Exam 2-4 Flashcards
What is a mutation?
It is a stable change in DNA structure.
It can happen via:
- Heritable change in DNA structure
- Permanent alteration of genetic information
- DNA damage that has escaped repair.
What are point mutation?
One base substituted for another.
Also known as base substitutions.
What are insertions and deletions?
The removal/addition of a base, causing a frameshift.
What are the two types of point mutations?
- Transition Mutations
2. Transversion Mutations
What is a transition mutation?
A type of point mutation.
A to G or C to T
Purine -> Purine
Pyrimidine -> Pyrimidine
What is a transversion mutation?
A type of point mutation.
A/G -> C/T
Purine to Pyrimidine
What are four types of consequences seen with point mutations?
- Missense Mutation
- Nonsense Mutation
- Silent/Sense Mutation
- Insertion/Deletion
What is a missense mutation?
It results in one wrong codon and one wrong amino acid.
What is a nonsense mutation?
A change in the base sequence that results in a STOP CODON. The protein will prematurely terminated.
What is a silent/sense mutation?
A change in the DNA sequence that results in a new codon that codes for the SAME amino acid.
What is frameshift mutation?
Causes the DNA to shift down or up a row.
What are three ways that mutations can arise?
- Mistakes in DNA replication.
- DNA subject to spontaneous damage (i.e. deamination or depurination)
- Induced damage- by environmental agents (i.e. chemical mutagens or UV radiation)
What are some examples of Deamination?
C -> U - now pairs with A
A -> Hypoxanthine - now pairs with C
C -> Xanthine - now pairs with C
What is depurination/depyrimidination?
It removes purine/pyrimidine bases from DNA.
What is oxygen radial damage?
Breaks sugar bonds.
What is non-enzymatic methylation?
Addition of carbon groups such as alkylation.
How does oxidative deamination occur?
Example is C -> U (spontaneous)
The primary amino groups of nucleic acids are somewhat unstable.
With the presence of H20, the amino group can be displaced changing C to U.
How does depurination/depyrimination happen?
It is spontaneous.
The glycosl bond linking DNA bases with deoxyribose is labile under physiological conditions. It happens via hydrolysis.
The loss if a purine or pyrimidine base creates an apurinic/apyrimidinic (AP) site (also known as abasic site)
What are four ways that DNA can be damaged due to environmental agents? (i.e. chemicals and energy)
- Nitrous Acid (HNO2)
- Alkylating Agents
- Intercalating Agents
- UV-induced pyrimidine dimers.
What can nitrous acid do to DNA?
Oxidative deamination.
What can alkylating agents do to DNA?
Adds a methyl group or ethyl group to bases; changing their chemical properties.
What can intercalating agents do to DNA?
Subsances such as ethidium bromide or acridine orange, distort the helical configuration of DNA, causing frameshift insertions or deletions.
These substances place themselves in between bases of DNA.
What can UV-light do to DNA?
It can cause pyrimidine dimers, which is structural damage that causes distortion.
How are pyrimdine dimers repaired?
Photolyases- they use light every to repair pyrimidine dimers.
This enzyme is not present in humans, we have to take chunks out of our DNA to remove dimers.
What is the 5-step mechanism of Photolyase?
- A blue light photon is absorbed by MTHF-polyGlu
- The excitation energy is passed to FADH in the active site.
- The excited FADH, donates an electron to the pyrimidine dimer to generate an unstable dimer radical.
- Electron rearrangement restores monomeric pyrimidines.
- Electron is transferred back to the flavin radical to regenerate FADH.
How is O6-methyl repaired back to guanine?
With the enzyme methyltransferase.
It has an active sulfur group that comes in and takes away the methyl group on guanine. This enzyme is only useable once, therefore it has to be degraded after one use. This makes it very expensive.
O-6 methyl guanine can lead to mutation because a Thymine is more likely to bind to it, instead of a Cytosine.
How is 1-Methyladenine or 3-Methylcytosine repaired back to normal?
They are through the same enzyme AlkB.
- Addition of OH group onto the methyl group.
- AlkB, Fe2+
- Alpha-kg + O2 ——> Succinate + CO2
- Intermediate step, loss of formaldehyde.
