Physiology 3140A Lecture Notes - Lecture 31: Reactive Oxygen Species, Dna Mismatch Repair, Microsatellite Instability
2 May 2018
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Cell Physiology Lecture 31
DNA Damage and Repair
- DNA damage and repair is localized in certain regions of the genome for efficiency
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 Every time you replicate DNA
o Transcription – proteins coming in and unwinding DNA
▪ Causes single or small nucleotide breaks
- Environmental agents:
o UV light
o Chemical exposure
o Chemotherapy
- Internal stress
o Inflammation
o Reactive Oxygen Species (ROS)
- Can lead to genomic instability, apoptosis, or senescence IF they are not fixed
- Genomic instability can predispose individuals to cancer, neurological disorders and cardiovascular
diseases
o Once you get genomic instability in the cell, you get a global phenomenon in the way the
genes are expressed
o Initiating factor in many types of cancer
What kind of DNA damage can occur?
- Types of breaks:
o Modified nucleotide
o Mismatched nucleotide
o Missing nucleotide
o Double stranded break
o DNA adduct – adjacent base pair interaction instead of opposing
- Each one type of DNA damage poses a unique problem to the cell that you HAVE to fix
- Multiple mechanisms for DNA repair
find more resources at oneclass.com
find more resources at oneclass.com
- DNA‐repair pathways. Several DNA‐repair pathways exist and deal with various types of DNA insults.
These pathways include (1) the direct reversal pathway, (2) the MMR pathway, (3) the NER pathway,
(4) the BER pathway, (5) the HR pathway, and (6) the NHEJ pathway.
- Double stranded repairs require non-homologous end joining th
What are the steps required for repairing DNA?
1. Identify the mistake – type, extent, etc.
o Recruit right types of proteins that are going to be there
o Recruit what is needed to repair
2. Remove the mistake – mismatch, unmatched base pair, non-cohesive ends
3. Trim back the DNA backbone
o Have a cleaner area to repair
4. Repair the sequence
o Want to restore it perfectly – many cases you do not
5. Ligate the backbone of DNA
o If it does not – becomes another site for DNA damage to occur
1. Direct Reversal:
- Stimulated by: inappropriate modifications
o NOT JUST METHYLATION
- Single step process with no excision of nucleotides
o ONLY DNA repair mechanism that does not recruit removing the nucleotide (no breakage to
DNA or excision of nucleotides)
- Multiple pathways that are specific to the modified nucleotide
o For every modification, there is a pathway that can do this = SPECIFICITY
▪ )f you are scanning many base pairs, you want more of a one size fits all do NER
or BER
- Mammalian O6‐methylguanine‐DNA methyltransferase MGMT removes DNA adducts by
transferring alkyl group to a cysteine
o Remove modification by taking an alkyl group off DNA and putting it to a recipient
o Causes methyl group to be transferred from the DNA to the cysteine group
- AlkBH can repair other modified nucleotides
o Transfers the methyl group to a hydroxyl group
- If it is a modified nucleotide, you can use direct reversal (DOES NOT ALWAYS WORK)
o When you are trying to remove a methyl group, you do not simply just modify the nucleotide
because 1) it is harder to identify & 2) requires other proteins
▪ NOT THE COMMON METHOD USED TO REMOVE METHYL GROUP (usually when
you are removing a methyl group you are doing it through a different pathway)
- INEFFICIENT PROCESS; if you had 20 different complexes going throughout genome is less efficient
that one that can recognize many
find more resources at oneclass.com
find more resources at oneclass.com
2. Mismatch repair:
- Repairs mismatched bp that may arise from mistakes in DNA replication
- Failure results in microsatellite instability
o Causes instability in that region of the genome
o Area is now targeted for more damage
o If it is in the middle of a gene or promoter – wipes out the gene
- 1) DNA mismatches are recognized by MUTS and MUTL
o MUTS + MUTL bind and recognize the mismatch + cause repair
- 2) MUTL causes an incision (nick) in the DNA
- 3) Exonuclease (EXO) removes the mismatched nucleotide
o EXO recruited and trims back one strand of the DNA allowing there to be a
template (other strand not digested, acting as template for repair)
o Exo = outside
▪ Backbone was not intact
- 4) DNA polymerase restores the correct nucleotide and then DNA ligase joins restores the
DNA backbone
- How do you recognize mismatch? Something runs along the DNA & when it got to a bump
due to wrong interactions (steric hindrance of improper interaction causes bump)
- WHAT CAN GO WRONG?
o Point mutation – complex does not know which right bp and right one is, just
comes in, removes and replaces
▪ MAY or MAY not have an impact
3. Base Excision Repair:
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)
A) Removal of the damaged nucleotide by glycosylases
- End up with a single base pair not align with anything
B) Strand incision by endonuclease APE1
- Endonuclease DNA where the backbone is still intact
- Endo = inside
C) Nucleotide(s) inserted by DNA polymerase
D) Ligation of DNA
Remove basepairs in response to more than just a mismatch
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Dna damage and repair is localized in certain regions of the genome for efficiency. Normal transcription, proliferation, etc: suggested that up to one million dna lesions occur every day. Great majority are fixed: every time you replicate dna, transcription proteins coming in and unwinding dna, causes single or small nucleotide breaks. Environmental agents: uv light, chemical exposure, chemotherapy. Can lead to genomic instability, apoptosis, or senescence if they are not fixed. Genomic instability can predispose individuals to cancer, neurological disorders and cardiovascular diseases: once you get genomic instability in the cell, you get a global phenomenon in the way the genes are expressed. Types of breaks: modified nucleotide, mismatched nucleotide, missing nucleotide, double stranded break. Each one type of dna damage poses a unique problem to the cell that you have to fix: dna adduct adjacent base pair interaction instead of opposing. Several dna repair pathways exist and deal with various types of dna insults.
