Mutation and Repair Flashcards
Skipped strand Mispairing
During DNA replication newly synthesized DNA containing repetitive sequences can misalign with template
Mispairing can occur on:
1. Newly synthesized strand
2. Template strand
***Mispairing is a mistake that can happen every time DNA repeats
Mispairing on Newly sythesized strands
Repetative regions in new strand can align with themselves instead of the temple – creates a bubble in dsDNA
Occurs if the same template region is repeated two times
Result: Get expansion in number of repeated sequences in the newly synthesized strands
What happens after new misaligned dsDNA
New dsDNA undergoes second round of DNA replication – NOW both strands act as template for DNA synthesis
One strand – n = 6 repeats –> Now a template and DNA polymerase will make 6 repeats
New strand – n = 8 repeats because of mispairing during replication –> If replicated then the new DNA will have 8 repeats
Expansion vs. Contraction
Expansion is more common than contraction
Contraction = lose repeats –> Occurs if misalign template
Expansion = get more repeats –> Occurs if misalign new sythesized DNA
Mispairing in Template
Mispairing can cause bubble in template = DNA polymerase can’t read part of the templaye (won’t read the part in the bubble
Result: Contraction – Newly synthesized DNA has fewer repeats
***In another round of replication = can get shorter track of repeats
What does expansion/contraction explain
Nucleotide repeat expansion/contraction = epxlains how many short polymorphic sequences in genome are provided
Polymorphic sequences
Sequence that has different forms/alleles in different individuals
Where do most nucleotide repeats occur
Most of the time nucleotide repeats occur in regions that don’t have genes = have little consequence on phenotype
Issue: Nucleotide repeats can occur in genes –> interfere with transcription/translation or protein function
- Repeats can occur in different regions of gene
Use of repeats
Used as molecular markers in lineage studying
Class of disorders from repeats
Trinucleotide repeat disorders –> caused by expansion of 3 BP repeats
Image:
If in coding sequence = get extra coding = get extra Amino Acids
If in introns = affects splicing = translation is affected
Huntingtons disease
Trinucelotide repeat disorder
Autosomal Dominant
Caused by Expansion of CAG sequence in HTT gene
- CAG = codon for Glutamine
Poly Q repeat
Abnormal HTT protein –> Leads to cell death
Symptons begin between 30 - 50 – Include Mood swings + Insteady Gait + Jerky Body movelments + Speech Loss + Dementia + Death
***Has varaible penetrance
Abnormality in Huntingtons
Normal – 5 - 35 HTT repeats (HTT gene has a small number of repeats = no symptoms)
Pathogenic – 36 - 250 Repeats
- Have varaible penetrace in the lower range
What determins huntingtons symptoms
Symtoms are correlated with the # of repeats
- Less repeats = may not have symtons = varaible penetance
More repeats = can develope more + More severes symtomes + might show symtoms earlier
- More repeats = more likley to have slippage = start having symtoms younger down pedigree + more severe
Huntington protein
Works in nueron in brain –> Expansion of CAG repeats = increase the number of codons for Glutamine (Glutamine = Q) –> Have Poly Q repeat
- Expansion of Glutamine resdiues = causes protein to misfold = brain cell death
Gene Anticipation
Describes the phenomon when a genetic disorder is passed on to the next generation and the symtoms become apparaent at an ealier age in each generation
- Individuals with lower numbers of repeats may not show symtoms or they may show them at an older age – with each generation there have been more opertunities for DNA replication and slipped strand pairing – leading to offsrping with more repeats + more sever symptomes + earlier onset
- Each time you replicate = increase the chance you will make a mistake = deeper in pedigree = more likley to make mistakes
- Slippage does NOT have to hapen each time but can
- More repeats = more likley to have slippage = start having symtoms younger down pedigree + more severe
Example – Huntington
Image – Start with 55 reaptes –> passes reapeats to half of kids (have slippage – get more repeats –> passed 85 and 75 repeats to kids) –> Kidds passed to kidds (Passed 90 and 250 – big expansion)
- Some individuals with repeats do not have symptoms BUT pass to kids that have expansions and have huntingtons
- Indiviuals with repeats pass to half their kids
- Pass down mutatnt allele BUT they often have expansion events = increase in number of repeats in kids with mutant allele
Where does Insulin come from
Insulin = produced by the pancreus
Diabetics = mutations in production of insulin –> they need to inject insulin
Human insulin can be made in E.coli + Yeast + human cells lines –> Have a plasmid (that can replicate itself) –> In the plasmid have heme fpr human insulin –> The cells make insulin – Can isolate insulin from the cells
- The E.coli are now considered recombinant organisms because they have DNA from other organsim