Yields Adenosine and cytosine.
What are the two types of excision repairs?
- Base Excision Repair (BER)
2. Nucleotide Excision Repair (NER)
What is base excision repair?
It fixes abnormal bases. (i.e. uracil, hypoxanthine, alkylated bases)
What is nucleotide excision repair?
It fixes large structural changes and helix distortion (i.e. pyrimidine dimers and bulky base adducts)
What are the four steps involved in base excision repair (BER)? Explain.
- DNA glycosylase (uracil glycosylase)
- cleaves the glycosidic bond between sugar and base.
- this cleavage produces an AP (apurinic/apyrimidic site)
- AP Endonuclease
- this enzyme rune across DNA to find any errors
- once it finds an error, it will cleave the phosphodiester bond next to the error
- this will produce a 3’ hydroxyl overhand (a substrate for DNA polymerase I)
- DNA Polymerase
- In this situation, DNA I doesn’t need a clamp; its going to find the 3’ hydroxyl group that is overhanding and remove bases right in front of it using 5’ -> 3’ exonuclease activity to chew up a subset of bases. Once it is repaired it will fall off. - DNA Ligase
- It will repair the nick that DNA polymerase created.
What is a good indicator that nucleotide excision repair might be used?
Distortion of the DNA molecule is a signal.
What is the 5-step mechanism of nucleotide excision repair (NER)?
- Recognition of damage by a protein complex.
- Example -> UvrA (the scanner), uses ATP hydrolysis - Two Exinucleases (excision endonucleases) bind the DNA at the site of the bulky lesion on top of the scanner that found the error.
- One cleaves the 5’ end and the other cleaves the 3/ end and the DNA segment is removed by a helicase.
- DNA Polymerase fills in the gap
- DNA Polymerase I to be specific, since it fixes short regions. - DNA Ligase seals the nick,
What is Xeroferma Pigmentosum?
It is a genetic disorder.
Sympthoms include, extreme sensitivity to sunlight and a >1000x higher risk of skin cancer.
The disorder is in the reapir of UV damage.
What will nucleotide excision repair fix?
Large structural changes and helix distortions.
i.e. pyrimidine dimers, bulky base adducts
What is a mismatch repair?
It is when both bases are OKAY, but the combo is not.
It fixes base-mismatches.
Mismatch repair in E.coli is dependent on what process and enzyme?
Mismatch repair in E.coli is dependent on DNA methylation with the use of DAM methylase.
Explain the mechanism for E.coli mismatch repair. There are 4 main steps.
Methylation directed mismatch repair in E. Coli occurs right after the replication of a new DNA strand.
- MutS recognizes mismatches and binds to the incorrect base. MutS undergoes an ATP-driven conformational change that allows it to move around the DNA molecule. MutL binds to MutS, stabilizing the complex.
- The MutS-MutL complex activates MutH, which locates a nearby methyl group on the parental strand. Since this is a newly synthesized DNA strand, it will be hemimethylated. MutH will incise the nearest unmethylated GATC from the incorrect mismatch of the new strand.
- DNA helicase II (UvrD) unwinds from the nick made by MutH in the direction of the mismatch and a single strand specific exonuclease cuts the unwound DNA.
- The gap is filled by DNA polyermase III and sealed by ligase.
What happens if DNA is not repaired before it is replicated?
It is going to cause a big problem that could lead to a mutation.
What type of repair would be needed for an unrepaired lesion?
Recombinational DNA repair
or
Error-prone Repair
DNA polymerase can’t read through damaged DNA so it will skip it, resulting in a single-stranded DNA molecule that is unpaired.
What type of repair would be needed for an unrepaired break?
Recombination DNA repair.
This would result in a double break of one DNA molecule.
What are error-prone polymerases?
They are a type of polymerase that adds on random nucleotides at the damaged area to just bypass the damage, so that DNA polymerase can continue on replication.
This is known as Translesion DNA synthesis (TLS)
TLS is part of what kind of response?
The SOS response - when DNA damage is extensive.
What is the mechanism of translesion DNA synthesis?
- DNA Polymerase III hits an area of damage, such as a pyrimidine dimer.
- The clamp and DNAPIII falls off.
