Topic 6 Flashcards
True or false: most mutations are harmful
True
- Cells have repairing mechanisms to correct changes in genetic materials
Mutations and repairing mechanisms have to maintain a balance between…
Organismal survival and biodiversity (variation in the DNA)
What are the 3 types of DNA damage?
- Gene mutation (spontaneous or induced by mutagens)
- Recombination (likely causing chromosomal arrangements)
- Transposable elements
Unequal crossing over leads to what?
Leads to the insertion/deletion of certain genes
What are the 2 mechanisms inducing DNA mutations?
- Replication errors
- Chemical modifications
Deamination (chemical modification that leads to DNA mutations)
Removal of an amino group
Depurination/depyrimidination leads to… (chemical modification that leads to DNA mutations)
Base loss
Oxidation (chemical modification that leads to DNA mutations)
Electron loss
Alkylation (chemical modification that leads to DNA mutations)
Transfer of an alkyl (CH3-) group
Nitrous acid (that is found in food preservatives) can induce what?
DNA damage
Energy-rich radiations cause what? (chemical modification that leads to DNA mutations)
Inter-base, inter-strand crosslinkings, and strand breaks
Intercalating agents can cause what?
DNA damage
Base analogs can cause what?
DNA damage
What 2 mutations are caused by replication errors?
- Point mutations (e.g. missense, nonsense mutations)
- Frameshift mutations
Slippage of template during replication leads to…
Deletion
Slippage of new strand during replication leads to…
Addition
Number the nucleotides on slide 9
In what tautomeric form are nucleotides usually found? (2)
Amino and keto
When amino form of nucleotide tautomerizes into imino form, what happens to the hydrogen bond donors and acceptors?
Hydrogen bond donor from amino becomes a hydrogen bond acceptor, while one of the deprotonated nitrogens becomes a hydrogen bond donor
When keto form of nucleotide tautomerizes into enol form, what happens to the hydrogen bond donors and acceptors?
Hydrogen bond acceptor on carbonyl becomes a hydrogen bond donor, while hydrogen bond donor on one of the protonated nitrogens becomes a hydrogen bond acceptor
Enol form of Thymine can base pair with…
Keto form of guanine
Enol form of guanine can base pair with…
Keto form of thymine
Imino form of adenine can base pair with…
Amino form of cytosine
Imino form of cytosine can base pair with…
Amino form of adenine
Transition mutation
purine -> purine or pyrimidine -> pyrimidine
Transversion mutation
purine -> pyrimidine or pyrimidine -> purine
Describe how substitution point mutations occur in two steps
Step 1: incorrect nucleotide is incorporated by DNA polymerase during replication -> mismatching (mismatch is called a lesion)
Step 2: Mismatched base is not repaired and undergoes DNA replication -> mutation
What does it mean if you read about an E6V mutation in a research article?
Glutamate mutates at codon (amino acid) 6 to Val
teg-1 (oz230) mutation changes AAG to UAG at codon 53. However, this corresponds to position 257 on genomic DNA, rather than 159. Why?
The position 259 includes all the introns
Frameshift mutation
A mutation changes the reading frame of codons during protein synthesis
Indel mutations that occur in multiples of 3 base pairs…
Preserve the reading frame of the gene
Slippage of DNA polymerase during DNA replication can cause…
Several diseases
What do Trinucleotide Repeat/Expansion Disorders involve?
Involve expansion of repeats of CAG, CGG, GAA and CTG
Huntington’s chorea
Expansion of CAGs (codon for glutamine)
Describe Fragile X syndrome
- The most common form of hereditary mental retardation with a frequency of 1/1500 males and 1/2500 females
- (CGG)n repeats in Fragile-X syndrome gene, FMR1
- Progressive expansion from generation to generation
How can triplet expansion diseases be diagnosed?
The number of triplet repeats in the fragile X locus can be determined by length analysis of PCR products
- because repeats increase the length of DNA
What are the 4 main types of chemical damage that can damage DNA?
- Hydrolytic reactions with H2O to damage nucleotides and the phosphodiester backbone of DNA.
- Electrophilic attack of DNA backbone by alkylating agents.
- Reaction with oxygen species, such as hydrogen peroxide, hydroxyl radicals, superoxide radicals, etc…
- Alteration of DNA structure by irradiation, such as X ray and UV
Cytosine deamination results in…
Uracil
Adenine deamination results in…
Hypoxanthine
Guanine deamination results in…
Xanthine
5-Methylcytosine deamination results in…
Thymine
Which modified bases can base pair with cytosine?
