Test1: Lect3 Amanda McCullough Flashcards
Define Mutagenesis:
A process by which genetic information is changed in a stable and heritable manner
Mutation:
- Germline define:
- Somatic define:
- Germline define:
1: In gametes, will be passed on
2: for a child a germline mutation is inherited - Somatic define:
1: In gametes, will not be passed on
2: for a child a germline mutation is acquired
Mutation:
Vs.
Polymorphism:
Mutation: Difference in DNA sequence of individual which is rare in a population and may be unique to the individual
Polymorphism: Difference in DNA sequence found in many individuals, at a specified frequency (usually > 1%)
SNPs:
single nucleotide polymorphism, occurs approx. 1/360 nucleotides.
monogenic disease:
- Define:
- Examples of:
- Define:
A disease caused by a one gene mutation - Examples of:
Hemophilia and sickle cell anemia
multifactorial disease:
- Define:
- Examples of:
- Define:
Multiple gene and environment interactions - Examples of:
Heart disease, diabetes
Gene-environment interactions:
- Define:
- Examples of:
- Define:
Susceptibility to endogenous agents and environment: - Examples of:
Smoking -> lung cancer
Sun exposure -> skin cancer
Note: there are genes which make people MORE susceptible to these.
Vicious cycle of aging:
Aging -> Accumulation of mutations -> Deregulation of transcription, impaired stress response -> Decline in DNA repair -> More mutations -> Aging -> etc.
Mutator phenotype:
- Define:
- Define:
1: A mutation in a gene involved in DNA repair or in ensuring DNA fidelity during replication, or in apoptosis (ability to kill yourself if dangerously mutated)
2: AKA a gene which when mutated will allow more mutants later
Senescence:
- Define:
- What can cause senescence:
- Define:
When cells appear to lose the ability to divide and grow. They just kind of sit there. - What can cause senescence:
1: Mutations.
2: Similar to the vicious cycle of aging.
Elephants are much larger than us, have many more cells than us, but get cancer less often or as often as us, why the paradox?
1: Elephants have 50 TP53 alleles, which results in about twice the cell apoptosis as us.
2: Simply, they kill their cells if they have cancer potential a lot sooner than we do, and don’t try to repair them.
Could you just stimulate apoptosis systemically?
Maybe, but in addition to killing cancer cells you may have diseases like Alzheimer develop, which also has high apoptosis.
Point mutation which doesn’t change the protein the codon is codes for:
Synonymous mutation.
Point mutation which changes the protein the codon is codes for, BUT the amino acid is chemically similar:
missense mutation (Conservative)
Point mutation which changes the protein the codon is codes for, AND the amino acid is chemically different:
missense mutation (nonconservative)
Uh oh! Did that mutation just cause a premature stop codon?
Nonsense mutation
Frameshift mutations come in two flavors, what are they?
Base insertion
Base deletion
Transition mutation define:
Purine to purine (A to G) Pyrimidine to pyrimidine (T to C) A - T to G - C or G - C to A - T
Transversion mutation define:
Purine to Pyrimidine (A to T) Pyrimidine to purine (C to G) A - T to T - A or G - C to C - G
Mechanisms of spontaneous mutations:
- Unrepaired Spontaneous lesions:
1: Depurination
2: Deamination
3: Oxidation - Replication
Depurination:
- Define:
- How does a polymerase deal with lack of base information?
- Define:
Loss of a purine or pyrimidine base (yes both same name) - How does a polymerase deal with lack of base information?
Most standard replication polymerases just add an A (the A rule)
Deamination of bases:
- Define:
- Common examples:
- Define: Amine to ketone - Common examples: (cytosine a problem child) Cytosine -> Uracil 5 methylcytadine -> thymine
Oxidative damage:
- Define:
- Common example:
- Sources of oxidative agents:
- Define: Damage caused by oxidative species (like oxygen) - Common example: 8 - hydroxyguanine (ketone at 8 position) - Sources of oxidative agents: Heterocyclic amines Oxygen Exercise Alcohol Tobacco
What do polymerases like to put across from 8 - hydroxyguanine?