What cells can be used to make insulin
E. Coli + Yeast + Human Cell lines
Use of PCR + Inulin + Plasmids
Use PCR to amplify the insulin gene from human DNA THEN clone it into a bacterial Plasmid
Overall – Insert the human insulin gene –> Amplify the gene then clone into bacteria
Steps:
Cut plasmid to produce sticky ends–> Insert Insulin gene – Glue the sticky ends together –> Put recombinant plasmid in E.Coli
dATP – Primer is extended 5’ –> 3’ – nucleotides get added to 3’ end of the primer
What does DNA polymerase need
Needs dsDNA and 3’ end –> Adds nucleotides to 3’ end
ANSWER: B and C
AT C – Goes 5’ –> 3’
AT B – Goes 5’ –> 3’
D = not right because would be 3’ - 5’ (5’ - 3’ would be awauy from yellow)
A = not right because 5’ - 3’ would be away from yellow
Fowards vs. Reverse primer
Both go inward towrds the sequnece you are amplifying
- Fowards = Comes from left (goes right)
- Reverse = Comes from right (Goes left)
Fowards = Identical to the 5’ - 3’ template strand (Identical to the top strand)
- Binds to bottom strand
Reverse = Identical to the 3’-5’ strand but in reverse = reverse compliment of the 5’ - 3’ strand (reverse complement of top strand)
- Reverse = binds to top strand
Example Reverse and foward primer
Number of strands after X number of PCR cycle
After 1 cyles – Have 2 strands of dsDNA (One with orginal + One with new strand)
After 2 cycles – Have 4 strands
- One of the strand
Length of building things on orginal template
Anything being built on orginal template = Longer than desired product BUT it is ok because these get diluted
- Because primer binds to ONE strand = will go on forcever on other side
Doesn’t happen from newly synthesized because dsDNA of ONLY newly synethesied = only stops at where the primers marked it = doesn’t go on forever
- Does not go on forever because stops because the newly synthesized are from the primer end = won’t go on forever
Restriction enzymes
Cut DNA at specific sequences – bind to specific sequence and cute DNA at that site
- Bind to dsDNA at specific sequences and cit the DNA leaving single stranded overhangs
WHEN they cut = they cut so there is overhang
- Cut = get 2 peices + have sticky overhang
Why have overhang
It is hard to stick 2 blunt ended peices of DNA together – hard to get blunt ends to stick together –> overhamgs are more likley to stick together = RE cut so have sticky ends
If we add a restriction enzyme recognition seqneces to the end of PCR product we will have a much easier time getting teh PCR product to stick in the plasmid
RE + PCR
If we add a restriction enzyme recognition seqneces to the end of PCR product we will have a much easier time getting teh PCR product to stick in the plasmid
- Restiction enzyme is used to cut at the end of the gene = get sticky ends = easy to get to stick together with plasmid
Different restriction enzymes
Different restriction enzymes recognize and cleave different sequnces
- All cut in different places
Some = produce sticky ends and some produce blunt ends
- Usually use ones that make sticky ends
Example application of question – if you wanted to add sticky ends at PCR = wanted to add in extra DNA that does not match template to get places for RE to cut
Answer: Add to the 5’ end of primer because DNA polymerase adds DNA to 3’ end of primer
- DNA polymerase adds nucleotoides to the 3’ end IF have a sequence that does not match template THEN DNA polymerase might not be able to add
Adding DNA that is not on template – add to the 5’ end of the primer – IF you have enough DNA at the 3’ end of the template – DNA polymerase can sit on the dsDNA and add nucleotides to it IF you do not give polymerase enough dsDNA eher it can add nucleotodes then it will not add nucleotides – SO you add DNA at the tail end of primer because as long as have enough primer to tenplate (enough dsDNA for polymerase to hold onto) = can get DNA polymerase to add
- Add to the 5’ end to give Polymerase enough dsDNA to be able to hold onto to be able to add more nucleotides
ANSWER: Ligase
Ligase = smoothers kinks after DNA replication
- Can glue PCR product into plasmid
Recombinant insulin plasmid (process image)
PCR –> Restriction Enzymes –> Ligase –> Ends get recombinant plasmid
PCR + restcition enzymes
PCR adds the restriction enzyme binding site THEN you are able to add the restriction enzyme
***Part of primer is still there after add restriction enzyme
DNA repair take home message
- DNA mutations occur during DNA replication and DNA damage – mistakes are a fact of life
- Every time replicate DNA introduce mistakes
- Mutations in DNA and repair pathways can lead to disease (Especially cancers) OR beneficial evolutionary changes
Halmark of cancers
Mutations
How many DNA repair pathways exist
At least 6 –> There is cross talk between these repair pathways
Understanding DNA repair mechanisms is an active area of research
What do gene editting technologies rely on
Gene editting technologies (SUCH AS CRISPR) rely on DNA repair pathways
Are all mutations bad?
