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MCB2023S > DNA Mutagenesis, Damage & Repair > Flashcards

DNA Mutagenesis, Damage & Repair Flashcards

1
Q

What are the causes of spontaneous mutations?

A
  1. Tautomerism
  2. Replication Errors
  3. Metabolism:
    o By-product = free radicals attack proteins, DNA, membranes etc.
  4. Depurination:
    o Apurinic sites don’t base pair normally, DNA Pol may insert wrong base
    o Results in ring opening in sugar moiety causing leaking of the DNA backbone = strand breaks
  5. Insertion of DNA via Mobile Genetic Elements
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2
Q

What are the causes of induced mutations?

A
  1. Physical agents:
    o Gamma radiation - Causes Reactive O Species (which cause strand breaks)
    o UV Radiation - UVB causes pyrimidine dimers
2. Chemical agents:
    o Base analogues
    o Base modifiers
    o Intercalating agents
    o DNA Crosslinkers
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3
Q

What are base analogues?

A
  • Type of induced mutation
  • Causes incorrect base pairing
  • Adenine analogue: 2AP (2 amino purine)
    o BP’s with C - cause AT-GC transition
  • Thymine analogue: 5-bromouracil (5BU)
    o BP’s with G - causes AT - GC transition
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4
Q

What are base modifiers?

A
  • Hydroxylamine:
    o Reacts with C (hydroxylation)
    o HC pairs with A (CG-TA transition)
  • Alkylating Agents:
    o Add alkyl group to base
    o Alkylation of G = 9-methylguanine (AlkG) which pairs with T
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5
Q

What are intercalating agents?

A
  • Insert into DNA backbone and change distance between bases
  • Ethidium Bromide:
    o Intercalates into DNA helix
    o Changes distance between bases
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6
Q

What are DNA Crosslinkers?

A
  • Crosslink base pairs
    o In same strand = intrastrand
    o Between 2 strands = inter strand
  • Mitomycin C & Cis Platinum:
    o Insert between strands of helix
    o Form strong covalent bonds
    o Strands cannot separate during replication
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7
Q

What are the 3 types of mutants?

A
  1. Auxotrophic Mutants
  2. Drug Sensitive/Resistance Mutants
  3. Conditional Lethal Mutants (partially active under certain conditions, e.g. temp sensitive)
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8
Q

What is an Ames Test?

A

Method to identify environmental mutagens using a mutational reversion assay.

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9
Q

What makes Salmonella Enterica strain so good for Ames Test?

A
  • Permeable Membrane
  • Lack DNA Repair Mechanisms
  • His auxotrophs
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10
Q

What is a reversion assay?

A

Reverts auxotrophic mutants back to wild type organisms.

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11
Q

What purpose does the liver extract play in the Ames Assay?

A

It converts potential carcinogens into derivates that react with DNA

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12
Q

When is a sample considered a positive mutagen?

A

When its MI is 2 for at least 1 of the test doses and the response is dose dependant

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13
Q

How to calculate the Mutagenicity Index

A

(Avg no. of his- to his+ revertants due to mutagen per plate)
______________________________________________
(Avg no. of spontaneous or natural his+ revertants of neg control plate)

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14
Q

What are the 3 classes of mutations?

A
  1. Microlesions
  2. Macrolesions
  3. Suppressor Mutations
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15
Q

What is the Philadelphia chromosome?

A

Result of translocation (macrolesion) between C9 and C22. abl gene translocated from C9 forms fusion abl-bcr gene on C22 leading to constitutive activation of Tyrosine signalling protein.

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16
Q

5 types of macrolesions

A
  1. Large scale deletions
  2. Chromosomal inversions
  3. Translocations (via transposons)
  4. Duplications (usually of repeated sequences)
  5. Insertions (e.g. via transposons)
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17
Q

2 Types of Microlesions

A
  1. Base substitutions (point mutations)

2. Frameshift mutations

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18
Q

Base Substitutions

A

Same-sense mutations:
o Physiologically conserved due to degenerate genetic
code.
o A synonymous codon is formed, so same amino acid is brought

Missense mutations:
o Different aa is coded for, can have differing levels of effect depending on where in the aa chain the mutation occurs (active site, co-factor binding site, protein folding, stability)
o Neutral mutation = Enzymatic activity of the product is not affected when the aa is functionally conserved

Nonsense mutations:
o Results in coding on stop codon
o Results in truncated protein
o UAG (amber), UAA (ochre), UGA (opal)

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19
Q

Frameshift mutations

A
  • Insertion or deletion of bases
  • Affects every subsequent codon
  • Has downstream effects in polycistronic operon
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20
Q

Suppressor mutations

A

Counter the phenotypic effect of a previous mutation by reverting or alleviating. Occurs at sites different from original mutation and can be either intragenic (on same gene) or intergenic (on different gene)

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21
Q

What are pyrimidine dimers?

