Genetics Flashcards
What is a mutation?
An accidental permanent change in a DNA sequence That is rarely advantageous
What is a nonsense mutation?
It is a single base substitution that introduces a stop codon
GGA ➡️ TGA, codon UGA
What is a missense mutation?
It is a single base substitution mutation where one amino acid codon is replaced with another
GGA ➡️ GTA, codon GUA (val) instead of GGA (gly)
What is a silent mutation?
It is a single base substitution mutation that changes the DNA sequence without altering the encoded protein. Basically the code on that results from the change ends up, giving you the same amino acid as the codon prior to the mutation
GGA➡️ GGT, codon GGU (gly) vs GGA (gly)
What is a frameshift mutation and what is it caused by?
A frame shift mutation is caused by an insertion or deletion that is not a multiple of three bases so it causes the open reading frame to shift forward or backward
This often leads to an early stop codon
Mutations that affect RNA splicing often generate frame shifts
What is an In-Frame deletion and why is it different from frame shift mutations?
It is a deletion of a multiple of three bases so basically it’s a deletion of a coat on which then causes a frame shift of the ORF backwards
What causes mutations?
DNA damage that can be spontaneous, or induced by environmental factors
What is deamination of DNA bases?
The removal of an amino group from a nitrogenous base which leads it to become a different base
This typically happens because of sodium nitrate and sodium nitrate, which is used in production of cured meats, tobacco products, pesticides, and cosmetics is also found in our gastric juices
Ex. C 👉🏼 U and (5-me C 👉🏼 T )
What is involved in oxidation of DNA?
It is another type of DNA damage where a reactive, oxygen species or ROS is generated by cellular respiration
So, a free radical OH inserts into either G or T
👉🏼 results in DNA strand breaks
What is depurination?
DNA damage caused by hydrolysis of the glycosidic bond linking a purine base to the sugar phosphate backbone
This leads to an abasic site or AP site (apurinic/apyrimidinic site)
What is alkylation of DNA?
Alkylating agents i.e. mustard gas covalently modify bases in DNA
This will damage the DNA by distorting the DNA double helix and can result in depurination
What is DNA damage that can be caused by radiation?
UV radiation can result in a cyclobutane ring between two adjacent perimeter rings; this results in common damage, such as a thymine dimer which kinks the access of the DNA helix, and therefore can have consequences for transcription
Ionizing radiation that releases electrons from atoms can generate ions that break covalent bonds meaning they can break the phosphate sugar backbone as a single strand break or double strand break
Examples of ionizing radiation are cosmic rays, x-rays, and radioactive materials
What is the general pathway of DNA repair?
RERaL
Recognition
Excision
Re-synthesis
Ligation
How do we do mismatch repair (MMR)?
First we recognize that there is a mismatched base pair, resulting from errors in replication
MutL-MutS recognizes the mismatch
MutH recognizes the template strand via methylated base and the newly synthesized strand …. It makes the un methylated strand via its endonuclease function
- Next we excise
Exonuclease digests from nick to the mismatch
- Then we resynthesize
DNA polymerase three fills the gap
- Lastly, we ligate
DNA ligase seals the nick
How do we do base excision repair?
- Recognize
Glycosylase and recognizes and leaves like glycolytic bond making an AP site
AP endonuclease makes a single strand break in DNA at the abasic site
- Excision
DNA polymerase one or beta uses exonuclease in a five prime to three prime direction to remove that
DNA
- Resynthesis
DNA polymerase 1 or Beta then fills in the gap
- Ligation
Ligase seals the nick
What is cancer the result of?
Of an accumulation of mutations in our genome
What do the accumulation of mutations result from and what do they lead to?
DNA damage and they lead to cancer
What is a mutation?
An accidental but permanent change in the Genome
What are the single base substitutions and what kind of mutations are they?
They are one base mutations, and they are categorized as nonsense
mutation, missense mutation, and silent mutation
What do nonsense mutations introduce?
A stop codon
It results in a truncated protein that probably has lost function
Where does the mutation happen?
The coding strand
Remember, the coding strand is the same as the mRNA in prokaryotes. The difference being that T’s in DNA are U’s in RNA.
What effect does the missense mutation have on protein function?
It depends on where the mutation happens so if it happens in the catalytic site, then that may completely destroy the proteins function, but if it is elsewhere and the protein, that doesn’t have a lot of important functions than the protein will be fine most likely
What proves that just because you have a mutation on the DNA level that you may not have a mutation on the protein level?
