Topic 6 Flashcards
What is dna damage and how can it occur
Any modification of dna that changes its coding properties or normal function in transcription or translation
Can happen spontaneously, induced by exposure to environmental factors like chemicals or radiation, or during replication
What can dna damage lead to
Are most mutations harmful
Mutations and genetic instability
Yes
What can cause changes in genetic material
Gene mutation (can be spontaneous or induced by mutagens)
Recombination (causing chromosomal rearrangements)
Transposable elements
Give an example of how recombination can change genetic material
If the chromosomes aren’t aligned properly during recombination
In one strand a deletion of a fragment can happen and a duplication in the other
What can cause gene mutations
Replication errors
Chemical modifications
What are the mutations that are caused by replication errors
What is the result of replication errors
Point mutations
Frameshift mutations
Mismatching, deletion, insertion, causing the reading frame to be same or diff
What are point mutations
A change in a single nucleotide
Can cause missense (change in nucleotide made diff amino acid) or nonsense mutations (premature stop codon
What are frameshift mutations
The slippage of the dna pol on the template causes a deletion in the new strand
Slippage on the new strand causes an addition in the new strand
Guanine and adenine structure and numbering
Both two rings
Guanine has c=o at c6
Adenine has c-NH2 at c6
In the five c ring numbers 7,8,9
In the 6 c ring the c 6 then go clockwise backward
Cytosine, thymine, uracil structure and numbering
Single ring for all
Cytosine has c-NH2 on c 4
Thymine has c=o on c 4 and methyl on c 5
Uracil same and thymine but no methyl on c 5
Number from 1 opposite to c 4 going clockwise
Which atom of purine and pyrimidines form bind with the sugar
Purine: N9
Pyrimidine: N1
What is a tautomeric shift
The dominant keto (c=o) and amino (c-nh2)
Turn to enol (c-oh) and imino (c-nh
Which nucleotides can have enol forms
Which can have imino
Guanine thymine and uracil
Adenine cytosine
What can enol form of thymine base pair with
Say how it changes
Keto guanine
T:A then T:G
after replication C:G
What can enol form of guanine base pair with
Say how it changes
Keto thymine
G:C to G:T
After replication A:T
What can imino form of adenine base pair with
Say how it changes
Amino form of cytosine
A:T to A:C
After replication G:C
What can imino form of cytosine base pair with
Say how it changes
The amino form of adenine
C:G to C:A
After replication T:A
What is the result of anomalous base pairing due to tautomeric shifts
Base pair transitions or transversions
What is a transition mutation
Transversion
Purine pyrimidines to purine pyrimidines (ex. G:C turns to A:T)
Purine pyrimidines to pyrimidines purine (ex. G:C turns to C:G)
Point mutations can be categorized into
Translation or transversion mitations
What are the two steps that point mutations occur in
At what pint is it actually a mutation
- Incorrect nucleotide is inserted during replication
- The mismatched base is not repaired and gets replicated
In step 1 it’s just a lesion/mismatch
In step 2 it’s a mutation
What is the teg-1 gene
A gene that when mitated causes tumour enhancement and over proliferation of mitotic cells
What is the mutation in teg-1 (oz230)
What is the mitation called
The AAG changed to UAG at codon 53 (position 257) , this causes a premature stop codon
Since UAG, called amber
What is the mutation in teg-1 (oz189)
What is the mitation called
UGG to UGA at codon 142
UGA means opal
What is the UAA stop codon labeled as
Ochre
What does K53X means
Lysine
53rd codon
X means a stop codon
If I have codon 53 how many nucleotides it this
But in reality it’s listed as higher (257) why?