A
  • Form a kink in the DNA backbone that blocks DNA replication
  • The covalent bond formed between 2 adjacent pyrimidine bases in the same DNA backbone forms due to UV irradiation
  • This forms either thymine dimer if it forms between 2 thymine bases or..
  • A 6,4- photoproduct which is formed between thymine and cytosine
  • These covalent links between the pyrimidine dimers cannot be replicated and are therefore lethal to the cell unless it is repaired
  • The repair of these pyrimidine dimers can also lead to mutations
  • Linked to cancers in mammalian cells, specifically skin cancers
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22
Q

Temperature sensitive mutants

A

Cannot form colonies at high temperatures

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23
Q

Cold sensitive mutants

A

Cannot form colonies at low temperature

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24
Q

Define Genotype

A

Genetic information on the genome. Includes plasmids and transposons in bacteria

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25
Q

Define phenotype

A

Physical characteristics in the environment.

Genotype + environment = phenotype

26
Q

Define Mutagen

A

A substance or agent that induces heritable change in cells or organisms.

27
Q

Define Carcinogen

A

Mutagen that induces unregulated growth processes in cells or tissues of multicellular animals, leading to cancer.

28
Q

Define genotoxicity

A

The property of chemical agents to cause damage to genetic information within a cell causing mutations which are not necessarily heritable.

29
Q

What are the consequences of mutations?

A
  1. General value:
    o Antibiotic resistance, pathogenicity
    o Industrial processes
    o Evolution
  2. Scientific Value:
    o Whole field relies on mutations
    o Useful tool in investigating gene regulation
    o Useful in assigning function to genes
    o Useful in understanding biological processes
30
Q

Penicillin Enrichment method

A

Isolates Auxotrophic mutants:

  • Start with population of bacteria in nutrient broth
    o Can add a mutagen to increase mutation frequency
  • Move bacteria into a minimal medium broth + penicillin.
    o Wild-type bacteria grow in minimal media, so they killed by penicillin
    o Auxotrophic mutants can’t grow so they aren’t killed by penicillin.
  • Plate what’s left onto nutrient agar master plate (has nutrients required for anything to grow, but only auxotrophs will)
  • Replica plate to differentiate between Auxotrophs to find the specific mutant you want
31
Q

How can you identify a mutant?

A

Replica plating:

  1. Sterile velvet is pressed on the grown colonies on the master plate
  2. Cells from each colony are transferred from the velvet to new plates (with media with an isolation factor)
  3. Plates are incubated
  4. Growth on plate is compared. A colony that grows on medium with histidine (e.g.) but can’t grow on medium without histidine is an auxotrophic histidine-requiring mutant
32
Q

What are the genotypes of Salmonela strains that are used in the Ames Test?

A
  • TA98: Frame shift
  • TA100: Base pair Substitution
  • TA102: Transition/Transversion
33
Q

What is the method of the Ames Assay?

A
  • Mix mutagen + liver cell extract + Salmonella his mutant
  • Control: liver extract + Salmonella his- mutant
  • Plate onto minimal media no his) and compare mutants that have reverted (his- to his+) to the control
  • Dose response between mutagen & number of his+ revertents (i.e. higher number of revertents when higher dose of mutagen)
  • The more reverted colonies as compared to the control the greater the mutagenicity of the mutagen = Mutagenicity index or Ratio
34
Q

