Topic 6 (DNA Damage and Repair) Flashcards
Define DNA damage
Any modification of DNA that changes its coding properties or normal function in transcription or translation
What can DNA damage lead to?
Mutations and genetic instability
True/False? Mutations arise solely by chance
False. They may be induced by chemicals and UV as well
What is the evolutionary tradeoff of mutations?
They may increase fatality or may increase biodiversity
What are the three types of DNA damage?
Mutation, recombination, and transposons
Why is recombination a way in which the genome can change?
Unequal crossing over results in gene deletion/duplication
What are the two mechanisms inducing DNA mutations?
Replication errors and chemical modifications
What are examples of chemical modifications to DNA which result in mutations?
Deamination, depurination/depyrimidination, oxidation, alkylation, nitrous acid, radiation, intercalating agents, and base analogs
What are the types of mutations that can occur due to errors in DNA replication?
Point mutations and frameshift mutations
Give 3 examples of point mutations
Missense, nonsense, and silent
Give 2 examples of frameshift mutations. How does each occur?
Deletion; slippage of template during replication
Insertion; slippage of daughter strand during replication
In what tautomeric form are Adenine and Cytosine found in usually?
Amino
In what tautomeric form are Guanine and Thymine found in usually?
Keto
How many hydrogen bond donors and acceptors are there in an amino to imino tautomerization?
Amino: 2 acceptors, 1 donor
Imino: 2 acceptors, 1 donor (different locations)
How many hydrogen bond donors and acceptors are there in a keto to enol tautomerization?
Keto: 2 donors, 1 acceptor
Enol: 2 donors, 1 acceptor (different location)
What changes about the hydrogen bond donors/acceptors in tautomerization?
Two donors/acceptors switch to the other kind (donor to acceptor and acceptor to donor), so number of donors and acceptors stays the same
The enol form of T can base pair with: __________. What does this form following 2 cycles of replication? Transition or transversion?
Keto form of G; TA to CG; transition
The enol form of G can base pair with: __________. What does this form following 2 cycles of replication? Transition or transversion?
Keto form of T; GC to AT; transition
The imino form of A can base pair with: __________. What does this form following 2 cycles of replication? Transition or transversion?
Amino form of C; AT to GC; transition
The imino form of C can base pair with: __________. What does this form following 2 cycles of replication? Transition or transversion?
Amino form of A; CG to TA; transition
What is a transition mutation?
Purine to purine/pyrimidine to pyrimidine (CG to TA)
What is a transversion mutation?
Purine to pyrimidine/pyrimidine to purine (CG to GC or AT)
What is a lesion vs a mutation?
Lesion is a single base change but its still paired with the correct base. Mutation is a double-stranded discrepency
What are the steps in creating point mutations?
- Incorrect nucleotide incorporated by DNA Pol (lesion)
- Mismatched base is not repaired and undergoes replication (mutation)
What is a K53X?
Lysine 53 changed to a stop codon (nonsense mutation)
What are the three stop codons? What colour name is associated with each?
UAA; ochre
UGA; opal
UAG; amber
Why is a mutation at codon 53 on the mature transcript at position 257 on genomic DNA?
Genomic DNA includes introns
What is an E6V mutation? What sickness is associated with this?
Glutamate 6 to Valine is a missense mutation; sickle cell anemia
What are indels caused by?
Aberrant DNA recombination or DNA Pol slippage during replication
What is a reading frame?
A contiguous, non-overlapping three-nucleotide codon in DNA or RNA
What is a frameshift mutation?
A mutation that changes the reading frame (3bp mutation/deletion preserve reading frame)
What are trinucleotide repeat/expansion disorders? Give an example
Expansion of repeats of CAG, CGG, GAA, and CTG; Huntington’s disease (expansion of CAG)
What is fragile X syndrome caused by?
Expanded CGG repeats in FMR1 (200-1000 results in disease)
What strategy can be used to determine the number of triplet repeats in the fragile X locus?
Gel electrophoresis (larger band = more repeats)
In what 4 ways can chemical damage occur to DNA?
- Hydrolysis of water (depurination/depyrimidination)
- Electrophilic attack of backbone by alkylating agents
- ROS (hydrogen peroxide hydroxyl radicals, etc.)
- Irradiation
What is deamination?
Removal of an amine group
Which bases can be deaminated? Why?