Hypoxanthine and Xanthine
Which nucleotide cannot deaminate?
Thymine
What results in base loss by depurination or depyrimidination?
Hydrolytic reaction cleaves glycosyl bond between the nitrogenous base and the deoxyribose to create an abasic (AP) site
- Under physiological conditions, depurination occurs at ~5000 bases/cell/day
The (open/closed) form of deoxyribose has an unstable 3’phosphodiester bond; therefore, hydrolysis may result in…
Open, may result in strand break and base loss
What produces reactive oxygen species (ROS)?
Cellular metabolism, such as cellular respiration in the mitochondria and detoxification in the liver, and ionization radiation
ROS can oxidize thymine to what? What does this result in?
Thymine glycol, this blocks DNA polymerase (due to steric hinderance)
ROS can oxidize guanine to what? What does this result in?
8-oxoguanine, this changes a GC base pair to an AT base pair because 8-oxoguanine can base pair with adenine.
Alkylating agents (CH3-) come from what?
Cigarette smoke and environmental pollutions
What positions does alkylation on Thymine occur most readily?
C=O of C2, N3 and C=O of C4
What positions does alkylation on Adenine occur most readily?
N1, N3, N7, amine on C6
What positions does alkylation on Cytosine occur most readily?
C=O of C2, N3 and amine on C4
What positions does alkylation on Guanine occur most readily?
N1, N2, N3, N7, C=O on C6
What are the highly reactive sites for alkylation in all of the bases?
N3 of A, O6 of G and N7 of G
S-adenosylmethionine (SAM)
The biological methyl group donor that can accidentally react with DNA to methylate A to give N6-methyladenine (m6A) -> DNA functions like RNA
Sodium nitrate (NaNO3) and bisulfite (HSO3-) used as food preservatives causes…
Deamination of guanine to xanthine, cytosine to uracil and adenine to hypoxanthine
True or false: guanine deaminating to xanthine causes a mutation
False, because hypoxanthine also hydrogen bonds with cytosine so there’s overall no change.
True or false: multiple potential chemical modifications can occur simulatenously on a single base
True
Cyclobutane pyrimidine dimers (CPD)
Condensation of two double-bonded C5=C6 atoms on adjacent pyrimidine bases that distort the DNA double helical structure (UV-induced)
Most common CPD
Thymine dimers
(6-4) pyrimidine photoproducts (6-4PPs)
Formation of a single covalent bond between the 6 position of one pyrimidine and the 4 position of the adjacent pyrimidine on the 3’ side (UV-induced)
CPD and 6-4 Photoproduct are (inter/intra) strand crosslinking
Intra
Ionizing radiations induce (inter/intra)base crosslinks
Inter
The formation of a pyrimidine dimer introduces a…
Bend or kink in the DNA
On encountering a pyrimidine dimer during replication…
The DNA polymerase stalls
Ionizing radiation causes…
The formation of excited and ionized molecules
- The most important are species formed by radiolysis of water, forming products capable of causing oxidative damage
Intercalating agents and the type of mutations caused by them
Chemical mimicking base pairs and intercalate between stacked base pairs -> stabilizing DNA helix -> indels that cause frameshift mutations
4 examples of intercalating agents
Proflavin, acridine orange, ICR-191, ethidium
Describe the hydroxylation of cytosine
Hydroxylation (i.e. addition of hydroxyl group) to the amino of cytosine -> hydroxylaminocytosine (HC)
- HC forms H-bonds with adenine to GC base pair changes to AT
5-bromouracil (5-BrU) is a _____ analog and exists as…
Thymine analog and exists in two forms: keto form and enol form
Keto form of 5-BrU base pairs with…
A
Enol form of 5-BrU base pairs with…
G
2-amino purine (2-AP) is an _______ analog
Adenine analog
2-AP base pairs with…
Thymine
Protonated 2-AP base pairs with…
Cytosine
Alkylation occurs most readily at…
Nucleophilic centres
Alkylation agents prefer _____ - rich regions
GC
Most common nucleophilic sites that are alkylated? (2)
N7 of guanine and N3 of adenine
Alkylated bases can mispair during…
Replication
5 examples of alkylating agents
- Ethylmethane sulfonate (EMS): CH3SO3CH2CH3
- Ethylethane sulfonate (EES): CH3CH2SO3CH2CH3
- N-methyl-N’-nitro-N-nitroso guanidine (MNNG)
- nitrosamine
- nitrosourea
O-6-Ethylguanine (alkylated version of guanine from EMS) base pairs with…
Thymine
O-4-Ethylthymine (alkylated version of Thymine) base pairs with…
Guanine
Interstrand and DNA-protein crosslinking can be caused by what 3 things?