153 in the processed sequence
This accounts for the fact that there are introns in the genomic sequence (unprocessed sequence)
What are indels and how are they caused
Insertions or deletions
Caused by bad recombination of dna or dna pol slippage during replication
What is a reading frame
What changes the reading frame
A contiguous non overlapping 3 nucleotide codon in DNA or RNA (has no introns)
Framshift mutations
What happens if an indel mutation affects a multiple of 3 base pairs
The reading frame of the gene is unchanged since since either only one amino acid is gone or added
What are trinucleotide repeat/expansion disorders
Give an example
When expansion of repeats of CAG CGG GAA CTG cause hereditary disorders even though no frameshift happens
Ex. CAG expansion in the gene encoding Huntington causes the Huntington chorea disorder
What is fragile X syndrome
Who does it affect more
What gene is it in
What is the normal number of repeat , number that makes you a carrier and the disease number
The most common form of mental retardation and Affects men more than women
Expansion of CGG repeats in the FMR1 gene (fragile x mental retardation)
Normal is 6-54
Carrier is 55-200
Disease is 200-1000
What is bad about fragile X syndrome
The fact that the CGG repeats get more and more expanded across generations means the offspring get worse and worse symptoms
How can the trinucleotide repeat disease get diagnosed using PCR
They can measure the number of repeats by the amount of BP in the PCr product
If 300 nucleotides there are 100 repeats
In chemical damage of dna what can cause this
Hydrolytic reaction with water to damage the nucleotides and phosphodiester backbone
Electrophillic attack of the DNA backbone by alkylating agents
Reaction with oxygen species like hydrogen peroxide, hydroxyl radicals
Alteration of the dna structure by irradiation from sunlight, x ray or uv
All in all, chemical damage to dna can happen why
Dietary and environmental stressors
In hydrolytic damage of nucleotides what is there
Deamination
Depurination
Depyrimidination
What is deamination
Removal of a amine group on a nucleotide
What gets Deaminated and what does it turn into
CAG
Cytosine turns to uracil
Adenine to hypoxanthine
Guanine to Xanthine
5-me-cytosine to thymine
After Deamination , what can xanthine and hypoxanthine pair with
What does this mean
Cytosine
When guanine gets deaminated to xanthine there isn’t a problem because it’s still pairing with C
What does uracil pair with
Adenine
When changing from A G to hypoxanthine or xanthine what changes
The amine becomes c=o and the n gets an h
Where would we see 5 methyl cytosine
In CpG islands
In hydrolytic damage How does depurination and de pyrimidation happen
The hydrolytic reaction cleaves the glycosidic bond between the base and the backbone sugar
This makes an abasic site (Ap site)
How often does dpurination occur
Under physiological condition 5000 bases per cell per day
What can be the result of the ap site
The backbone is unstable
When is becomes unstable the sugar is in the open form and vulnerable to a nucleophile attack
This leads to cleavage/breakage of the backbone
What causes oxidative damage of nucleotides
Give examples
Cellular metabolism like respiration can make reactive oxygen species (ROS)
The ROS are OH radicals, O2 radicals, and h2o2
What can ROS do to which nucleotides
What does each turn into
Oxidize T and G
T turns to thymine glycol
G turns to 8-oxoguanine
What does thymine glycol and 8-oxo-guanine look like
Thymine glycol has two OH
8-oxoguanine has two c=o
What do thymine glycol and 8-oxoguanine result in
Thymine glycol is bulky due to the two OH so it stall the dna pol during replication
8-oxoguanine pairs with adenine
What causes alkylating damage of nucleotides
Alkylation’s agents (ch3-) from cigar smoke and environmental pollution
The agent acts as a nucleophile
What are the highly reactive sites of the nucleotides that can get alkylated
N3 of A
O6 of G
N7 of G
What’s a special case of methylation of a nucleotide
S- adenosylmethionind (SAM) , a methyl donor to adenine can turn adenine
Into N6 methyladenine (m6A)
This makes the modified adenine behave like rna
What causes Deamination
In food preservatives , the sodium nitrate naNO3 and bisulfite HSO3- and nitrous acid (HNO2)
cause Deamination
How can bisulfite help in bisulfite sequencing in studying epigenetics
The unmethylated cytosine gets deaminated into uracil
So after replication it goes from C:G to T:A
But methylated stays the same
This is how you can see which were methylated
What is special about guanine in terms of the modifications it can have
It’s can have alkylation Deamination and oxidation all at the same time on that one base