Silent mutations
The coding strand may have a different codon from the template strand, but the amino acid that’s expressed from that different codon will be the same as the one expressed from the template strand
Is a frame shift mutation, the same as an in frame deletion?
No, the frame shift mutation is an Indel that is not a multiple of three bases. It does usually lead to an early stop codon.
The inframe deletion is a deletion of a multiple of three bases
Do infra deletions usually have deleterious effects?
No, they do not because many times these mutations just affect one coat on, but if it is done consistently, that’s enough to result in a phenotype
What is an exception to inframe deletions that normally do not cause deleterious effects?
Cystic fibrosis because when the specific amino acid phenylalanine, which is critical for its folding is deleted then the chloride pump doesn’t function which results in this disease
This all happens in the CFTR gene
What is DNA damage?
Abnormal chemical structure of DNA
When will DNA damage result in mutations?
When it is not fixed, then it can result in these permanent changes in the genome
What causes DNA damage?
Spontaneous changes
Environmental factors
What are types of DNA damage?
——-Deamination of DNA bases- you are moving in amine group
Ex cytosine changing to uracil in our DNA where it does not belong
Five methyl cytosine changing to thymine is wrong because thymine does not belong where five methyl cytosine is (CpG Islands - promoter proximal regions where C’s can be methylated —— methylation changes how transcription factors interact with that region)
—— oxidation of DNA
oxygen gets added onto our bases when they should not be there, and that will mess up the base pairing between the template strand and the coding strand of the DNA and it can cause a polymerase to add the wrong base
—- ex hydroxide free radicals
——depurination
Creates a site without a purine
By breaking the glycosidic bond between the base (which is released) and sugar
- this is therefore an abasic site (aka apurinic, or apyrimidic sites)
——-alkylation
Adding alkyl groups to Dna
Ex s-adenosyl methionine adding methyl group to guanine making 7 methyl guanine
——thymine dimers
Caused by ultraviolet or UV radiation
-it forms a cyclobutane ring because it rearranges the electrons between both thymines
—— DNA strand breaks
These are caused by ionizing radiation
Breaks a phosphodiester bond either in one strand of the DNA for single-stranded break or two phosphodiester bonds in both strands of the DNA causing a double stranded break
Why are CPG islands known as mutational hotspots?
Because we commonly see mutations in these islands
- these mutations are due to deamination, which is the event that is so common there
What kind of event changes adenine into hypoxanthine and guanine into xanthine?
How often does this event occur?
Deamination of DNA bases
This event does not occur often like the deamination in CpG islands or deamination of Cytosine to Uracil
What typically causes deamination?
Sodium nitrate and sodium nitrite that come from production of cured meats, tobacco products, pesticides, some cosmetics, our own gastric juices and leafy vegetable consumption (lettuce, spinach, etc.)
What is a permanent change that can no longer be detected by our repair systems?
Mutations that result from DNA damage that once could be detected (ex deamination, etc)
Once DNA damage, such as deamination of five methyl cytosine to find in the CPG islands is not fixed by our repair systems and goes through replication to form new coding strands from the template strand, where the mutation occurred. Then that mutation becomes undetectable because the system doesn’t see a problem with the new base pairing between the template, strand and the coding strand
Example, the event changes, the five methyl cytosine to thymine which is clearly wrong because the thymine is bonding with guanine
——-However, when this is not corrected and replication occurs, then the coding strand creates a complementary base for that I mean, which is adding, and so the system no longer sees the problem that it once would have detected
What is the most common reactive oxygen species that we see in our cells? Where do they insert?
Hydroxide free radicals
They insert into our G’s and T’s in our DNA
Oxidation of C’s and A’s can also happen but at much lower rates
Besides causing the DNA to have abnormal chemical structures, what can oxidation of it cause?
It can cause DNA strand breaks
Why does depurination happen more often than depyrimidization?
Because the glycolytic bond is stronger in pyrimidines than it is in purines
True or false polymerase can work with abasic sites?
False it won’t know what to do with a site that doesn’t have a base for it to link with its base that it’s carrying
How does alkylation of DNA happen? What causes it?
This can happen spontaneously in the cell
Ex S-adenosyl methionine can take its methyl group and transfer it onto genes in our DNA making 7-methyl guanine
True or false seven methyl guanine belongs in our genome
False. it is used as a cap that is added post transcriptionally
What does calculation do to the DNA structure to cause its damage?
It distorts the DNA structure because it causes kinks and bends in the DNA so when the polymerase goes through this region, it will either stall or it will incorporate the wrong bases
What is mustard, gas, and example of as far as DNA damage?