CAG all have amine groups
Deamination of C into (product) causes a (transition/transversion) into (new pairing)
U; transition; CG to TA
Deamination of A into (product) causes a (transition/transversion) into (new pairing)
Hypoxanthine; transition; AT to GC
Deamination of G into (product) causes a (transition/transversion) into (new pairing)
Xanthine; no change; no change
Deamination of 5-Me-C into (product) causes a (transition/transversion) into (new pairing)
T; transition; CG to TA
What is the biproduct of deamination?
Ammonia
DNA hydrolysis causes what kind of chemical damage?
Depurination/depyrimidination
What causes depurination/depyrimidination? What is the product?
Hydrolysis of the glycosidic bond; apurinic/apyrimidinic site
What is the result of a depurination/depyrimidination?
Base loss or base pair transition
What may occur in an abasic site?
Sugar backbone isomerizes to the open aldehyde form, which is unstable and vulnerable to nucleophilic attack
What produces ROS?
Metabolism, cellular respiration, radiation
Oxidation of T by ROS produces (product) which (impact)
Thymine glycol; blocks DNA Pol due to steric hindrance
Oxidation of G by ROS produces (product) which (impact)
8-oxo-G; pairs with A (transversion), GC to TA
What are the highly reactive sites of A and G for alkylating damage?
N3 and O6, respectively
Where are alkyl groups added to bases?
Nucleophilic positions
Methylation of A by SAM results in?
DNA functioning like RNA
Nitrous acid causes:
Deamination of CAG (NOT 5-Me-C)
Sodium nitrate and bisulfite are _______ that cause _______
Food preservatives; deamination
True/False? Chemical modifications cannot occur simultaneously on a single base due to steric hindrance
False. Multiple modifications may occur simultaneously on a single base
Which modifications may occur to a single nucleotide base simultaneously?
Oxidation, alkylation, and deamination
What is the likelihood of a T-T CPD forming over a C-C CPD?
T-T»T-C>C-T>C-C
What causes CPDs?
UV radiation
In the case of T-C and C-C dimers, what are these called instead of CPDs?
6-4PPs, or 6-4 pyrimidone photoproducts
What is a CPD?
Cyclobutane pyrimidine dimer caused by the condensation of two double-bonded C5:C6 atoms on adjacent pyrimidine dimers
What is the most common CPD?
T-T (thymine dimer)
What does it mean when CPDs and 6-4PPs are called intra-strand crosslinks?
They form chemical bonds within their own strand, not the complimentary strand
What happens to the shape of the DNA when a thymine dimer forms?
It introduces a bend/kink in the DNA
What happens to DNA replication machinery upon encountering a pyrimidine dimer?
DNA Pol stalls
In the presence of radical O2, how is DNA radiosensitivity effected?
Increased
What does ionizing radiation cause the formation of? What is the most important species and how is it formed?
Excited and ionized molecules; ROS (H2O2, OH radical, O2 radical) formed by radiolysis of water
What can ROS cause?
Base loss, strand breaks, and DNA-protein crosslinks
What are intercalating agents?
Chemical that mimics base pairs and inserts between stacked bases, which destabilizes the DNA helix, which then causes frame shift mutations by making indels
Provide 4 examples of intercalating agents
Proflavin, acridine orange, ICR-191, and ethidium bromide
What is hydroxylation?
The addition of a hydroxyl group to the amino of cytosine
Hydroxylation of cytosine produces:
Hydroxylaminocytosine (HC)
Hydroxylaminocytosine forms hydrogen bonds with (base), resulting in a (base pair) after two rounds of replication. This is a (transition or transversion)
A; C-G to T-A; Transition
The keto form of 5-BrU base pairs with (base), causing a (base pair) after two rounds of replication. This is a (transition or transversion)
A; no change (T-A); neither
The enol form of 5-BrU base pairs with (base), causing a (base pair) after two rounds of replication. This is a (transition or transversion)
G; T-A to C-G; transition
What is 5-bromouracil?
A thymine analog
What is 2-amino purine?
An adenine analog
2-AP base pairs with (base), causing a (base pair) after two rounds of replication. This is a (transition or transversion)
T; no change (A-T); neither
Where does alkylation most readily occur?
Nucleophilic centers
True/False? Electrophilic centers are subject to attack by alkylating agents
False. Nucleophilic centers
The protonated form of 2-AP base pairs with (base), causing a (base pair) after two rounds of replication. This is a (transition or transversion)
C; A-T to G-C; transition
True/False? G-C rich regions are preferred for alkylation
True
Which atoms of guanine and adenine, respectively, are the most commonly alkylated?