What is the cause of uv induced damage on dna
What is the most common result
It can cause cyclobutane pyrimidines dimers (CPD)
The most common result is thymine dimers
On which atoms can the thymine dimers form
Formed by one bond between the c6 of one thymine and c4 of the other (6-4)
Called 6-4PPs
Or two bond between the c5 and C6 of the two thymines
(6-5)
What is the likelihood of dimer formation in UB induced damage
TT»_space; TC > CT > CC
What is the result of dimer formation due to uv light
Causes a kink in the DNA so DNA pol stalls and cant replicate
What is a diesease caused by TT dimer formation due to UV light
Skin cancer
Aside from forming dimers what can ionizing radiation also do
The high energy causes the radiolysis of water which makes ROS
These ROS can cause base loss (AP sites), strand breaks
And dna protein cross links (dna and transcription factors are cross linked which stops gene expression)
What are intercalating agents and what do they do
They are chemicals that mimic the base pairs and intercalate between them
They cause the dna pol to skip that region of dna and make indel and frameshift mutations
Also change the dna topology by stretching them
What are examples of intercalating agents
Proflavin, ethidium bromide
What is hydroxylation and the result
Adding a hydroxyl group to the amino of cytosine
Makes hydroxylaminocytosine (HC)
HC binds to adenine
So C:G to T:A
What are the base analogs
5-bromouracil
2-amino purine
What is 5-bromouracil (5-BrU)
What does the keto form pair with
What does the enol form pair with
A thymine analog which exists in the keto and the enol form
Keto form pairs with adenine (no effect since stil T:A)
Enol pairs with G
What is 2-amino purine (2-AP)
An adenine analog
When 2-AP is protonated at N1 it pairs with C
What are alkylating agents
Where does alkylation most readily occur
They are thing that introduce alkyl groups
Occurs most readily at nucleophillic centres of the nucleotides
Prefer GC rich regions to attack
N7 guanine O6 GUANINE N3 adenine
When an aplkylating agent alkylates a base, when does it have the most affect
After replication, that’s when it becomes a mutation and bases get mismatched
Then through more replication the mutations can get amplified
What are alkylating agents examples
EMS
EES
MNNG
Nitrosamine
Nitrosourea (used to introduce mutations)
What can also get alkylated by ems
O6 of guanine
O4 of thymine
When researchers use EMS to alkylate something what type of mutation it it and why is it helpful
It’s usually a substitution mutation (GC to AT)
Can induce a mutation using EMS to the organism and observe the outcome of the phenotype of the organism then attribute the change in phenotype to the function of the gene
What naturally occurring agents can be used to introduce inter strand and dna protein cross linking
Uv, ionizing radiation, bifunctional alkylating agents (such as psoralens)
What is psoralen
A naturally occurring compund from plants that is highly sensitive to light
When added to dna, you expose it to light, the psoralen gets activated
Cross linking agent that cross links the pyrimidines bases between two strands, links
Stops cell division
How can psoralen be used as a treatment
Since it stops cell division though making kinks
It’s used in PUVA (psoralen uv a) treatment of skin problems like psoriasis eczema and vitiligo
What dna damaging agents can be used in chemotherapy
Alkylating agent
Crosslinker like cisplatin (which is an alkylating agent)
What does cisplatin do in chemotherapy
It’s causes dna damage by adding alkyl groups to the bases
Damages the mitochondrial dna (its own type of dna)
This activates cyt c which triggers the apoptosis pathway which causes the cell to die
This kills the cancer cells since they are more likely to take up cisplatin
What are the classes of repair mechanisms
Direct reversal of DNA damage
What are the classes of repair mechanisms
Direct reversal of dna damage
Base excision repair
Nucleotide excision repair
What is the direct reversal of DNA damage used for
What is included in it
Small common damage
The mismatch repair system
Photoreactivation of pyrimidines dimers
Removal of a methyl group (from alkylation) by methyl transferase
What is included in. Base excision repair BER
The nitrogenous base is removed, then repair happens
Specific for one type of damage base
What is included in nucleotide excision repair
The nucleotides is/are removed, then repaired
General to many types of damage and is used to repair a larger region
what organisms have MMR systems
When can the MMR happen
It’s conserved in prokaryotes and eukaryotes
It repairs simple dna lesions (so first Gen mismatch), so once the lesion is replicated it can’t repair