- UV
- ionizing radiation
- bifunctional akylating agents
Psoralen
- Naturally occurring compound
- Intercalates adjacent pyrimidines and is sensitive to light -> psoralen is activated by light and links pyrimidines between strands to introduce a kink
How can psoralen treat certain skin conditions?
Used in PUVA treatment for skin problems, such as psoriasis, eczema and vitiligo because it cross-links pyrimidines which stops cell division/proliferation.
What are two DNA-damaging agents with potential in chemotherapy? Give an example for the second one
- Alkylating agents
- Cross-linkers (e.g. cis-platin)
Cis-platin is a… (2)
- Alkylating agent
- Cross-linker
Describe how cis-platin can be used as a chemotherapy agent
Cis-platin alkylates nuclear DNA and damages it, as well as damages mtDNA. This causes apoptosis of cells because cytochrome c is released (by MOMP aka mitochondrial outer membrane permeabilization)
What are the three classes of DNA repair mechanisms?
- Direct reversal of DNA damage
- Base excision repair
- Nucleotide excision repair
Direct reversal of DNA damage only occurs for…
Very common types of damage
3 types of direct reversal of DNA damage
- Mismatch repair system
- Photoreactivation of pyrimidine dimers
- Removal of methyl group by methyltransferase
Describe base excision repair in general
Base is removed, then repair occurs - specific for type of damaged base
Describe nucleotide excision repair in general (2)
- Nucleotide(s) is removed, then repair
- General to many types of damage
MMR system is (different/conserved) in prokaryotes and eukaryotes
Conserved
When is the MMR system used?
When only the new strand carries the mismatched base (i.e. not a mutation yet)
What is a limitation of the MMR system?
Misinsertions and short indels cannot be repaired by the MMR system
In E.coli, DNA pol ___ is used to replicate DNA strand
III
Dam methylase function in DNA recognition
Dam metylase is an enzyme that methylates adenine residues in the palindromic sequence GATC on the parental strand of the DNA. Transient hemimethylation of a DNA duplex following replication distinguishes the parental strand from the daughter strand.
After methylation of the daughter strand, _____ recognizes the mismatched distortion and forms a complex with ____
MutS, MutL
The MutS-MutL complex forms a ____ loop, due to _____ scanning
The MutS-MutL complex forms a DNA loop, due to bidirectional scanning
MutS-MutL complex recruits ____ to cleave ______
MutS-MutL complex recruits MutH to cleave unmethylated GATC sites
MutS-MutL complex recruits ____ to _______
(not MutH)
MutS-MutL recruits helicase II (aka UvrD) to unwind DNA in the direction of the mismatch and an exonuclease degrades the displaced DNA strand to create a single-strand gap
When cleavage at a hemimethylated GATC is on the 3’ side of the mismatch, a ____ is recruited (e.g. ____)
When cleavage at a hemimethylated GATC is on the 3’ side of the mismatch, a 5’->3’ exonuclease is recruited (e.g. RecJ or exonuclease VII)
When cleavage at a hemimethylated GATC is on the 5’ side of the mismatch, a ____ is recruited (e.g. ____)
When cleavage at a hemimethylated GATC is on the 5’ side of the mismatch, a 3’->5’ exonuclease is recruited (e.g. exonuclease I or exonuclease X)
The single strand gap from the exonuclease in MMR is…
Coated with single-strand DNA-binding protein (SSB), filled in by DNA Pol III, and sealed by ligase
True or false: eukaryotic cells also use MutS and MutL
False
- eukaryotic cells have several proteins that arestructurally and functionally analogous to bacterial MutS and MutL
True or false: eukaryotic cells contain homologs to bacterial MutH and Dam methylase
False
- Eukaryotic cells lack homologs to bacterial MutH and Dam methylase and do not use methylation to distinguish between new and old strands -> further research needed!
Structure of CPD photolyase
Single polypeptide chain of 454 to 614 amino acids found in bacterial, archaeal, and eukaryotic cells, except the cells of placental mammals
- Contain two noncovalently bound chromophores
CPD photolyase reaction
Catalyze monomerization of thymine dimers:
All photolyases bind FADH and contains either MTHF (5,10- methenyltetrahydofolylpolyglutamate) or 8-HDF (7,8-dimethyl-8-hydroxy-5-deazariboflavin). MTHF (or HDF) absorb light and transfer energy to FADH to cleave the cyclobutane ring.