NO – NOT all mutations are bad
- Mutations are how we learn how cells workd
- Can be used for evolution
Mutations between parents and offspring
There are 100 nucleotide mutations between patents and offspring due to mistakes in DNA replications – MINOR source of mutations
- Mistakes due to DNA polymerase
Mutations due to DNA polymerase
Sometimes DNA polymerase makes mistakes
- Minor source of mutations
MOST enzymes do one job BUT DNA polyymerase does more than one job – It makes DNA + senses mistakes + Has EXOnucleoase activity = NOT a major source of mutations
DNA Polymerase correction
Proof reads + Exonuclease activity – Polymerase has repair mechansims – looks to see if the nucleotide is right – IF there is a mistake it can move to another domain of the enzyme –> can cut off mistake –> Add new nucelotides
Proofreading – DNA polymerase inspects each nucleotide that it inserts
Exonuclease activity – IF Polymerase senses an error it will remove that nucleotide and try again
***Sometimes errors get through
Errors in repetative regions
Can occur if DNA slips – expands and contracts DNA
Error = slippage –> Slippage gets incorporated into the next generation
In Nasacnt DNA – the new ssDNA can bind to instelf –> form a hairpun loop (stem loop)
- New DNA binds to itself NOT The template
- Result: Expands DNA
- region binds to complementray on same ssDNA –> Get loop of DNA – WHEN that DNA is used again it will become a template and NOW has extra nucelotides
Expansion Vs. Contraction
You can get slippage in the new strand or template strand
Expansion = because of Newly synthesized DNA
Contraction = because of template (lose repeats)
Expansion = MORE COMMON
UV Light + DNA
UV light = causes distortions to DNA helix + Interferes with DNA replication + Gene expressions
Occurs when have 2 T next to each other –> When UV light hits = causes covalent bonds between aromatic rings
Causes BULGE in DNA
ROS
Reactice oxygen species –> Natural byproduct of mitocondrial Pxydative Phosphorylation (because the electrons go other places – the electrons that espace react with H2O or O2 –> get radicals –> radicals damage DNA
- INflamation can increase mitocondrial ROS production
DNA damage by ROS:
1. Modified bases (Example – get C –> U)
2. Nicks in phosphate backbone – get breaks in backbone
3. Double strand breaks – get breaks in backbone (Very common)
- Big problem – lose of chunk of DNA
Double strand breaks
Very common – 10-50 every day
Causes:
1. ROS
2. Ionizing radiation (UV)
3. Chemicals
4. Physical stress on chromosomes
5. Errors in DNA repair
DNA repair pathways
- Mismatch repair (MMR) – cut out a bunch of nucleptides –> refill area –> Seal nicks
- Direct Repair – If have damage to nucleotide – gets rid of modification
- Nucelotide Excision Repair (NER) – cut out a bunch of nucleptides –> refill area –> Seal nicks
- Base Excision Repair (BER) – Have nucleotide you don’t want –> cut it out –> Put new nucelotide in –> Seal Nick
- Non-homologous end joining (NHEJ) – when have double strand breaks –> takes 2 blunt ends and glues together
- If have overhang will fill over hang to get blunt ends
- Homolgous recombination (HR) – Use recombination to copy from other chromosome (copy what is on dad chromsome onto mom’s chromosome)
Mismatch repair
Fixes Mismatches (Example G-T)
Process:
1. A mismatch on the new strand is first detected
2. Restrictino enzymes cut the DNA – EXOnucleoases resect the wrong nucleotides and a few more – leaving a gap
3. DNA polymerase resythesizes the DNA in the gap
4. Ligases seal the gap to remove the nick
How do MMR enzymes know which strand is new and which strand is old?