What is the limitation of MMR
Misinsertions and short indels can’t be repaired
Only small changes
In every _____ nucleotide dna pol causes ___ mismatch
But in ecoli how does the MMR help
Every 10^6
Cause 1
Is makes it so every 10^9 or 10^10 nucleotides has a mutation because MMR repairs it
What is the first step in MMR system in e.coli
If a mismatch by dna pol 111 happens
There is a palindromic sequence in the ecoli strands : GATC
The newly made strand gets recognized by DAM methylase
DAM methylase methylated the adenine residue in the GATC sequence in the parent strand of the dna
There is a split second where the daughter strand is not methylated (this is hemimethylation)
This lets the repair enzymes distinguish the parent strand from the daughter strand
If muts is mutated what happens
You get lots of mutations in. Ecoli because it’s essential for the MMR system
What are steps 2-5 of the MMR system in ecoli
MutS forms a complex with mutL on the dna
The MutS/L complex scans the dna bidirectionally to find the mismatch and forms a loop where the mismatch is
Once they find the mismatch, the complex recruits MutH to cleave at the unmethylated GATC (on the daughter strand)
Then MutS/L complex recruits UvrD (helicase II) to unwind the dna in the direction of the mismatch
When unwound, the strand with the mutH cleavage is dangling and an exonuclease comes and chews off the end with the mismatch
In MMR, when cleavage of the daughter strand at the 3’ end of the hemimethylated site (on the parent strand) happens what exonuclease gets recruited
A 5-3 exonuclease (since the end of the nick is the 5’ end on the daughter strand)
RecJ or exonuclease VII
In MMR, when cleavage of the daughter strand at the 5’ end of the hemimethylated site (on the parent strand) happens what exonuclease gets recruited
A 3-5’ exonuclease since the end of the nick is the 3’ end of the daughter strand
Exonuclease 1 or exonuclease X
What is steps 6 and 7 of MMR system (after the exonuclease degrade the mismatch)
The single stranded gap in the daughter strand is coated with single stranded DNA binding protien (SSB)
This lets dna pol III come in and fill in the proper sequence, and the nick is sealed by ligase
What is the MMR system like in eukaryotes
They have proteins that are homologous to MutS and MutL But
They lack homologs to MutH and DAM methylase
They dont use methylation to distinguish between parent and daughter strands
What are CPD photolyases
What is the chain length
Where are they found
Where are they not found
An enzyme which catalyzes the monomerization of CPD dimers
A single polypeptide chain of 454 to 614 amino acids
Found in bacterial, archeal, and eukaryotic cells.
Not found in placental mammals
What is the structure of CPD photo Lyase
Has two noncovalently bound chromophores
These chromophores absorb sunlight , meaning it needs the sun to be active and pull apart the dimers
How does the E. coli has CPD photo Lyase actually use sunlight to pull apart the dimers
It binds FADH
Also has MTHF or 8-HDF chromophores
MTHF or 8-HDF absorbs light and transfer electrons from the light to FADH
Then FADH cleave the cyclobutane ring (dimers)
What gene encodes the CPD photolyase
What domains does it have
What is its expression in wildtype cells
phr (photoreactivstion) gene
Aplha helical domain, alpha beta domain, a secondary pocket (for FADH to bind)
Low expression of phr
What happens if the phr gene is mutated
No CPD photolyase activity
Sensitive to UV exposure (since cant repair the dimers)
So dna replication stalls: grow slowly and have low viability
What domain of the photolyase can harness the sun energy
The alpha helix domain which has the MTHF region
What is the process of CPD photolyase direct reversal repair
The photolyase binds to the thymine dimer in the presence of light
Once bound it flips the TT dimer into its active site pocket using the phosphate backbone as a hinge
The MTHF absorbs a photon from light, then transfers the energy to FADH in the catalytic site
The excited FADH transfers electrons to the dimer, splitting it into two T (pyrimidines)
The electron returns to the flavin radical and forms FADH. The CPD photolyase enzyme leaves
What type of reversal mechanism is the CDP photolyase repair
One step
What is o6 methyl guanine
What does it pair with
What mutation does it cause
So what need to happen
A guanine that has been alkylated at O6
T
GC to AT transition mutations
Before the lesion turns into a mutation is need the because fixed
How does direct reversal of O6 methylguanine happen
What organisms is it in
The methyl group of o6 meG is transferred to a cysteine residue
In the enzyme O6 meG dna methyl transferase
This corrects the o6 me guanine to regular guanine
