DNA Modification And Repair Flashcards
Form of normal DNA modification
Methylation
What are the major methylated bases in prokaryotes?
Adenine and Cytosine
-happens after replication
N6 methyladenine
N4 methylcytosine
What is the role of methylation in bacteria?
Methylation in bacteria occurs at specific sites
Protects the bacteria’s DNA from cleavage by restriction endonucleases
What is the functional importance of methylation of adenine in E.coli?
Methylation of adenine residues in the sequence GATC is involved in mismatch error correction
Can be on either A or C or both
Cytosine in Eukaryotes
The only normal base modification in eukaryotes
5-methylcytosine
3-5% cytosine content of most DNA
Where is the methylation present in Eukaryotes?
5-methylcytosine is usually found in C residues 5’ to G
When a C in one strand is methylated, the C in the complementary strand is also methylated
What does it mean for methylation in eukaryotes to be heritable?
1) Sites of new methylation can be selected during gametogenesis by a de novo methylase
2) Not all C’s are methylated
3) fertilization and replication
4) Methylation is carried out by a maintenance methylase after replication
How does eukaryotic methylation leave room for error recognition?
Daughter strands not immediately methylated after replication –> so you can do error repair earlier on
How does methylation control gene expression?
Unmethylated promoters expressed to produce the protein
Methylated promoters are inactive and are not expressed
What is 5-azacytidine?
Inactivity of methylated genes reversed by treatment of 5-azacytidine
5-azacytidine is a cytosine analog that can be metabolized into dCTP and incorporated into DNA
How does 5-azacytidine work as a potential treatment for beta-thalassemia?
1) a normal methylated DNA goes through replication in 5-azacytidine
2) The cystosine in the daughter strands are actually 5-azacytidine
3) Replication of these new DNA leads to methylation after
4) one daughter DNA is normally methylated (due to the original methylation on that strand)
5) one daughter DNA has a loss of methylation leading to gene expression
Factors that cause DNA mutation
- Mistakes during replication
- Reactive oxygen species (ROS)
- Chemical damage
- Radiation
- ionizing agents
- Deamination of a cytosine or 5-methylcytosine
What base does a deamination of 5-methylcytosine (5mC) lead to?
It become thymine (Bc of that CH3 on C5)
Would become uracil if not methylated cytosine
How does 5-methylcytosine cause a transition mutation?
1) Deamination of methylated cytosine
2) Changed to a thymine
3) Replication of this leads to a daughter DNA which makes an originally what should be a GC base pair become an AT base pair
4) one daughter DNA is normal
Why is Deamination of 5-methylcytosine seen as dangerous if not repaired?
Thymine is not seen as an abnormal base by repair mechanisms but it’s legit changing the gene sequence that the DNA should be encoding
What is a transition point mutation?
Purine-pyramidine base pair changed into a different purine-pyramidine base pair
Ie: CG –> TA
What is a transverse on point mutation?
A purine-pyramidine base pair is changed into a pyrimidine-purine base pair
Ie: AT —> TA
Deletion or insertion mutations- causes and examples
Addition or removal of one or more base pairs
Caused by intercalating agents that fit in between adjacent base pairs of the double helix
Ie: ethidium bromide used in labs or doxorubicin used to treat lymphoma
Can also be caused by transposable elects, errors in replication, and repeating elements such as the triplet repeats
How does an insertion/deletion mutation work?
DNA is a 3 base code in which 3 bases code for 1 amino acid
Insertion/deletion of 1 nucleotide into a gene shifts the reading frame
What does photodimerization result in?
Results in infra strand dimerization of adjacent thymines
Catalyzed by UV light
Usually dimerizes pyrimidines: TT, CC, CT
What are the 2 sorts of dimers that can result?
1) cyclobutane thymine dimer (bonds are 2 vertical lines between C6-C6 and C5-5 on adjacent thymines
2) 6-4 Photoproduct (bond is diagonal between C6-C4 on adjacent thymines)
What is the main target for base damage/modification?
Primarily Purines
Can be lethal if not treated
How does O6-alkylguanine work?
Has a high probability of being base paired with thymine during replication
This leads to a GC to AT transition if not repaired
Repair of O6-alkylguanine? - prokaryotes and eukaryotes
O6-alkylguanine [or methylguanine] DNA methyl transferase (MGMT) transfers the methyl from O6- methylguanine to itself
MGMT self alkylates and restores the guanine
if you can change the guanine before replication, you can change the base pairing back to C
Excision repair- basic idea
Damaged DNA is recognized, removed, and then replaced by DNA polymerase
Nucleotide excision repair (NER)
Repairs intra strand thymine dimers caused by UV irradiation
1) UvrAB complex scans DNA 3’ –> 5’
2) Damange DNA site causes UvrAB complex to stop
3) UvrAB complex bends DNA
4) Dissociation of UVrA and binding of UvrC to UvrB
5) UvrBC complex endonuclease activity cuts both sides of the dimer
6) Helicase unwinds damaged DNA
7) Polymerase 1 fills in the gap
8) backbone rejoined by DNA ligase
Function of DNA-N-glycosylases
They remove incorrect bases in DNA
Ie: Uracil that can appear as a result of Deamination of cytosine Deamination –> can cause a GC to AT transition mutation
Base Excision Repair of mismatched uracil by Uracil DNA N-glycosylase (prokaryotes)
1) Recognition of damage
2) Base removed from back bone by Uracil-DNA N-glycosylase
3) Endonuclease cute backbone 5’ to the damage
4) Nick translation by DNA polymerase I–> DNA pol I ends up replacing more than 1 base because of nick translation
5) Nick in backbone sealed by DNA ligase
What is a apyrimidinic site?
A site without a pyrimidine base Bc it easy removed as seen in base excision repair for mismatched uracil
2 BER pathways for eukaryotes
Short patch BER similar mechanism to prokaryotes
Long patch BER takes over if 5’ to abasic site is refractive to cleavage by DNA pol epsilon
How does mismatch repair work?
During replication DNA proofread for mistakes by DNA polymerase III epsilon subunit
Nacent DNA also scanned for errors- mismatched bases and single base insertions or deletions
If found corrected by mismatch repair.
Mismatch repair- MutHLS system
1) Recognition of damage by MutS
2) Using ATP hydrolysis for energy, MutS pulls DNA in from both directions until reaching a GATC sequence, the sequence in E.coli where A is methylated
3) MutH Endonuclease cleaves the unmethylated DNA strand 5’ to the G in the GATC sequence
4) DNA Helicase II unwinds the DNA back past the mismatch
5) An exonuclease then removes the damaged DNA
6) DNA polymerase III and DNA ligase then fill the gap and seal the nick
MutS, MutL, MutH
MutS- DNA mismatch/damage recognition
MutL- molecular matchmaker; Endonuclease; termination of mismatch-provoked excision
MutH- strand discrimination
CMMRD
Constitutional mismatch repair deficiency
Rare autosomal recessive syndrome caused by homozygous mutations in mismatch repair genes
Nucleotide excision repair in eukaryotes- Global genome
Probes the genome for helix-distorting lesions
Nucleotide excision repair in eukaryotes- transcription coupled
Removes transcription blocking lesions to permit unperturbed gene expression