Gene that encodes CPD photolyase
phr (photoreactivation) gene
phr expression in wildtype cells
Low
Phr- mutant phenotype (4)
- UV sensitivity
- Lack of CPD photolyase activity
- Slow growth rate
- Low viability
What are the 2 main domains of CPD photolyase?
- α-helical domain
- α/β domain
What are the 5 steps of UV-induced CPD photolyases?
- CPD photolyase binds to thymine dimer in the presence of light
- Thymine dimer is flipped into the active site pocket, using the phosphate bakbone as a hinge
- MTHF (aromatic molecule present in the enzyme) absorbs a photon, transfers excitation energy to FADH- in the catalytic site
- Excited state FADH- transfers electron the thymine dimer; splits dimer into two thymines.
- Electron returns to flavin radical to re-form FADH-. Enzyme dissociates.
O6-methylguanine (O6-meG) tends to pair with __ rather than C, resulting in what type of mutation?
Tends to pair with T rather than C, resulting in GC to AT transition mutations
How is O6-meG repaired?
By transferring the methyl group of O6-meG to a cysteine (Cys) residue in the enzyme O6-methylguanine-DNA methyltransferase
- O6-methylguanine-DNA methyltransferase is then degraded after being methylated (it is a suicidal enzyme)
- mechanism found in all organisms
What other DNA repair mechanism can also repair thymine dimers?
Nucleotide excision repair (NER)
- involved in more extensive mutations (e.g. indels).
What does prokaryotic base excision repair (BER) repair?
Repairs a single damaged nucleotide that distorts DNA very little and ssDNA breaks that lack a ligatable junction
In bacterial BER, recognition of the damaged base is performed by a…
DNA glycosylase
DNA glycosylase
Enzyme that hydrolyzes the N-β-glycosyl bond between a nucleotide base and a pentose, creating an abasic site in the DNA
During BER,the ssDNA is cleaved at the abasic site by….
AP endonuclease
AP endonuclease
An enzyme that cleaves the DNA backbone at an AP site to create a nick with a 3’ hydroxyl and a 5’ deoxyribose phosphate
During BER, a segment of DNA is removed and patched by…
the nick translation activity (can remove and patch nucleotides) of Pol I, and DNA ligase seals the remaining nick
The uracil DNA glycosylase (UDG) acts only on…
DNA (from cytosine deamination); it does not remove uracil from RNA
All cells contain (different/the same) glycosylases that…
All cells contain different glycosylases that recognize different types of damaged bases
How do the first two steps of eukaryotic BER differ from bacterial BER?
They don’t; they are the same
In long patch repair in eukaryotic BER, flap endonuclease…
Explain why
Is recruited to remove the displaced 5’ terminus and replace region with new nucleotides (and ligase ligates nicks)
- Flap endonuclease is recruited because eukaryotic DNA polymerases lack 5’ -> 3’ exonuclease activity
Short patch repair replaces only…
The damaged nucleotide base
- Ligase ligates nick
In a double helical structure, the nitrogenous bases are facing inward; therefore, how can a DNA glycosylase recognize the damage?
The enzyme scans the minor groove of the helix to look for a “kink” in the damaged base and then “flipping” the damaged base out of the DNA duplex into the enzyme’s active site.
- The DNA helix is bent somewhat but adjacent base pairs are not disrupted
Nucleotide excision repair uses an _______, an enzyme that…
Excinuclease, an enzyme that cleaves a phosphodiester bond in the DNA on either side of a bulky lesion in DNA, to remove the lesion
In E. coli, what 4 enzymes are involved in nucleotide excision repair?
UvrA, UvrB, UvrC, UvrD
- SOS response enzymes
Which enzymes in E.coli scan a DNA helix for damage in NER?
UvrA and UvrB form a complex and scan DNA helix for damage
What happens to E.coli NER enzymes when a lesion is encountered? Describe specifically what happens to the Uvr enzymes
UvrA leaves and UvrB melts the two DNA strands and recruits UvrC excinuclease to make incisions in the DNA backbone on the 5’ and 3’ sides of the damaged nucleotide(s) to create a ~12-13 nt ssDNA gap. The UvrD enzyme (a helicase) then releases the 12-13 nt fragment. DNA pol I and ligase fill and seal the gap.
What enzyme recognizes the lesion in NER in eukaryotes?
XPC
Which proteins are recruited to the lesion to separate DNA to form a single stranded DNA bubble during eukaryotic NER?
XPB and XPD are recruited to the lesion to separate the DNA
After XPD and XPB are recruited to the lesion in eukaryotic NER, what protein binds to the bubble? What does this protein do?