tRNA Suppressor Mutations

A
  • Common in bacteria
  • Intergenic suppressors where a gene coding a mutant tRNA reads a mutated codon either as:
    o The original codon coding for the same amino
    o A codon coding for a functionally conserved amino acid
  • The tRNA suppressors are named as nonsense or missense depending on the nature of the original mutation
  • Function:
    1. Primary mutation changes a codon in an mRNA so that the protein product is no longer functional
    2. Secondary suppressor mutation in tRNA gene changes the anticodon of a tRNA so that it recognizes the mutant codon caused by the primary mutation
    3. The amino acid that is now inserted restores protein function
  • Negative effects of tRNA Suppressor mutations
    o Typically, tRNA suppressor mutations are associated with alleviating nonsense mutations – therefore also known as nonsense suppressors
    o However, tRNA suppressors/nonsense suppressors results in problematic side effects as all identical stop codons in the genome will also be suppressed to the same degree – stopping translation of those genes becomes an issue
35
Q

Damage reversal strategies

A
  1. Enzymatic Photoreactivation (Light Repair)

2. Methyltransferases

36
Q

Enzymatic Photoreactivation mechanism of action

A
  1. Photolyase scans DNA mol for pyrimidine dimers and binds to dimer close to catalytic site together with redox co-factor FADH> Dimer is flipped out from the DNA
  2. FADH in the Photolyase/dimer complex undergoes photoexcitation (energy generated from absorption of quantum of light)
  3. This causes electron from FADH to be transferred to the dimer which breaks the covalent bond between the dimers, and it splits.
  4. An electron returned back to FADH, and the photolyase is released from the DNA
37
Q

Enzymatic Photoreactivation General Info

A
  • Light dependant
  • Enzyme catalysed repair of cyclobutyl pyrimidine dimers
    o Monomerization of pyrimidine dimers
  • Wavelengths (320-500nm)
  • Enzyme = DNA photolyase
    o Light harvesting properties
    o Chromophore structure – increases absorption area
    o Flavin adenine dinucleotide (FAD) – the cofactor – gets photoexcited
  • phr genes code for photolyases (
38
Q

Methyltransferases

A
  • Alkylation of guanine and thymine gives rise to 06-methylguanine and 06-methylthymine respectively
  • 06-methylguanine and 06-methylthymine can bind to thymine and guanine respectively
  • Methyltransferases can transfer methyl group from alkylated base to itself and allow bases to resume normal pairing
  • Once methyltransferases accepts alkyl group it is inactivated and degraded
  • ada genes code for methyltransferases
39
Q

Damage Excision Strategies

A
  1. Nucleotide Excision Repair
  2. Base Excision Repair
  3. Methyl-Directed Mismatch Repair
40
Q

Nucleotide Excision Repair genes

A

UvrABC & Mfd

41
Q

NER Mechanism of Action

A
  1. UvrA and UvrB protein recognizes damaged DNA due to distortion of double helix
  2. UvrA is released and UvrB remains, UvrC is recruited to the site of the lesion
  3. UvrC & UvrB cleave several bases away on the 3’ and 5’ sides respectively, flanking the damaged site, excising a ±12-15 oligonucleotide
  4. UvrC released and UvrD (a helicase) removes the damage-containing oligonucleotide from the dsDNA mol
  5. Resulting gap is filled by DNA Polymerase I and sealed by DNA ligase.
42
Q

What is a disease related to the NER system?

A
  • Xeroderma Pigmentosum
  • Multi-gene disease related to the NER system
  • Intensity of disease differs depending on the mutation
  • Sensitivity to sunlight, pigmentation abnormalities, prone to skin cancers
43
Q

Transcription-Coupled NER Mechanism of Action

A
  1. RNA polymerase stalls at DNA lesion on DNA strand being transcribed
  2. Mfd protein complex is recruited to the site before the stalled RNA polymerase
  3. ATP hydrolysis by the Mfd complex translocates the complex forward releasing the stalled RNA polymerase
  4. This allows the DNA lesion to be recognized by UvrA and recruit the NER to repair the lesion
44
Q

What is a disease related to the TC-NER system?