It is a type of alkylation of the DNA which covalently links the two strands preventing them from undergoing transcription because those strands cannot be separated, and then that leads to the cell not being able to make proteins which then causes it to die
True or false in finding diamonds, these fly means are now covalently linked to each other
True the rearrangement of electrons happens because of UV radiation, which causes a cyclobutane ring to form between both thymine, which is really a covalent bond between them
What do thymine dimers cause as far as DNA damage?
Causes large distortions, kinks in the DNA
When a polymerase comes, it is either going to stall or probably put the wrong base across from this region
When we talk about DNA strand breaks what is incorporated in this understanding?
Besides the break in the phosphodiester bond or bonds all the bases near the brakes are also going to be damaged
We see deamination events, oxidative events, and depurination
So ligase cannot work with these bases, meaning it cannot just ligate the two ends of the sugar phosphate backbone back together
What are DNA strand breaks caused by and what are examples of it?
Ionizing radiation, which is high energy radiation that I can release electrons from atoms generating ions, which can break covalent bonds
Examples are cosmic rays, x-rays, and radioactive materials
What is the general pathway of DNA repair?
- Recognition of the lesion or DNA damage.
- Removing that DNA damage through excision.
- Filling in the gap with DNA.
- Migration of loose ends, which would involve DNA ligase.
When is Mismatch repair - MMR pathway of DNA repair used?
It is used right after replication has happened to check the DNA polymerase‘s work
—-therefore mismatch repair is looking for mistakes caused by the polymerase
What happens when we have mutations in the MMR proteins in humans?
It results in a disease state (very aggressive hereditary Non-Polyposis Colorectal Cancer)
Because these mutations lead to the MMR proteins becoming dysfunctional so that they cannot participate in the MMR or mismatch repair pathway —- i.e. whenever polymerize makes a mistake. There’s nothing else that can detect it.
What is the MMR pathway??
***DNA would’ve been just replicated
—- we know this because the parental (template) strand was just methylated …. The bottom strand is not methylated so it is obviously the new strand (has not been methylated yet)
- Recognition: Complex of MutL-MutS will follow replication machinery, looking for mismatches
—— when it finds a mismatch, it will bind to it
—— they then recruit MutH (another mismatch protein)
——MutH binds to the parental strand at the methylated site and behaves like an endonuclease by breaking a phosphodiester bond (clean single-stranded break) directly across from that methyl group in the newly synthesized strand - Excision : exonuclease comes in and is going to start at the single stranded break and chew up the DNA through the region of the mismatch to remove that mismatch
- Re-synthesis: DNA Pol 3 will still be around since application just finished so it will refill the gap by synthesizing DNA five prime to three prime
- DNA ligase will come in and ligate the strands together, making it a continuous piece of DNA which fixes the mismatch.
What is the pathway for base excision repair BER DNA repair?
- Recognition: first step is removing the nitrogenous space.
- DNA glycosylase (many types in eukaryotes ) cleaves the glycosidic bond making an abasic site (damaged nitrogenous base is removed) - Excision: AP endonuclease (apurinic ) binds to abasic site and breaks the phosphodiester bond creating a single stranded break
—— in bacteria DNA Pol 1 is going to come in and use its five prime to three prime exonuclease activity to remove that damaged site and some other bases - Re-synthesis: the same DNA Pol 1 is going to synthesize new DNA filling up that region
- Ligation: then DNA ligase comes in and ligates them together.
What kind of DNA damage does BER primarily focus on?
It targets DNA damage focused on the nitrogenous base
so
deamination, oxidation, depurination all of these things can be fixed with BER
What is nucleotide excision repair - NER DNA repair?
It repairs lesions that distort the structure of the DNA double helix, such as thymine dimers or alkylation
So we end up removing the entire nucleotide vs just a nitrogenous base in BER
- Recognition: Excinuclease - prokaryotes use UvrABC excinuclease and eukaryotes use XP excinuclease
- Excision: Helicase -
Prokaryotes use UvrD Helicase and eukaryotes use TFIIH
—-it breaks hydrogen bonds and release the fragment that has DNA damage on it
—- the fragment will just be degraded or recycled into nucleotides - Re-synthesis: DNA Pol 1 (Ecoli) or DNA Pol Epsilon (eukaryotes) will come in and fill the gaps
- Ligation- DNA ligase ligates the strands together
What is special about an excinuclease?