N7 and O6 of guanine, N3 of adenine
Provide 7 examples of alkylating agents
EMS, EES, MNNG, nitrosamine, nitrosurea, cigarette smoke, and environmental pollution
The O-4-ethylthymine base pairs with (base), causing a (base pair) after two rounds of replication. This is a (transition or transversion)
G; TA to CG; transition
The O-6-ethylguanine base pairs with (base), causing a (base pair) after two rounds of replication. This is a (transition or transversion)
T; GC to AT; transition
Interstrand crosslinking and DNA-protein crosslinking can be caused by:
UV, ionizing radiation, and bifunctional alkylating agents, such as psoralens
What is psoralen?
A naturally occurring bifunctional alkylating agent that intercalates adjacent pyrimidines and is sensitive to light
What is psoralen used in? Why?
Psoralen UVA (PUVA) treatment for skin problems, such as psoriasis, eczema, and vitiligo. It stops all cell division when photoactivated
What are two types of DNA damaging agents with potential in chemotherapy? Provide an example for each
Alkylating agents (psoralen) and cross-linkers (cis-platin)
True/False? Nucleotide excision repair (NER) is general to many types of DNA damage
True
When does direct reversal of DNA damage occur?
Occurs for very common types of damage, like for mismatches, pyrimidine dimers, and methylation
Describe the pathway of cis-platin
Cancer cells (fast-dividing cells) are more likely to pick up cis-platin. It is taken into the cell, and either causes DNA damage, mitochondrial DNA damage (causes leakage of CytC, which causes apoptosis)
When does base excision repair (BER) occur?
Cytosine deamination to U
Why is the mismatch repair system (MMR) highly conserved?
Mismatches can occur in both prokaryotes and eukaryotes, so it’s needed in both
What is the limitation of MMR?
Indels cannot be repaired
Which strand carries the lesion recognized by MMR?
The daughter strand
What is the replication error rate of DNA Pol III (prokaryotic)?
10^-6
What is the function of Dam methylase?
Identifies newly synthesized strand and methylates adenine residues on the sequence GATC on the new strand
The sequence (GATC) Dam methylase recognizes is:
Palindromic
How is the parental strand distinguished from the daughter stand?
Transient hemimethylation (daughter strand is not methylated but parental is)
Describe the sequence of events in the MMR system in prokaryotes
- DNA Pol III synthesizes new strand
- MutS recognizes mismatched distortion and forms a complex with MutL
- Formation of a DNA loop and bidirectional scanning by the MutS/L complex
- Complex recruits MutH to cleave the nearest unmethylated GATC sites (endonuclease)
- MutS/L recruits UvrD (helicase II) to unwind DNA in the direction of the mismatch and an exonuclease degrades the displaced DNA strand to create a single-stand gap
- ssDNA is coated in single-stranded binding proteins (SSB) and DNA Pol III and ligase fill and seal the gap
Which exonuclease is recruited when the hemimethylated GATC is on the 3’ side of the mismatch?
(hint: which strand is the methylated GATC located on?)
5’ to 3’ (RecJ or exonuclease VII)
Which exonuclease is recruited when the hemimethylated GATC is on the 5’ side of the mismatch?
(hint: which strand is the methylated GATC located on?)
3’ to 5’ (exonuclease I or X)
True/False? Eukaryotic cells have MutS and MutL
False. They have structural and functional analogs, but not MutS/L themselves
Which MMR machinery do eukaryotic cells lack homologs for?
MutH and Dam methylase
True/False? Eukaryotic MMR uses relative strand methylation to distinguish between new and old strands
False. Further research needed
What is a CPD photolyase?
A single polypeptide chain found in all cells except placental mammals (us) that contain 2 non-covalently bound chromophores that utilize UV to catalyze monomerization of thymine dimers
What wavelength light is used in direct reversal of thymine dimers?
400nm
What coenzymes are used by CPD photolyase?
FADH and either MTHF or 8-HDF
What is the function of the coenzymes used in CPD photolyase?
MTHF or 8-HDF absorb light and transfer the energy to FADH to cleave the cyclobutane ring
Which gene encodes CPD photolyase?
phr (photoreactivation)
What level of phr expression is seen in wildtype cells? LOF mutants?
Low expression; very low expression
What is the phenotype of LOF phr mutants?