RPA binds to the bubble and positions XPF and XPG nucleases on either side of the lesion.
During eukaryotic NER, what do XPG and XPF do?
XPG cleaves on the 3’ side and XPF cleaves on the 5’ side of the ssDNA bubble
- 24-32 nucleotide fragment is displaced
After the 24-32 nucleotide fragment is displaced in eukaryotic NER, what happens next?
The PCNA clamp recruits a DNA pol to fill the gap, which is then sealed by a ligase
In Transcription-Coupled DNA Repair (TCR), what is the damage recognized by?
The damage is recognized by the RNA polymerase that is stalled at the lesion
In TCR, what does a stalled transcription complex lead to?
A stalled transcription complex recruits nucleotide excision repair proteins, which release RNA polymerase and repair the DNA
Which genomic regions will be subject to the greatest DNA repair?
The genomic regions that are most highly transcribed
Xeroderma Pigmentosum (XP) mode of inheritance
Autosomal recessive
What is xeroderma pigmentosum caused by? What does this result in?
A defective NER system -> cells are not able to repair UV damage (e.g. pyrimidine dimers)-> skin cells are extremely photosensitive -> early onset of skin cancer
What 6 types of glycosylases are in mammals?
- UNG (uracil DNA N-glycosylase)
- TDG (thymine DNA glycosylase)
- MPG (methyl purine DNA glycosylase)
- OGG1 (8-oxo-guanine glycosylase)
- MYH (MutY homolog)
- NTH1 (endonuclease three homolog)
What mutations does UNG (uracil DNA N-glycosylase) fix? (3)
ssU; U:G; U:A
What mutations does TDG (thymine DNA glycosylase) fix? (1)
GT mismatches
What mutations does MPG (methyl purine DNA glycosylase) fix? (4)
7-meA; 3-meA; 7-meG; 3-meG
What mutations does OGG1 (8-oxo-guanine glycosylase) fix? (1)
8-oxo-G
What mutations does MYH (MutY homolog) fix? (2)
A:G, A:8-oxoG
What mutations does NTH1 (endonuclease three homolog) fix? (2)
T-glycol; C-glycol
Carriers having a chromosomal inversion usually (do/do not) show a mutant phenotype
Do not, due to a balanced rearrangement
Paracentric inversion
Middle genes in a chromosome are inverted, but inversion does not include the centromere
Crossing over of paracentric inversion loops produces…
Unbalanced acentric or dicentric chromosome, which may not lead to viable offspring.
Gametes formed after the 2nd meiosis after a paracentric inversion usually contain…
Either a normal or balanced copy of the chromosome because the acentric and dicentric products of the crossover are inviable
Pericentric inversion
Middle genes in a chromosome are inverted, and the inversion includes the centromere
Crossing over of pericentric inversion loops produces…
Unbalanced gametes with chromosomes segments that are both duplicated and deficient
Gametes formed after the 2nd meiosis after a pericentric inversion may contain…
Normal, balanced, or unbalanced chromosomes.
Unbalanced gametes after pericentric inversion contain…
A copy of the chromosome with duplication or a deficiency of the material flanking the inverted segment.
In general, what can repair damaged DNA (double-stranded break repair)?
Explain how in general
Homologous recombination
- An allele on the homologous chromosome is used as a template to replace the sequence on the damaged chromosome.
When does Non-Homologous End Joining (NHEJ) occur?
NHEJ occurs if a sister chromosome is unavailable for high-fidelity homologous recombination repair
Describe the general steps for non-homologous end joining (NHEJ) (2)
- Ku70/80 binds at each DNA end
- The other components of NHEJ are recruited, and the two ends are ligated together
- The repaired ends don’t always reproduce the sequence before the double-stranded break; therefore, indels may be incurred
In repair by translesion DNA synthesis (TLS), what happens to the replisome complex?
When the DNA polymerase stalls as it encounters a lesion at the open replication form, the replisome complex dissociates from DNA
After the replisome complex dissociates during translesion DNA synthesis (TLS), what is recruited to the lesion?
An error-prone polymerase of the Y family (e.g. pol IV) is recruited to polymerize across the lesion
- The polymerase must be low fidelity and lacks a proofreading 3’ -> 5’ exonuclease activity, so that it can still synthesize DNA no matter what the lesion is (survival vs. death, mutations may be produced by the cell doesn’t die)
After the error-prone polymerase synthesizes DNA across the lesion during TLS, what occurs?
The replication complex reassembles and continues
Translesion DNA synthesis repairs what three types of DNA damage?
- Pyrimidine dimer
- Apurinic site
- Bulky adduct on base