All organisms
What happens to the methyltransferase enzyme after direct reversal of O6 me guanine
Since it has been modified by taking in the methyl, it degrades itself
What is included in excision repair
BER
NER (repairs indels, more nucleotides)
More simple lesions can be repaired by
MMR
BER
direct reversal of dimers or o6me guanine
What does BER repair
Single damaged nucleotides
SsDNA breaks
How is prokaryotic/bacterial BER done
The damaged base is recognized by DNA glycosylase
DNA glycosylase hydrolyzes the N-beta-glycosyl bond (glycosidic bond) between the base and the agitate of the back bone
This makes an AP site (a basic site)
The ssDNA at the AP site is cleaved by AP endonuclease
AP endonuclease (since inside the strand) cleaves the DNA backbone at the AP site and makes a nick and released the 3’ OH and 5’ DRP (deoxyribose phosphate)
In prokaryotic BER what happens after the AP endonuclease makes a nick
The segment of the DNA is removed and sealed by the nick translation activity of Pol I
Then dna ligase seals the remaining nick
What is the nick translation activity of Pol I
How can it be used to label the dna
The dna pol 1 has two activities
Able to be an exonuclease to remove nucleotides (in this case 5-3 since 5’ end is floating up)
Also adds nucleotide from the 5-3 end
It’s a technique that use to label the dna fragment since the dntp that it adds during nick translation can be labelled
Then when repairing these get added and the dna is labelled
The DNA glycosylase is _____
What does this mean
Conserved
Meaning all organisms have some type of glycosylase for BER that recognize diff types of damage bases
If cytosine gets deaminated and turn to uracil
What bacterial glycosylase will do BER on it
Uracil DNA glycosylase (UDG)
It works only on dna since lesion that make uracil only happen on dna
Not work on RNA
What are each of the human glycolsylases and what do they fix
UNG (uracil dna N glycosylase) :
ssU, U:G, U:A
TDG (thymine DNA glycosylase)
MPG (methyl purine DNA glycosylase)
OGG1 (8-oxoguanine-glycosylase):
8-oxo-G
MYH (MutY homolog):
A:G, A:8-oxoG
NTH1 (endonuclease 3 homolog)
T-glycol, C-glycol
What happens in eukaryotic BER
The first two step are the same
But after the AP endonuclease makes the nick there is two pathways:
Long patch repair
Short patch repair
What is the eukaryotic BER long patch repair
the eukaryotic polymerase doesn’t have the 5-3 nick translation exonuclease activity
So the 5’ DRP end peels up
And flap endonuclease chops it off and fills in the gap
Then ligase seals the nicks
What is the eukaryotic BER shirt patch repair
The polymerase beta only fills in the gap from the damaged nucleotide
The ligase seals the nicks
Since the nitrogenous bases in BER are facing inward
How can the DNA glycosylase recognize the damage and flip out the damaged base
It scans the minor grooves of the helix in search of kinks
Kinks caused by the damaged base not matching perfectly with the other base
When lesion is recognized the dna bends a bit then the base gets flipped out
But the adjacent bases are not disrupted
What does NER repair
Bulky damaged bases
Indels
Generally big lesions
What how does nucleotide excision repair work in prokaryotes
How many nucleotides is the gap
The UvrA and UvrB complex scans the DNA helix for damage
When a lesion is encountered, UVRA leaves, UVRB melt the two dna strands
UVRB recruits UVRC excinuclease (excision endonuclease)
UVRC makes a 5’ and 3’ nick on either side of the damaged nucleotides (damage in between both ends of cut)
This makes a 12-13 nt ssDNA gap
UVRD helicase releases the 12-13 nt fragment by unwinding the dna
DNA pol 1 and ligase fill and seal the gap
What is the biggest difference in NER
UVRC Making two cuts
Helicase unwinds the helix to remove the entire fragment
How does NER happen in eukaryotes
How long of a gap is there
XPC scans for and recognizes the lesion
XPB AND XPD are recruited to the lesion and separate the DNA to make a ssDNA bubble
After bubble, RPA binds to the bubble and positions XPF and XPG on either side of the lesion
XPF and XPG are nucleases
XPG cleaves on the 3’ side and XPF the 5’ side
A larger 24-32 nt fragment is displaced
The PCNA clamp recruits a DNA pol to fill in the gap which is then sealed by ligase
What is transcription coupled DNA repair (TCR)
When there is unrepaired dna damage and transcription is starting
The damage is recognized by the RNA polymerase
The rna pol stalls at the site of the lesion
When stalled, the pol leaves and recruits the NER proteins
What region of DNA would have the most TCR repair
The regions that are most highly transcribed
Is the RNA pol proofreading ability as strong as dna pol proofreading?