An adenine (fits better than cytosine)
Environmental (induced) mutagenesis:
- Define:
- Examples:
- Define:
Exposure to substances or wavelengths in environment which cause mutagenesis. - Examples:
Smoking Tobacco (Increases oxidative species)
Heterocyclic amines (browning meat)
Acrolein (gasses produced in air)
Radiation and chemical (induced mutagenesis):
- Define:
- Examples:
- Define:
This is really a form of environmental mutagenesis. But different because you could have a high isolated exposure, chemical spill, etc. - Examples:
Mustard gas
What is it important to remember when working with DIFFERENT mutagens on a cell line?
- Ideal survival percent?
They are not all created equal. You have to use equally toxic amounts of the two (which will likely be different amounts, depending on their toxicities)
- Ideal survival percent?
>35% survival, so you don’t just kill them all
Cisplatin:
- What is it?
- Why is it effective?
- What is it?
A chemotherapeutic drug - Why is it effective?
Crosslinking agent
Types of crosslinking DNA-damage:
- Define them as well:
Intrastrand crosslink: Covalent crosslinking between bases on the same strand
Interstrand crosslink:
Crosslinking of bases on two different strands (but usually the same duplex?
Which type of crosslinking is the worst?
Why?
Interstrand crosslinking.
Cannot even pull the strands apart
Name a type of intrastrand crosslinking:
Thymine-thymine dimerization from UV
What induces DNA interstrand crosslinks?
Many chemotheuraputics, like cisplatin
Complementation:
Defined previously.
production of a wild type phenotype when 2 genomes bearing a different recessive mutation are united in the same cell (cell fusion/crosses)
Epistasis:
- Define:
- Conclusion:
- Define:
I knock out gene 1 (g1), and my phenotype drops from 10 to 5.
I knock out gene 2 (g2), and my phenotype drops from 10 to 7.
I knock out g1 and g2, and my phenotype drops from 10 to 5 (same as g2). - Conclusion:
The genes are in the same pathway. It doesn’t get worse with the double knockout, because the pathway is fried when g1 is knocked out.
Additivity:
- Define:
- Conclusion:
- Also called:
- Define:
I knock out gene 1 (g1), and my phenotype drops from 10 to 5.
I knock out gene 2 (g2), and my phenotype drops from 10 to 7.
I knock out g1 and g2, and my phenotype drops from 10 to 2 (lower than both). - Conclusion:
The genes are in different pathways. It gets worse in the double knockout, so i probably fried to seperate pathways which support the phenotype. - Also called:
Non epistatic.
Name repair pathways (asterisk by one not being taught this course):
Mismatch repair Base excision repair Nucleotide excision repair Damage bypass (translesion synthesis) * Double-strand break repair
Mismatch Repair (MMR):
- Responsible for:
- Linked to:
- Responsible for:
Repairing mismatched bases and small “loop outs” (IDLs) - Linked to:
Replication, occurs in a post-replication repair pathway
How does E. coli identify the new strand from the template strand for purposes of mismatch repair?
1: It methylates adenines.
2: There is a delay in methylation post replication.
3: The methylated strand is right.
MMR in bacteria
MutS:
(in bacteria)
Searches for/recognizes mismatched pairs
(Link most of time searching)
MMR in bacteria
MutL:
(in bacteria)
Coordinator protein (helps keep MutH in check) (You need something to link)
MMR in bacteria
MutH:
(in bacteria)
Endunuclease for strand nicking, cuts opposite of the methylated strand
(Hiya to cut it, Link goes Hiya)
How do bacteria do mismatch repair?
Delay in methylation!
MutS identifies mismatch ->
MutL works to position and confirm MutH ->
MutH cuts the strand ->
Helicase and polymerases come in and make a new strand.
Methylation occurs
What if a bacteria over-expressed DAM methylase? How would this effect mismatch repair?
We would see less delay between new strand synthesis and methylation. Repair proteins would have a hard time identifying the template strand.
Do humans have a mutH homolog?
No. They use a endonuclease which complexes with PCNA. Theorizes PCNA can help with recognition of nascent strand vs template strand.
How is mutS different in humans?
to proteins in a heterodimer (they are different proteins) will form the initial recognition process
What is the current model for how strand specificity is determined for MMR in humans?
The mutL homolog complexes with PCNA.
The mutH homolog interacts with PCNA for interaction.
This could impart knowledge of which strand is nascent (newly synthesized)
On lagging strand do you need a mutH homolog to create a nick?
No, there are already nicks. More importantly nicks are on the nascent strand.