It breaks 2 phosphodiester bonds in the same strand of DNA surrounding the lesion site
Besides, NER, what is TF2H used in?
As a helicase, it is not just used to remove damaged DNA, but it is used as a helicase to separate the strands of our DNA during transcription for our transcription machinery
What is the implication of using the same Helicase (TF2H) for NER and transcription in eukaryotes?
It means that NER is directly coupled to transcription
Therefore, anytime we transcribe a gene this nucleotide excision repair pathway is going to be following behind, checking the DNA and looking for any of this nucleotide damage
What is the Non-homologous end joining pathway?
It is the predominant mechanism in G0 and G1 for repair of double strand breaks in mammalian cells (prokaryotes have a similar mechanism)
If these are not fixed there will be weird chromosomal rearrangements that contribute to cancer
- Recognition: ku70/80 (2 molecules of each part of the complex binds to the ends pf the double stranded break)
——ku70/80 binding recruits PKcs which is a kinase that initiates bridging of the broken ends
2.The bridge (PKcs) will recruit other proteins including exonucleases that will chew back the damaged bases resulting in a wider gap (widening the double stranded break)
- Re-synthesis: error prone terminal transferases - are unique polymerases (DNA Pol micro, and DNA Pol theta) - 5’ to 3’ antiparallel strands they will add random bases to each end if the DNA because there is no template for them to use as a reference
We then have endonucleases come fix overhangs etc
- ligation: DNA ligase comes in and ligates the strands together… this fixes the double stranded break
True or false non-homologous and joining is error prone repair
True.
This means we will probably introduce mutations into the cell by using this DNA repair pathway
This is still better than retaining a double stranded break
When is non-homologous end joining taking place in the cell cycle
?
Before replication because G0 and G1 happened before S phase where replication takes place
In G0 and G1 we do not have identical copy of each chromosome yet
What is usually the cause of a double stranded break?
Ionizing radiation
…. Remember, these are not clean brakes and therefore ligase cannot just come in and like get them back together because there will be a lot of damage, including deamination, oxidative damage, and depurination.
What is PKcs acting as for a double stranded break and what recruits it?
It acts as a bridge and is recruited by Ku70/80
Why are we losing genetic information during non-homologous and joining of the double-stranded break??
Because during excision when the Exonuclease comes in and chews up the damaged bases around the bridge this results in loss of genetic info
What kind of area are we creating by using a prone terminal transfers that put in random bases in between the area of the double strand breaks?
We are creating an area of micro homology where there are some G and seas that base pair to each other and some teas and A’s that base pair to each other. This way we get hydrogen bonding between the two strands of DNA.
Why shouldn’t we worry statistically about using non-homologous and joining that will probably introduce mutations into our DNA strands?
Because only about 1.5% of our genome actually encodes protein, the rest of it is basically non-coding sequences so if there’s a greater probability that a non-coding area of DNA at this double-stranded break that we are trying to repair
When can homologous recombination be used and what is the process of this DNA repair pathway?
It can be used when we have an identical copy of each chromosome
Therefore, this DNA repair can be used during S and G2 phases because replication would have already occurred
—— therefore we will use the intact chromosome as a template for the broken chromosome
Just remember it is like non-homologous end repair but we use recombination to reference an intact template strand in order to do error free creating of new bases and therefor hydrogen bonding etc
True or false homologous recombination is an error-prone pathway like non-homologous end repair
False it is a non-error-prone pathway or error, free pathway Because we are using a template strand to replace the DNA that we lost so the bases that we replace are based on a reference to the template strand
What is similar about MMR and NER and what is different about them?
MMR scans DNA after DNA replication and NER scans DNA after transcription
What is another way to handle DNA damage besides pathways such as MMR or NER
?
Cell says “Sound the alarm”
Usually happens when there is a lot of DNA damage
Pathway:
- Activate kinases
- Kinases phosphorylate transcription factor, p53
- p 53 goes into nucleus and binds DNA sequences and activates expression of certain genes
3a. Firstly activating genes involved in cell cycle arrest —- keeps cell from replicating DNA
3b. Secondly it activates genes involved in DNA repair —— repair proteins fix damage
3ci: if damage is very extensive then p53 will activate genes involved the process of Senescence (forces cell back into G0 phase permanently (no more dividing or growing))
3cii if damage is overwhelming due plethora of mutations then p53 will activate genes involved in apoptosis which is programmed cell death
Why is P 53 known as the “Guardian of the Genome”?
It is an important transcription factor for DNA damage signaling
