Mutation and Repair Flashcards
Phenotype vs genotype
Phenotype: observable properties of an organism
Genotype: Sequence of DNA
What is a mutation?
Inheritable change in the DNA sequence
(damage to DNA can’t be inherited so not mutation)
What are point mutations? what are the two kinds?
when a basepair in DNA is changed to a different basepair
Transition: a transition mutation is where a purine base (A & G) changes to another purine base and a pyrimidine base (C & T) changes to another pyrimidine base
Transversions: a transversion mutation is where a purine base (A & G) changes to a pyrimidine base (C & T) and vice versa
Missense mutations
point mutations in the coding region of a gene can cause a change in the protein where one amino acid is replaced by another
Nonsense mutations
a point mutation in the coding region of a gene can change a codon into one of the 3 nonsense codons that specify a STOP to translation (UAA, UAG, or UGA) causing production of a truncated, and usually, an inactive version of the gene product
Frameshift mutation
a high percentage of all spontaneous mutations are frameshift mutations – this kind of mutation arises when small insertions or deletions of one or a few basepairs occurs – when this happens in the coding region of a gene the reading frame of the gene is disrupted downstream of the frameshift mutation
Because the reading frame is disrupted the gene product will be truncated, usually within 20 amino acids, downstream of the frameshift
Frameshift mutations usually inactivate the gene product
What is triplet expansion
a disease caused by the insertion of many additional copies of a repeated codon triplet into a gene due to template slippage in the DNA replication process.
what is fragile X syndrome
triplet expansion disease
What is a deamination
removal of amino groups from biomolecules such as nucleotides
Deamination of nucleotide bases by spontaneous hydrolysis
A hydrolysis reaction in which water is added to cytosine, resulting in deamination to uracil and ammonia
What is the origin of point mutations
oxidative damage
what is oxidative damage
bacteria with aerobic metabolism and pathogens exposed to an inflammatory response triggered by the innate immune system are exposed to reactive oxygen species (ROS) that can damage DNA by chemically modifying the bases of the DNA
A common oxidative modification occurs to G to produce 8-oxo-G which can sometimes mispair with A instead of C inducing GC to TA and CG to AT transversions
How is nitrous acid a source of mutation
Deamination by nitrous acid.
Nitrous acid can deaminate C and A residues, causing them to base-pair with the wrong nucleotide during replication
C after deamination turns into U which pairs with A instead of G, a CG basepair becomes a TA (a transition mutation). A after deamination turns into hypoxanthine which pairs with C instead of T, an AT basepair becomes a GC basepair (a transition mutation).
how can chemicals produce mutations
The two-step process of a point mutation (b) The parental DNA contains a damaged adenosine base (X). The damaged A more readily pairs with a C than a T, so replication over the damaged base results in a mismatched nucleotide in one of the first-generation cells. Further replication can result in a permanent mutation in a cell of the second generation.
DNA repair processes that act to remove the damage, damaged base or the resulting mismatch lower the mutation rate that would arise from these damaged bases.
how can sunlight cause DNA damage
One type of reaction caused by UV light results in a cyclobutane ring, which involves C-5 and C-6 of adjacent pyrimidine (in this case, thymine) bases
An alternative reaction results in a 6-4 photoproduct that links C-6 and C-4 of adjacent pyrimidines.
T’s stacked on each other in the same strand can become crosslinked by excitation afforded by UV spectrum light.
How can DNA damaging agents be used to treat cancer? What are some agents?
Chemotherapeutic DNA-damaging agents. Chemotherapeutic agents act by preferentially damaging the DNA of fast-dividing tumor cells
Cisplatin, bleomycin, doxorubicin
how does cisplatin and bleomycin work
Cisplatin is a cross-linking reagent that reacts with N-7 of two G residues to form intrastrand or interstrand cross-links.
Bleomycin binds to DNA and forms reactive oxygen species that cause strand breaks. Iron normally binds bleomycin, but cobalt was used to prevent DNA modification in the crystal structure shown here
how does doxorubicin work
Doxorubicin is a DNA intercalator, inserting between adjacent nucleotide residues. Intercalators can cause frameshift mutations during replication, or they can block replication forks, making the forks susceptible to nucleases that cause strand breaks.
mismatch repair (first 3 steps)
In E. coli, MutS2MutL2 binds a mismatch and scans the DNA for a GATC site. MutH nicks the DNA at the nearest unmethylated GATC site, facilitating repair of the mismatch on the newly synthesized strand by excision, filling in of the gap, and ligation.
Recall, once DNA has just been copied the newly synthesized strands (nascent strands) lack dam methylation for a while. This allows the MMR system in E. coli to distinguish between the parental strand and the nascent strand. Since mismatches from errant replication are more likely to occur on nascent strand, repair is directed towards the nascent strand.
Mismatch repair (steps 4-7)
Direct repair of pyrimidine dimer mechanism
E. coli photolyase has two chromophores (light-absorbing groups) that work in sequence to use the energy of light to repair a pyrimidine dimer.
Photoreactivation was discovered in the study of UV damaged phages. It was discovered that prior exposure to sunlight allowed ‘reactivation’ of some phages inactivated by exposure to UV light. Phage infected cells kept in the dark prior to UV inactivation did not undergo this reactivation after UV exposure
Base excision repair (bacterial
In bacteria, a glycosylase excises a damaged nucleotide base, then an AP endonuclease nicks the backbone at the a basic site. Nick translation by Pol I excises the 5′ deoxyribose phosphate (5′-dRP) and some dNMPs, and synthesizes a new strand. Ligase seals the gap
Base excision repair (eukaryotes)
Eukaryotic BER, after the first two steps (similar to those in bacteria), can take either of two paths. In long patch repair (left), a DNA polymerase extends the DNA strand from the 3′ terminus, displacing the 5′ single-stranded DNA; this is followed by cleavage by a flap endonuclease and ligation. In short patch repair (right), only one nucleotide is inserted (by Pol β) prior to ligation.
UvrA, UvrB, UvrC, UvrD, Pol I, DNA ligase roles in NER
Uvr
NER mechanism in E. coli
The NER pathway uses several proteins, including UvrA (red), UvrB (purple), and UvrC (green), that recognize the lesion and make incisions on either side, allowing UvrD (helicase II) to displace a section of lesion-containing DNA. The single-strand gap is filled in by Pol I, and the DNA is sealed by ligase. A transcription-coupled repair (TCR) path can also be taken, in which RNA polymerase stalls at the lesion on the coding strand (not shown). After the RNA polymerase is displaced, the reaction proceeds as shown here, using UvrA–D, Pol I, and ligase.
Which 3 E. Coli polymerases are capable of translesion synthesis? What family are they in
Double-strand break repair mechanism? What does it require?
Accurate repair of a DSB requires an undamaged source of duplicate genetic information—that is, homologous double-stranded DNA. The broken ends are first processed to generate 3′ single-stranded extensions, and both ends are used for strand invasion of the homologous double-stranded DNA to form D-loops. The invading 3′ ends are extended by DNA polymerases.
RecA mechanism
forms filaments on ssDNA
RecA and other recombinases in this class function as filaments of nucleoprotein. (c) Filament formation proceeds in discrete nucleation and extension steps. Extension occurs by adding RecA subunits so that the filament grows in the 5′→3′ direction. When disassembly occurs, subunits are subtracted from the trailing end.
Example of damage repair and notable enzyme for each of the following processes
Mismatch repair
Base excision repair
Photoreactivation
Nucleotide excision repair
Double-strand break repair
Translesion synthesis