How do you use mismatch repair for somatic hypermutation (useful for antibody generation):
AID enzyme creates G - U mutant ->
These are recognized by mutS analogs ->
PCNA and other are recruited, but PCNA is MONOUBIQUINATED ->
Monoubiquinated PCNA recruits a low fidelity polymerase (pol eta) instead of a high fidelity one (as it normally for MMR) ->
Low fidelity polymerase makes mistakes ->
More errors occur
- Name a disease caused by failure of mismatch repair:
- What are the symptoms:
- Name a disease caused by failure of mismatch repair:
HNPCC (hereditary nonpolyposis colon cancer), aka lynch syndrome (link could fight a lynch… if he wasn’t broken ;P ) - What are the symptoms:
High chance of colorectal cancer (~50%), high chance of undometrium cancer (~50%), and high chance of ovarian cancer (~10%)
To cause lynch syndrome what mutation must occur in MMR?
Any mutation which causes failure of a protein in the process.
What is microsatellite instability (MSI)?
- How is it caused?
Somatic increase of small repeat sequences
- How is it caused?
NUMBER 1 is more common in lynch syndrome!
1: Repeat sequences form small hairpin structures, on the nascent strand this results in elongation, the Pol will read the sequence again
2: Repeat sequences form small hairpin structures, on the template strand this results in shortening of the strand, the Pol will read the sequence again
In what disease is microsatellite instability a characteristic sign?
- Why?
Lynch syndrome
- Why?
MMR repair is what would normally remove the small hairpin structures, failure to do so results in microsatellite instability
What broader category is microsatellite instability apart of?
Replication slippage. Whenever the polymerase skips a sequence (slips over it) or slips back on a sequence and repeats it.
Base Excision Repair (BER):
- Responsible for:
- Linked to:
- Some examples of types of damage:
- Responsible for:
Alterations to bases, including some alterations which result in mismatches (There is overlap with some enzymes of BER and MMR) - Linked to:
Nothing specific, this is a housekeeping pathway (Base, homebase, housekeeping), it is always on. - Some examples of types of damage:
Oxidation, depurination, etc.
Why is BER important in the brain?
It is not associated with replication, since the brain is not replicating (usually), it is one of the few pathways for repair which works in the brain
How does BER work?
Error in base recognized ->
Glycosylase removes the base ->
AP site now exists, AP nuclease makes a cut ->
1 - 6 nucleotide gap is made by removal of nucleotides ->
Resynthesized ->
Ligase seals nick
ap site define:
apurinic/apyrimidinic site
Some differences between MMR and BER?
MMR replication linked, BER constitutionally active
MMR cuts out larger sequences, BER cuts out smaller
BER and MMR recognize different types of damage
Why are glycosylases called glycosylase?
- Why aren’t glycosylase knockouts embryonic lethal?
Cut bond between sugar and base
- Why aren’t glycosylase knockouts embryonic lethal?
There is likely a redundant pathway which fixes these issues
What two pathways can BER take?
Short patch:
Long patch:
Short patch BER:
All the same, but only 1 to 2 nucleotides are removed and specialized polymerase which only adds 1 to 2 nucleotides replaces them (pol beta)
Long patch:
1: replacement of nucleotides is accomplished by normal high fidelity replication machinery (pol delta, pol sigma, PCNA)
2: 5’ end cut, nucleotides moved out of the way as replication occurs, 3’ end cleaved, excised. Minimizes exposure of ssDNA to mutagenic cellular environment.
3: 2 - 12 nt covered
Why is repair regulation so tightly regulated?
You don’t want to start a job unless you have all the proteins to finish it (don’t want to leave gaps).
Example of BER Deficiency:
- Cause:
- Symptoms:
MUTYH Polyposis Syndrome (type of colon cancer) - Cause: biallelic mutations in MUTYH (mutY) - Symptoms: Colon cancer (fastest replicating cells)
What does MUTYH (mutY homolog) do?
1: It removes A opposite an 8 - hydroxyguanine.
2: Doesn’t remove the 8-hydroxyguanine, so doesn’t fix the actual problem, just delays it
3: It must also be able to recognize strand specificity, because it doesn’t remove 8 - hydroxyguanine triphosphates added opposite an A during replication
IDLs:
- Stands for?
- Associated with?
- Stands for?
Insertion deletion loops (microsatellite) - Associated with?
MMR