- A different polymerase (DNAP IV or V) hooks on the damaged area and places random bases at the area of damage.
- The polymerase then disassociates with the DNA.
- DNAP III then reattaches with its clamp to continue synthesis.
What pathway would be used for a double stranded break if you have a sister chromatid available?
Homology-Directed Repair (using a homologous chromosome)
This will utilize a similar copy to repair the break.
This repair will most likely occur right after S phase where the cohesions are keeping the sister chromatids close to each other.
What pathway would be used for a double stranded break if you were in the G1 phase?
Non-homologous End-Joining (NHEJ)
Since you are in G-phase, a sister chromosome will not be available to help with the repair. This repair is very inaccurate.
What happens if DNA damage is extensive?
You will turn on the SOS response. A response with a complex of many proteins and enzymes that are typically not expressed.
What is RecA?
RecA is a protein that binds to a single-stranded DNA break to initiate the SOS response.
Describe the steps in order of the SOS response.
You start with a single stranded DNA molecule, which is a signal for severe damage.
- RecA recognizes this damage and coats the single-stranded DNA in what can be called as a RecA DNA polymer.
- This structure will lead to the activation of all of these different gene expressions (SOS response) - The function of RecA produces the release of the LexA repressor from its promotor site in which the SOS genes are now activated and expressed.
- The LexA undergoes proteolysis or self-degradation. - Once the DNA is repaired, LexA will bind back to its promotor site, turning off those genes.
What is the LexA repressor?
It is a protein that sits at the promotor sites of SOS genes which inhibit their expression until severe damage is detected by RecA.
It is the kEY SOS protein.
What are the four steps in the repair of ssDNA or a dsDNA break?
- Create ssDNA with a free 3’ OH
- Find homolog by strand exchange
- Extend region of strand exchange beyond initial homology.
- Resolve junction of dsDNA’s to reestablish 2 separate chromosomes.
What is main enzyme that catalyzes the production of a ssDNA with a free 3’ OH?
RecBCD Helicase/Nuclease
What roles do the subunits of RecBCD have?
Rec C -> finds the dsDNA breaks
Rec B -> Helicase (3’ -> 5’) with a nuclease
Rec D -> Helicase (5’ -> 3’)
Step 1:
Explain what RecBCD helicase/nuclease complex does.
Rec C hooks up onto the area where there is a dsDNA break or a ssDNA already present.
Both Rec B and D work at an extremely fast pace, unzipping the DNA molecule. Meanwhile this is happening, Rec B is cutting up the DNA strands via nuclease. It cuts the strand going 3’ -> 5’ at a faster rate as compared to the 5’ -> 3’ strand.
Once it reaches an area known as the “Chi site” or the “Recombinational hotspot,” it leaves that strand alone, and continues to degrade the other strand. Rec C binds to this Chi site, which will inhibit the function of Rec B, in which Rec B will now serve in protecting the Chi site.
The goal of this is to produce a molecule that is single stranded having an extension of the 3’ OH end (does not apply to ssDNA breaks).
What is the Chi sequence?
5’ - GCTGGTHH - 3’
Step 2:
Explain Holliday Junctions and finding the homologous chromosome.
RecA is going to bind to the ssDNA in search of a homolog on the other DNA molecule. RecA binds to DNA via ATP.
The RecA polymer will act to untwist the ssDNA molecule.
The untwisting of the ssDNA will allow the homologous chromosome to be integrated within it’s secondary binding site.
Base-pairing till occur until there is homologous base pairing.
This will produce a D-loop.
Once there are at least >50 base pairs, you will have DNA extension with a DNA polymerase. At the same time, the displaced strand (normal strand) will find homology with the other strand and extend as well.
This crossing over between strands creates what is called a Holliday junction.
Step 3:
Explain the extended region of strand exchange beyond initial homology.
Rec A -> starts branch migration
RuvA and RuvB -> drive the migration
RuvC -> processes the Holliday junctions into recombination products by nicking the junctions at specific sequences.
In eukaryotes, instead of RecA, what do we use?
Rad-51. It coats the ssDNA.
It cut the strand, ATM-kinase activate exo-nucleases that produce ssDNA.
