DNA Repair Flashcards
1
Q
DNA damage vs DNA mutation
A
- DNA damage is initial alteration of normal DNA structure
- If cells fail to repair damage -> become DNA mutation
- Cells cannot recognize DNA mutation as incorrect and remain permanent change in sequence -> can become inherited
2
Q
Possible outcomes when cytosine deaminates to uracil
A
- Successful DNA repair: Repair proteins recognize that uracil does not belong and attempt to repair before DNA replication. If successful repair, replace with correct nucleotide
- Unsuccessful DNA repair: Undergoes round of replication, one daughter DNA uses non-damaged strand as template and other uses damaged strand as template -> adenine to guanine mutation -> repair proteins cannot recognize adenine but recognize uracil and replace with incorrect nucleotide (T)
- No DNA repair: Undergoes second round of replication. One daughter DNA uses damaged strand as template -> mutation in newly synthesized strand -> unsuccessful repair. Second daughter DNA use mutation as template, result in parallel mutation in newly synthesized strand -> recognize as normal and no repair takes place
3
Q
Depurination and depyrimidation
A
- Endogenous and spontaneous
- Spontaneous hydrolysis of nucleotides N-glycosidic bond (links sugar to base) -> cleavage releases base from nucleotide
- creates apurinic/apyrimidic site (AP)
- Loss of information -> if polymerase encounters AP site, replication stop (relies on H-bonding properties of bases)
- Use trans-lesion synthesis polymerase (TLS) to bypass abasic site -> incorporates random nucleotide, high error rate not ideal
4
Q
Cleavage of DNA backbone
A
- Can be caused by abasic sites
- Cyclic form turned into linear aldehyde form
- ## Linear aldehyde form subjected to beta elimination: cleavage of DNA backbone, breaking of phosphodiester bond of abasic site -> yields 3’ aldehyde terminated product, no available 3’-OH group to facilitate repair process
5
Q
Deamination
A
- Endogenous and Spontaneous
- Occur on A, G, C: convert amine group to carbonyl group
- Affects base pairing properties, amine group act as hydrogen bond donor and carbonyl group act as hydrogen bond acceptor
- Can lead to incorrect base pairing during replication and cause mutations
6
Q
Oxidative damage
A
- Endogenous and spontaneous
- Reactive oxygen species produced from mitochondria during oxidative phosphorylation
- Electrons that leak out of ETC bind to oxygen and form superoxide anions -> restricted to mitochondria
- Superoxide dismutase (SOD) convert superoxide anions to membrane permeable -> reduce to form hydroxyl radical which can cause DNA damage
7
Q
Effects of oxidative damage: 8-oxo-G
A
- Oxidized form of guanine
- Addition of carbonyl group to 8 position of guanine (not used as H-bond acceptor or donator)
- Causes steric clashing with ribose sugar -> favors syn conformation -> base pair with adenine
8
Q
UV damage
A
- Exogenous
- Occurs when pyrimidine bases absorb UV light causing double bond in pyrimidine ring to open
- If two pyrimidines adjacent, form cyclobutene pyrimidine dimer(CPD)
- If single bond formed between two adjacent, 6-4 photoproduct is formed
- Shortening of distance cause generation of noticeable kink
- TLS inserts AA opposite to any pyrimidine dimer
- If dimer made of TT, no mutagenesis
- If made of CC, CT, TC, mutation formed
9
Q
Alkylation damage
A
- Exogenous
- Alkyl group transferred to nitrogen or oxygen atom of nitrogenous base
- Cause alteration in base pairing properties
- Transfer of methyl group from donor (SAM) to base
10
Q
O6 methyl guanine
A
- O6 normally acts as hydrogen base acceptor to cytosine. Methylation causes it lose ability to hydrogen bond
- N1 transitions from hydrogen bond donor to hydrogen bond acceptor
- Allows to form two hydrogen bonds with either cytosine or thymine via hoogsteen base pairing
- Randomness in DNA replication cause 50% chance of mutation
11
Q
How prevalent is DNA damage in cell
A
- Depurination: 10,000 lesions/cell/day
- Depyrimidation: 500 lesion/cell/day
- Deamination: 100 lesions/cell/day
- Alkylation: 5000 lesions/cell/day
- UV radiation: 100,000 lesions/cell/day
12
Q
Direct Repair
A
- Chemically reverse damage done to nitrogenous bases
- O6-methylguanine repaired by MGMT
- MGMT flips base out of double helix into active site through interdigitation. Opposite base pair becomes “orphan nucleotide”. MGMT’s arginine residue stabilizes double helix while O6-methylguanine undergoes repair
- Sulfur atom of key cysteine 145 residue binds to methyl group and removes it. Guanine base reinserted
- S methyl bond formed in double helix is irreversible so MGMT is degraded, referred to as suicide enzymes
13
Q
Base Excision Repair
A
- Can recognize individually damaged nucleotides such as deamination, methylation, and oxidation
- Damaged bases removed from DNA and replaced with new nucleotide using different enzymes
- Single base modified due to DNA damage
- specific DNA glycosylase recognize damaged base and cleave N-glycosidic bond to remove base
- 5’AP endonuclease makes nick in DNA backbone to generate 3’OH and 5’deoxyribose monophosphate
- Template strand identifies missing base and DNA polymerase adds onto free 3’OH. DNA ligase seals gap
14
Q
Nucleotide Excision Repair
A
- Recognize distortions in during DNA helix structure such as pyrimidine dimers
- Repair bulky alkylations, dimer formations, and novel lesions
- XPC-HR23B recognizes and bind to DNA double helix, flips out several bases in distorted region increasing severity of DNA kink -> attracts TFIIH
- TFIIH composed of XBP and XPD and unwinds DNA strands in opposite directions using ATP. Result in formation of single stranded DNA loop, one subunit encounters lesion and stop, other subunit keep unwinding for 20 nucleotides
- Undamaged strand protected by single strand binding protein RPA
- XPG + XPA nuclease binds 3’ of loop and XPF binds 5’ of loop. Excise lesion and additional nucleotides on either side
- Replisome fills gap
15
Q
Mismatch repair
A
- Fixes mistakes from DNA polymerase
- Use methylation (epigenetic mark) after replication complete
- Unwind DNA -> endonucleases cleave phosphodiester bond -> replisome replaces nucleotides -> DNA ligase seals phosphodiester backbone
