DNA repair Flashcards
Why is DNA repaired?
Well you only have 2 copies of genomic DNA per cell versus RNA/proteins where you have many copies so you can dispose and replace or just make new ones from the DNA.
DNA IS IRREPLACEABLE.
Change can lead to mutations 2 ways…
- ) Uncorrected errors in DNA replication
- ) Damage to replicating or non-replicating DNA… like oxidative damage, depurination, deamination, UV.
for extra fun: mutations in germline can be passed onto offspring
How does cancer do it?
Mutations are extra bad in components of DNA repair machineries or DNA damage sensing/signaling pathways… because this leads to more mutations and more instability…
Accumulation of changes to the DNA.
If cell can’t kill itself or stop dividing. CANCER.
Types of DNA repair (broadly)
A.) Direct reversal of the damage
B.) Excision repair: NER, BER, and MMR
C.) Lesion Bypass
D.) DSB’s repaired via NHEJ or HR
Direct Reversal of Damage
Examples:
- Ligate a break in phosphodiester backbone with DNA ligase
- Repair of O6-methylguanosine by MGMT (O6-methylguanosine methyltransferase) (this is reversing methylation of guanine bases)
You don’t need a template. You don’t break the strands. The process is specific to the type of damage.
Excision repair
Excise damaged region, precisely replace.
- ) Remove damaged area
- ) Gap filled in by DNA polymerase that uses undamaged sister strand as a template.
- ) Ligation
All excision requires: endonuclease and/or exonuclease, DNA polymerase, and DNA ligase.
Nucleotide Excision Repair (NER)
Removes DNA lesions that distort DNA structure and block RNA or DNA polymerase movement on the DNA.
ex: thymine dimers caused by UV and DNA adducts caused by carcinogen exposure
1. ) Damage recognized by a multi-protein complex that has endonuclease activities
2. ) Cut one strand through backbone on either side of the lesion
3. ) DNA helicase unwinds the DNA and releases the single-strand oligonucleotide with the lesion
5. ) Fragment eaten by exonuclease
6. ) DNA polymerase fills in complimentary nucleotides
7. ) Ligase seals it
You need 30 diff proteins for this! Lesions are either recognized via: (but rest of process same)
- Global genome NER (distorting lesions anywhere)
- Transcription-coupled NER (distorting lesions in areas actively transcribed)
Examples of NER genes in disease
Cockaynes
Xeroderma Pigmentosum (XP)
Trichiothiodystrophy
Base Excision Repair
Removes DNA lesions missed by NER, but do NOT necessarily block polymerase fx or do NOT distort DNA structure.
Requires: GLYCOSYLASES (each recognize a specific type of altered base).
Ex: uracil glycosylase recognizes uracils in DNA that come from cytosine deamination, 5-methylcytosine-DNA glycosylase recognizes 5-methylcytosine to initiate DNA demethylation
Glycosylase flips the altered base out from the double-strand helix and hydrolyzes the N-glycosidic bond (between base and sugar) to REMOVE the damaged base
Produces apurinic or apyrimidinic (AP) site
AP site is removed by an AP-specific endonuclease and an AP lyase
Resulting gap is filled by DNA polymerase
Nick sealed by ligase
Mismatch repair (MMR)
Fixes errors in nucleotide incorporation made by DNA polymerase during DNA replication.
Mismatched base pair is recognized shortly after DNA synthesis by the MutS and MutL proteins in bacteria (in humans it’s MSH and MLH)
How do bacteria know which strand is new? it’s not yet methylated.
Endonuclease cleaves phosphodiester backbone of new strand. Exonuclease chews away the new DNA strand including mismatched while helices helps unwind.
DNA polymerase repairs single strand gap with complimentary base pairs, DNA ligase seals the phosphodiester backbone.
SUPER CRITICAL to remove the wrongly inserted base on new strand and not its mismatching partner on parents strand. So this is why it only removes portion of newly synthesized with the bad base.
How do humans know which one is new? DNA nicks are more abundant on the newly replicated strand
Disease caused by MMR issues?
HNPCC
Lesion Bypass
So much damage of the kind that normally blocks DNA replication (like UV thymidine dimers) that cell can’t fix it all, cells use lesion bypass (same as translation synthesis)
It allows cells to keep replicating/dividing in face of damage. But it is very mutagenic because
alternate DNA polymerases that LACK 3’ to 5’ proofreading exonuclease activity are used to replicate past the lesion.
Result is an error rate 100-10,000 higher than normal dan replication!
DSB’s and NHEJ and HR
Homologous recombo: requires extensive sequence homology between broken ends and template DNA, is generally accurate.
NHEJ: need no sequence homology b/w 2 broken ends. Which often leads to insertion or extensive deletion of nucleotides at the breakpoint.
Choice between HR and NHEJ varies depending on cell types and cell cycle stages. (which proteins are present in what cell type…)
HR and NHEJ may play overlapping roles. Cooperate to maintain genome integrity/promote survival.
DNA damage checkpoint
surveillance mechanism that halts cell cycle progression to give time for repair.
Signaling pathway made of damage sensors, signal transducers, and effectors.
CENTRAL to lesion detection is a pair of homologous protein kinases: ATM and ATR.
They are recruited to damage site and initiate sequential recruitment and activation of downstream proteins.
Get activated in human cells undergoing earlier stages of tumorigenesis to delay/prevent cancer.
Mutations in ATM/ATR: instability. Malignancy.
3 steps for cancer?
Mutation or mismatching event in gene of self-proliferation
Repair mechanisms miss it or are overwhelmed by too many of these events
Self-destruction pathways can’t be activation (mutated)