Old DNA = often methylated (has CH3 groups) –> MMR proteins use methylation state to decide which is the correct template strand
IMAGE = knows to cut strand with G because methylation patterns
Nucleotide excision repair
Overall: recognize bulges and misshapen DNA structure
- Fixing multiple bases NOT just one
Process: A segment of the DNA containing the misshapen is removed THEN refilled
Direct repair
Fixes chemical modification on nucleotide bases –> Enzymes recignize + remove these enzymes
NOTE – DOES NOT INTERUOPT THE PHOSPHATE BACKBONE
Base Excision repair
Overall: Fixes mismatches or chemical modifications to a base by removing and replacing a SINGLE nucleotide
ONLY 1 nucleotide is removed and replaces
CUTS AND REPAIRS PHOSPHATE BACKBONE
Issue in Base Excision Repair
BER is not always good at determining which nucleotide is the correct nucleotide = sometimes the fix leads to a permanent mutation
***New permanent mutation can be introduced
What can double strand breaks make
ds breaks can produce different types of overhands
- 5’ overhangs
- 3’ overhans
- 5’ and 3’ overhangs
NHEJ
Overall: Fixes double strand breaks
Process:
NHEJ fills in or removes overhangs to produce blunt ends –> THEN NHEJ will ligate the blunt ends together
Enzymes = Polymerases + Nucleases (exonuclease) + Ligases
Homologous repair
Overall: Fix double strand breaks
Process:
1. resect a larger portion of DNA
2. Using homologous chromosome as a template for new DNA synthesis
3. The repaired chromosome now contains a sequence that is identical to homologous chromosome
IMAGE – A part of the blue chromosome now contains a copy of the sequence from the red chromosome
***ALSO called Homologous direct repair
What can occur during homologous recombination
Crossovers can occur = reorganizes maternal and paternal alleles –> Form of mitotic recombination
What repair pathway fixes deamination
Base Excision Repair –> Have glycosylases – each one recongnizes a certain mismatch
- Glycosylases = makes a hole
BER –> Glycosylases = takes out one base – makes a hole –> Another enzyme sees hole –> takes out backbone –> polymerase fills in nucleotide in hole –> Ligate smooths gaps
- To cut nucleotide backbone = need endonuclease (because in middle)
Can’t have direct repair or mismatch repair –> Choose BER first but if mismatch finds it it will do it – can have overlap
Inversions
When regions of chromosome are reordered – often the result of mistake in DNA repair
Pericentric vs. Paracentric inversion
Pericentric = around centromere
Paracentric = only on one side of centromere – on one arm of a centromere
Position effect in inversions
When gene regulation is alterted due to inversion or transolation
Inversion can have an affect if interupts a gene or a gene regulatory region
- If affects gene regulatory region = gene is there but not being turned on correctly
Potential affect of inversions
Inversions can affect fertility – inversions often lead to gametes that are not viable = reduced fertility
IF have inversion = homologous chrosomes can’t recombine well –> can get a loop (loop around to match –> THEN they align with a loop in the chromosome and then recombination happens
END - get a gamete with extra/missing genes
- Get deletions + duplications in offspring
- Not an issue in mom/dad (inversion does not affect them) BUT get gametes that might not be viable
2nd affect – can get recombination that produces dicentric chromosome – has 2 centromeres
- The dicentric chromosome = doesn’t work in cell division –> chromosome will break
Translocations
Cut and exchange –> No cost or gains JUST peice from one chromosome going to other chromosome
- Was on one chromosome and is put on the other chromsome
Familial downsyndrome
Caused by transolaction that fuses chromosome 14 and 21 –> get 3 copies of chromsome 21 = downs syndrome
- Caused by translocation
Have chromosome 21 and 1 area of chromsome 21 and have chromsome 14 –> fuse together –> the indiviuals is normal but 3 chromosome can’t line up correctly –> have 3 possibilities for gametes (IMAGE)
Can get 2 copies of 14 or 1 copy of 14 or 3 copies of 14 –> ALL are not viable
- Carrier - has reduced fertility and 1/3 of kids will have downs syndrome
- Carrier = phenotypically normal
Weird that the individual got liver disease because they are Bb
When sequence the person – we see they are bb
What happened – they had homologous recombination –> had a ds break and fixed it by homologous recombination –> used the other chromsome as a template for repair (coped sequence from other chromome)
- End = got rid of B alleles – fixed double strand break by copying other chrosmome – THEN they became homozygous bb = got liver disease
- b is copied in place of B
Non-allelic homologous recombination
Sometimes homolgous recombination can occur between the “wrong” repetative sequneces – results in deletions and duplications (CNVs)
- Not recombing at the right spot
Simple vs. Reciprical translocations