No
What is xeroderma Pigmentosum (XP)
A autosomal recessive disease
It has a defective NER system where uv damage can’t be repaired
So the skin cells are very photosensitive
Leading the early onset skin cancer
What causes unequal crossing over of chromosomes and what does it lead to
If the chromosomes are not aligned properly during recombination, there will be unequal crossing over
This causes a duplication on one chromosome and deletion in the other
Leading to loss or enhancement in the phenotype
What causes unequal crossing over of chromosomes and what does it lead to
If the chromosomes are not aligned properly during recombination, there will be unequal crossing over
This causes a duplication on one chromosome and deletion in the other
Leading to loss or enhancement in the phenotype
In a paracentric chromosme what is the long arm and what is the short arm called
Long is Q
Short is P
What are balanced rearrangements and the types
Paracentric inversion
Pericentric inversion
What is paracentric inversion
What is the result
An inversion of genetic content in the chromosome(so before A BCD now A CBD)
But the genetic content is the same so in the parent (carrier) there is no phenotype change
But during recombination in the offspring, four types of gamete are made
Balanced: ABCD, A CBD
Inviable: ABCA (a repeat) with two centromeres or DBCD (d repeat) with no centromere
In para centric inversion what invisible chromosomes are made and what is the cause
Either acentric (none) or dicentric (2) chromosomes
Lead to unstable chromosomes and unviable offspring
What happens in pericentric inversion
Crossing over of pericentric chromosomes causes unbalanced gametes to be made
They make duplicated or deficient segments of the genes near the inverted segment (ABCA OR DBCD)
So it can make four gametes with balanced, balanced, unbalanced, unbalanced chromosomes
But still one centromere in each
What is DSBR (double stranded break repair)
What is a backup to it
Double stranded break happens due to radiation, If not repaired cell will die
Since diploid cells, homologous recombination with the other chromosome can repair the damaged DNA
So an allele on the homologous chromosome can be used as a template to replace the sequence on the damaged chromsome
How does DSBR get fixed
The double stranded broken chromosme goes through a set of cleavage
This cleavage makes 3’ overhangs
The 3’ overhang invades into the template strand (the homologous chromosome)
This lead to two outcomes:
SDSA or DSBR
What is SDSA in DSBR
Synthesis dependent strand annealing
The two homologous chromosme are separated and there is no crossing over event
What is DSBR in DSBR
Invasion happens and the dna gets repaired
But either non crossover or crossover dna is formed
What is NHEJ (non homologous end joining)
What is the problem
The homologous chromosme is not always there to act as a template
KU70/80 binds at each end of the DNA the is broken
Other proteins are recruited and the two ends are ligated together
Since forcing the two ends together, if the breakage caused deletion of DNA that deletion would stay
MUTATIONS STILL PERSIST
What is translesion DNA synthesis
The dna is replicating a damaged dna strand
DNA pol stalls during replication due to a lesion at the replication fork
The replisome complex dissociates from the DNA
Another DNA pol belonging to the Y family is recruited
What happens in translesion DNA synthesis (TLS) when the Y family polymerase is recruited
This dna pol has doesn’t have proof reading ability so forgiving to dna damage
They will continue the replication with the mutation
The mismatch can be repaired later
What is the end result of TLS
The damage is still there in the dna
But the cell doesn’t die since if it just stopped replicating it would have died
Allows survival with the mutation rather than just dying