A
  • Cockayne Syndrome:
    o Caused by mutation in the eukaryotic TC-NER machinery
    o Rare fatal autosomal recessive neurodegenerative disorder characterized by growth failure, impaired development of the nervous system, abnormal sensitivity to sunlight, eye disorder and premature aging.
45
Q

Base Excision Repair Mechanism of Action

A
  • Certain forms of base damage are recognized by DNA glycosylases that catalyse excision of the damaged base from the sugar-phosphate leaving an AP (apurinic/apyrimidinic) site in the DNA.
  • AP site recognised by a 5’ AP endonuclease which cuts the phosphodiester backbone on one DNA strand on the 5’ side of the AP site leaving a 5’ terminal deoxyribose phosphate moiety (‘’flap’’).
  • The gap is filled by DNA polymerase while DNA deoxyribophosphodiesterase (dRpase) catalyses the excision of the 5’ sugar-phosphate moiety (‘’flap”).
  • The resulting nucleotide gap following DNA polymerase dissociation is filled by DNA ligase.
46
Q

BER General Info

A
  • DNA Glycosylases: excise bases by hydrolysing the N, N-glycosidic linkage between the base and the sugar phosphate backbone leaving a AP (apurinic or apyrimidinic) site.
  • Class 1:
    o DNA glycosylase without AP Endonuclease activity
    o General recognition of all alkylated bases
    o E.g. 3 Methyladenine DNA glycosylase II
  • Class 2:
    o DNA glycosylase WITH AP Endonuclease activity
    o E.g. pyrimidine dimer endonucleases
  • Types of damage: (change chemistry of bases changing their binding scpecificity to other bases)
    o Oxidative damage
    o Alkylation damage
    o Hydrolytic damage
47
Q

Methyl-Directed Mismatch Repair Mismatch Recognition

A

o Strand discrimination is only possible during replication before daughter cell is methylated
o GATC sequence on parental DNA strand is hemi-methylated (parental is methylated but not daughter strand) by DAM methylases with Adenine methylated at C6

48
Q

Methyl-Directed Mismatch Repair Mechinsm

A
  • MutS scans the DNA for mismatches and binds together with MutL to the mismatch in the DNA.
  • MutH recognises methyl groups on hemi-methylated strand and then binds MutL/MutS proteins to form a complex
  • MutH nicks the non-methylated daughter strand at GATC.
  • Exonucleases remove nucleotides from the non-methylated cut daughter strand including the mismatch.
  • DNA Polymerase III fills in the gaps using the methylated parental strand followed by ligation to seal the end.
49
Q

Disease related to Methyl-Directed Mismatch Repair

A

Hereditary Nonpolyposis Colorectal Cancer

50
Q

Damage inducible genes and the functions of there products

A
  • recA RecA: General recombination; co-protease
  • lexA LexA: SOS repressor
  • uvrABC UvrABC: Endonuclease in excision repair
  • recBCD Recombination repair
  • umuCD UmuCD: Mutagenesis
  • sfia SfiA: Cell division inhibitor
51
Q

SOS Response General Info

A
  • Includes:
    o NER
    o Homologous recombination
    o umuD mutagenesis (Error-prone DNA repair)
  • Consists of different DNA repair systems under the same global control via SOS boxes (cis sites) in the promoter region
  • The various genes of the SOS system are expressed at different levels, even in the repressed state, depending on affinity of the repressor for SOS box
  • It is a UV Inducible system where induction is dose dependant
    o Higher UV doses induce the system while lower doses do not induce it fully.
  • Consists of 3 phases:
    o Early phase: mostly dominated by accurate DNA repair (NER)
    o Middle phase: involve recombination repair (RecA; RecBCD)
    o Late phase: characterized by elevated mutation levels caused by error-prone DNA replication polymerases (SOS Mutagenesis)
52
Q

SOS regulation

A
  • SOS Regulation dependant on 2 gene products:
    o LexA repressor and RecA protease
    o LexA binds to SOS boxes and represses genes
    o Operators that bind LexA weakly are the first to be fully expressed – LexA inactivation can sequentially activate different mechanisms of repair.
53
Q

Agents and Treatments that induce SOS Response

A
  • Agents and treatments that induce SOS Response (Dose dependant)
    o UV and X-rays
    o Alkylating agents
    o Nalidixic acid
    o Any treatment that causes halt in DNA replication
54
Q

SOS Response Phenotype

A

o Enhanced survival after damage
o Enhanced mutation rate
o Filamentous cells

55
Q

Activation of RecA

A
  • Protease activity of RecA acrivated by DNA damage (ssDNA)
  • Binds to ssDNA to form RecA filament
  • RecA filament can then cleave LexA allowing expression of genes
56
Q