UV sensitivity, lack of CPD photolyase activity, slow growth rate, low viability
Describe the sequence of events following CPD photolyase activation
- CPD photolyase binds thymine dimers in the presence of UV
- Dimer is flipped into the active site pocket
- MTHF absorbs a photon, transfers excitation energy to FADH- in the catalytic site
- Excited state FADH- transfers an electron to dimer and splits it into 2 T’s
- Electron returns to flavin radical to form FADH- and the enzyme dissociates
Describe the direct reversal of O6-meG by DNA methyltransferase
Transfer of methyl group to a Cys residue on O6-meG DNA methyltransferase
Methylation of the O6 on guanine results in a (base pair), which is a (transition/transversion) mutations, which is repaired by (enzyme)
GC to AT, transition, DNA methyltransferase
True/False? Direct repair of O6-meG is found in all organisms except placentals
False. All organisms
What kind of enzyme is DNA methyltransferase in terms of it’s processivity?
Suicide, it can only perform the reversal once before degradation, so it has low processivity
What kind of mutations is base excision repair used to repair?
Single damaged nucleotides and ssDNA breaks that lack a ligatable junction
Describe BER in prokaryotes
- DNA glycosylase recognizes damaged base and hydrolyzes the N-beta-glycosyl bond between the base and pentose, creating an abasic site
- ssDNA is cleaved at the abasic site by AP endonuclease
- A segment of DNA 3’ of the break site is removed and patched by DNA Pol I and ligase
Where does AP endonuclease cleave the DNA relative to the abasic site?
5’
Describe what is meant by the nick translation activity exhibited by DNA Pol I. When is this activity used?
Movement of where the nick is located on the DNA (first made at the abasic site and is moved 3’ of the original site after polymerization); prokaryotic BER
True/False? After BER in prokaryotes, ligase seals the cut a couple of nucleotides 3’ of the initial DNA damage site
True
True/False? There is only one type of “universal” DNA glycosylase that can recognize any type of damaged base
False. There are different glycosylases for different bases; ex. Uracil DNA Glycosylase (UDG) operates specifically to remove uracil from DNA
UNG detects:
ssU, U:G, and U:A
TDG detects:
Thymine
MPG detects:
7-meA, 3-meA, 7-meG, 3-meG
OGG1 detects:
8-oxo-G
MYH detects:
A:G, A:8-oxo-G
NTH1 detects:
T-glycol, C-glycol
Describe BER in eukaryotes
- DNA Glycosylase detects
- AP endonuclease nicks
3a. Long patch: flap endonuclease is recruited to remove the displaced 5’ terminus
4a. DNA pol fills the gap and ligase seals it
3b. Short patch: Pol beta removes only the abasic pentose
4b. Pol beta fills the gap and ligase seals it
Why do prokaryotes and eukaryotes have different BER pathways?
Eukaryotes lack 5’-3’ exonucleases
How does DNA glycosylase recognize DNA damage if the bases face inwards?
The presence of a kink in the minor groove can be detected, so the damaged base is then flipped into the active site of the enzyme
True/False? DNA glycosylase bends DNA when it is removing a damaged base. This temporarily disrupts the adjacent base pairs
False. They are not disrupted because the DNA isn’t bent significantly
What is different about the type of DNA damage repaired in BER vs NER?
BER repairs single nucleotides while NER repairs bulky bases
Describe NER in prokaryotes
- UvrA and UvrB complex scans DNA helix for damage
- UvrA dissociates when damage is detected and UvrB melts the two strands and recruits UvrC excinuclease
- UvrC makes incisions on the backbone on the 5’ and 3’ sides of the damaged bases to create a 12-13 nucleotide ssDNA gap
- UvrD is a helicase and releases the 12-13nt fragment
- DNA Pol I and ligase fill and seal the gap
What type of enzyme is UvrC?
Excinuclease
What type of enzyme is UvrD?
Helicase
Describe NER in eukaryotes
- XPC recognizes the lesion
- XPB and XPD are recruited and separate the DNA to form an ssDNA bubble
- RPA binds to the bubble and positions XPF and XPG nucleases on either side of the lesion
- 24-32 nt fragment is displaced and PCNA clamp recruits DNA Pol and ligase to fill and seal
Describe transcription-coupled DNA repair (TCR)
- RNA Pol recognizes damage and stalls
- Stalled complex recruits NER proteins, which release RNA Pol and repair the DNA
Which regions are subject to the greatest DNA repair by TCR?
Genomic regions that are most highly transcribed
What is xeroderma pigmentosum (XP)? What is it caused by?
