB) and DNA polV (umuCD) are error-prone DNA polymerases that can synthesize DNA over short gaps without a template and lack proofreading ability.
What genes in the SOS regulon? What controls them?
recA, uvrABC, dinB, umuCD, lexA, and others are regulated by LexA.
How does LexA regulate the SOS regulon?
All SOS repair genes are partially repressed by LexA under normal conditions to provide sufficient DNA repair under normal conditions.
What is the SOS regulon pathway?
- DNA suffers extensive damage, leading to the accumulation of ssDNA which binds and activates RecA
- RecA stimulates LexA to undergo autoproteolysis, which degrades LexA.
- Repression of LexA on dinB, uvrA, umuCD, and lexA on the SOS regulon is relieved to carry out DNA repair response.