Mechanism of Sequential response

A
  • Early response: NER (uvrABC) is the first SOS repair mechanism to be induced whose aim is to fix DNA damage without commitment to a full-fledged SOS response.
  • Middle Response: If NER does not fix the damage adequately (if amount of damage is greater than ability of NER system), the LexA concentration is further reduced by RecA activity (more damage means more ssDNA so more protease activity etc.), so the expression of genes involved in recombination repair (RecBCD) are activated.
  • Late response: If the damage cannot be repaired via NER and recombination, cell division is stopped (sfiA) coupled together with lower LexA levels and mutagenesis prone mechanisms (umuCD) are activated.
    o As a result of these properties, some DNA repair genes may be partially induced in response to even endogenous levels of DNA damage, while other genes appear to be induced only when high or persistent DNA damage is present in the cell.
57
Q

Transcriptional Regulation via Regular

A
  • Regulon = group of genes (scatters throughout genome) controlled by one common regulator – protein repressor LexA
  • LexA recognizes a particular operator sequence in the promoter (SOS box) and binds to it
58
Q

Homologous Recombination

A
  • The exchange of DNA sequences between DNA strands that contain identical or near identical sequences in their length (Middle SOS Response)
  • Genetic recombination is involved in DNA repair and allows cells to retrieve DNA sequences lost through DNA damage – these could be via homologous sequences on the genome or on MGEs
  • The homologous region that can recombine with each other can be as few as 50-100bp to a whole chromosome and they need various recombination proteins (rec) to carry out the recombination
  • The greater the region of homology the higher the frequency of the recombination
  • RecA is required for homologies >1000bp and binds to ssDNA and the RecA-ssDNA filament can bind to unwind another dsDNA to promote base-pairing (assimilation)
  • RecBCD is a protein complex with helicases/exonuclease activity that can unwind and degrade ss & ds DNA
59
Q

RecBCD Pathway

A

Pathway of Homologous Recombination

  • A Chi site or sequence is a short stretch of DNA in the genome of a bacterium near which homologous recombination is more than likely occur.
  • In E. coli and other enteric bacteria, the Chi sequence is 5’-GCTGGTGG- 3‘ and can occur up to 1000 times in the genome.
  • Chi sites serve as stimulators of DNA Double-strand break Repair in bacteria, which can arise from DNA damage.
  • RecBCD enzyme is a large complex of three polypeptides with both DNA-unwinding (helicase [B&D]) and DNA hydrolysis (nuclease [C]) activities. The unwinding activity is faster than the nuclease activity
  • When RecBCD moves past the Chi sequence, nuclease activity stops and unwinding continues. The generated 3’ ssDNA is loaded with multiple RecA protein molecules. RecA promotes the exchange of this ssDNA for its equal in an intact homologous DNA molecule.
60
Q

Error-prone DNA Repair

A
  • Error-prone DNA polymerases (umuD operon) are produced only in the presence of high DNA damage via the SOS response.
  • These specific DNA polymerases attracted to stalled replication forks due to unrepaired DNA damage and replace normal polymerase.
  • Sloppy DNA polymerase adds random nucleotides to the strand being synthesized opposite the damaged bases (template)
    o Termed ‘Translesion DNA Synthesis’
    o Restores proper base only 1⁄4 of the time. (i.e. ¾ times it introduces mutations)
  • After translesion synthesis, sloppy polymerase replaced by normal polymerase.
  • No halt in replication but daughter cells carry new mutations.
61
Q

Non-Homologous End-Joining (NHEJ)

A
  • Repairs double-stranded breaks (DSB) in DNA
  • Involves three main proteins (Ku70, Ku80, DNA PKcs) and accessory proteins
  • DSB is recognized by Ku dimer (Ku70–Ku80) and DNA-PKcs
  • The dimer and proteins above allow the two DNA ends to be repaired (synapsed).
  • Other accessory proteins are recruited and DNA-PKcs allows accessory proteins to process DNA ends
    o Overhanging nucleotides at end that do not have complimentary nucleotide to pair are ‘cut back’ (resect) and ends ligated by DNA ligase IV
    o Resection results in NHEJ losing DNA – error in DNA
  • Key feature of Crispr Gene editing

MCB2023S (9 decks)

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