An autosomal recessive disorder resulting from a defective NER system, which means cells can’t repair UV damage, so skin cells are extremely photosensitive (early skin cancer)
What are the seven types of chromosome rearrangement?
Terminal deletion
Interstitial deletion
Unequal crossing over
Ring
Isochromosome
Robertsonian translocation
Insertion
What is terminal deletion?
Deletion of the end of a chromosome
What is interstitial deletion?
The deletion of a fragment within the chromosome
What is a ring rearrangement of chromosomes?
Ends are deleted and new ends are joined
What is an isochromosome?
One end of the chromosome replaces the other end (longer arm replaces the shorter arm so now you have a chromosome with two long arms)
What is Robertsonian translocation?
Translocation of a chromosome arm onto another arm
What is insertion?
A fragment from one chromosome translocates to another
Why do carriers of a chromosomal inversion not show a mutant phenotype?
They have a balanced rearrangement
What is a balanced arrangement?
Genes stay the same but change locations within the chromosome
What produces unbalanced chromosomes?
Crossing over of a paracentric inversion
What makes offspring with unbalanced chromosomes inviable?
Some may be acentric or dicentric, which causes issues in cell division, and they may have gene duplication and/or deletion
Describe normal vs. balanced
Normal: genes occur in specific order (ABCD)
Balanced: genes occur in a different order but retain function (ACBD)
How many balanced:unbalanced:inviable gamates are produced after crossing over of paracentric inversion loops?
2:0:2
How many balanced:unbalanced:inviable gamates are produced after crossing over of pericentric inversion loops?
2:2:0
Carriers of balanced arrangements usually produce gametes containing _______ chromosomes
Unbalanced
Describe balanced vs. unbalanced chromosomes
Balanced: same number of genes, same or different order as wildtype, retain one centromere
Unbalanced: Have gene deletion/insertion, but retain one centromere
Where are the gene duplications/deletions found on unbalanced chromosomes?
At the ends of the chromosome
Paracentric vs pericentric inversion loops?
Pericentric includes the centromere, paracentric does not and occurs between the same arm of the chromosome
What are the two repair mechanisms for a double stranded breaks that result in full repair?
Synthesis dependent strand annealing (SDSA) and double-stranded break repair (DSBR)
True/False? SDSA can only result in a noncrossover event
True
True/False? DSBR can only result in a noncrossover event
False. Both crossover and noncrossover
When does NHEJ occur?
When a sister chromosome is unavailable for DSBR
Describe the steps of NHEJ
- Double-stranded break occurs
- Ku70/80 binds at each DNA end
- DNA PKcs are recruited and the two ends are ligated together
Why are indels common with NHEJ?
Repaired ends don’t always reproduce the sequence before the break
What is TLS?
Translesion DNA synthesis
Describe TLS
- DNA Pol III stalls when it encounters a lesion at the replication fork and dissociates
- Pol IV is recruited to polymerize across the lesion
- Pol IV dissociates and the replisome is rerecruited
Why is Pol IV recruited during TLS?
A low-fidelity polymerase that lacks 3’-5’ exonuclease activity is needed so that the DNA can still be synthesized and the mutation is repaired later
Summarize the damage type and enzymes used for MMR
Replication errors; MutS, MutL, MutH (E. coli), MSH, MLH, and PMS (humans)
Summarize the damage type and enzymes used for photoreactivation
Pyrimidine dimers; DNA photolyase
Summarize the damage type and enzymes used for BER
Damaged base; DNA glycosylase, AP endonuclease
Summarize the damage type and enzymes used for NER
Pyrimidine dimer and bulky adduct on base; UvrA, UvrB, UvrC, UvrD (E. coli), XPC, XPA, XPD, XPF, and XPG (humans)
Summarize the damage type and enzymes used for DSBR
Double stranded breaks; RecA (E. coli)
Summarize the damage type and enzymes used for TLS
Pyrimidine dimer, apurinic site, or bulky adduct on base; Y-family DNA Pol (UmuC or Pol IV)
Alkylation can be repaired by which mechanisms?
BER, alkyltransferases (ex. DNA methyltransferase), and NER
Depurination can be repaired by which mechanisms?
BER
Pyrimidine dimers can be repaired by which mechanisms?
CPD photolyase, NER
Deamination can be repaired by which mechanisms?
BER
Double stranded breaks can be repaired by which mechanisms?
DSBR, NHEJ, SDSA
Direct reversal of DNA damage can be seen in which mechanisms?
MMR, photoreactivation (photolyases), and removal of methyl groups (methyltransferases)
