Physiology 3140A Lecture Notes - Lecture 7: Dna Mismatch Repair, Dna Adduct, Stress (Mechanics)
22 May 2018
School
Department
Course
Professor
Physiology 3140
Dr. Pin
DNA Repair
DNA DAMAGE AND REPAIR
Sources of DNA damage:
- Normal transcription, proliferation, etc.
o Suggested that up to one million DNA lesions occur every day. Great majority are fixed
o Transcription (you are always opening up the DNA to do transcription and that is going
to cause single nucleotide breaks)
- Environmental agents
o UV light (radiation)
o Chemical exposure
o Chemotherapy
o Free radicals
- Internal stress
o Inflammation
o Reactive Oxygen Species (ROS)
- Errors in replication
- Can lead to genomic instability, apoptosis, or senescence
- Genomic instability can predispose individuals to cancer, neurological disorders and
cardiovascular diseases
What kind of DNA damage can occur?
- Each one of these DNA damages that could happen
opposes a unique problem to the cell
- DNA‐repair pathways
- Several DNA‐repair pathways exist and deal with various
types of DNA insults.
- These pathways include
1. the direct reversal pathway
▪ for modified nucleotides
2. the MMR pathway
▪ for DNA mismatches
3. the NER pathway
▪ for DNA adducts - where you get the adjacent bp interacting with eachother as
opposed to the opposing bp interacting with eachother
▪ this also happens if there is an insertion of a bp that shouldnt be there
4. the BER pathway
▪ for missing nucleotides
5. the HR pathway (when there is homologous combination)
6. the NHEJ pathway
▪ for double stranded breaks
What are the steps required for repairing DNA? (EXAM QUESTION!!!!!)
1. Identify the mistake – type, extent, etc.
2. Remove the mistake – mismatch, unmatched base pair, non-cohesive ends
3. Trim back the DNA backbone (not in all cases – but in most cases this is done so you have a
cleaner sequence to repair)
4. Repair the sequence (in the ideal situation, you repair the sequence perfectly, but in many cases,
you dont, you dont – you just slap something in there and hope for the best)
5. Ligate the backbone of DNA
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find more resources at oneclass.com
Direct Reversal
- Usually stimulated by an inappropriate modification
o So it doesnt just go along and recognize methyl groups that are there (a lot of the
example show this as what it is focusing on but its not just that)
- Single step process with no excision of nucleotides
- Only DNA repair mechanism that doesnt require removing a nucleotide or breaking the DNA
- Multiple pathways that are specific to the modified nucleotide
o For every single type of modification that there is, there is a pathway with a specific
enzyme that can do this
o This is great for specificity but sometimes when you are scanning the bp, you want more
of a one size fits all
- This is an inefficient process
o If you had 20 diff complexes going through the genome trying to figure out what is
wrong, that is much less efficient than having one that can recognize multiple
- Example: mammalian O6‐methylguanine‐DNA methyltransferase MGMT removes DNA adducts
by transferring alkyl group to a cysteine
o So in the image, there is an inappropriate methyl group →MGMT comes in and causes
the methyl group to be transferred from the DNA onto the cysteine group and then it is
fixed
- Example: AlkB can repair other modified nucleotides – instead of transferring it onto a cysteine,
it transfers it to a hydroxy group
o There are many different types of single modifications that can occur this way
Mismatch repair
- How do you recognize the mismatch?
o You probably had something that was running along the DNA and it got to a bump in the
road (bc the DNA is set up nice and smooth but the bump is caused bc there is wrong
interactions – theres steric hinderance)
- Repairs mismatched bp that may arise from mistakes in DNA replication (might have an
inappropropriate insertion as you are bringing in bp)
- Failure to repair the mismatch results in microsatellite instability
o The reason why G interacts with C and A interacts with T is because that is the most
stable interaction
o If you start putting in other things, you are creating this instability that is going to create
this small area that will be targeted for more damage
o If this area happens to be in the middle of a gene or a promoter it can wipe out that
entire gene
- MUTS and MUTL bind and recognize the mismatch DNA
- MUTL and MUTS causes an incision (nick) in the DNA and recruit EXO1
- Exonuclease (EXO) removes the mismatched nucleotide (trims one strand of the DNA)
- EXO strips down that one strand of DNA and allows there to be a template for repair
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find more resources at oneclass.com
- DNA polymerase restores the correct nucleotide and then DNA ligase joins restores the DNA
backbone
- What can go wrong here?
o There can be a point mutation – DNA replacement
▪ If you use the template of the right sequence, then you get the right sequence
(the original sequence)
▪ But this complex doesnt know which direction, it doesnt know which is the
correct bp, it is just coming in and replacing
▪ So this is where you can put in a point mutation (change in DNA)
▪ Point mutations may have no effect or they may have an effect
Base Excision Repair
- Removes bp in response to something more than a mismatch (repaired modified basepair of
nucleotides)
- Can be in 2 different ways: in a short way where it only removes 1 or 2
nucleotides or it can remove a long section
- Repair of modified base pairs or nucleotides which is the most common DNA
insult
- Typically repairs a single nucleotide (short patch), but can replace up to 13
nucleotides (long patch)
o Recognition and removal of the damaged nucleotide by glycosylases
o You now end with a single base pair (this causes instability) so you get
recruitment of endonuclease APE1
▪ Note: endonuclease cuts inside and exonuclease cuts outside
o So the endonuclease APE1 recognizes something where the backbone is
still intact (this is why we use an endonuclease instead of an
exonuclease)
o Strand incision by endonuclease APE1
o Nucleotide(s) inserted by DNA polymerase
o Ligation of DNA
find more resources at oneclass.com
find more resources at oneclass.com
