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BMSC320 > DNA Repair and Mutations > Flashcards

DNA Repair and Mutations Flashcards

1
Q

what are 3 types of DNA repair?

A
  1. Nucleotide selectivity: correctly pairing nucleotides (accounts for most of low error rate)
  2. Proofreading: DNA pol being able to check bases they incorporate and only proceed if correct, otherwise re-incorportate the correct one
  3. Mismatch repair: make fidelity of genome better by correcting mistakes
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2
Q

What are some descriptions of mutations

A
  • negative, neutral, or positive
  • small or big
    -spontaneous or mutagenized
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3
Q

small or big mutations

A
  • individual nucleotide changes
  • small insertion/deletions
  • large scale genomic rearrangements
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4
Q

spontaneous or mutagenized mutations

A
  • chemicals can cause mispairing, base alteration
  • radiation can break the backbone, create free radicals, and fused nucleotides
  • viruses can cause insertions
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5
Q

What causes mismatch repair?

A
  • mistakes in synthesis
  • polymerase 3’ to 5’ exo proofreading removes most misincorporated bases but mistakes occur at 1x10^-7
    -fortunately, stable H-bonded mismatches won’t give proper base-pair distances/angles
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6
Q

If the fit is poor, how come mistakes don’t get fixed by the 3’ to 5’ exonuclease?

A

mispairing by transient tautomerization (keto-enol shift)
- nucleotides have additional forms so mispairing can escape surveillance proofreading

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7
Q

What are the 3 chemistry terms for the different forms?

A
  1. Resonance - two forms coexist and one molecule switches back and forth readily
  2. Isomers - two forms exist but don’t switch back and forth
  3. Tautomers - two forms exist (one major, one minor) but switching is rare
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8
Q

Describe keto-enol shift/tautomerization

A
  • H-bond donors and acceptors are modified
  • H-bonding between bases is still really good which is why it may skip surveillance
  • the incorrect shapes are unstable so when they shift back, things get deformed which signals that there is a misincorporated base
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9
Q

Describe the 4 steps of mismatch repair in E. coli

A

occurs after replication is completed
1. detect/sense mismatch
2. determine which DNA strand is new (aka wrong)
3. cut and remove new DNA
4. resynthesize correctly

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10
Q

What is Mut S?

A
  • an ATPase
  • dimers that bind and scan the DNA backbone; essentially a sliding clamp bound to DNA
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11
Q

How is Mut S involved in mismatch repair detection?

A
  • finds small insertions, deletions, and mismatches due to their structural distortions
  • uses the change in protein conformation binding a mismatch to enhance ATP binding
  • if ATP is bound the protein stops in place with a half-life of 10 seconds which allows the next step to occur
    (ATP binds better at mismatch than if there is no mismatch)
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12
Q

How are Mut H and Mut L involved in mismatch repair?

A
  • when mismatch is bound, Mut S recruits Mut L (dimeric matchmaker in E. coli)
  • Mut L recruits Mut H (an endonuclease that determines old vs. new strand)
  • Mut H can only cut at unmethylated GATC sequences on new strand
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13
Q

What is Dam?

A
  • DNA Adenine Methylase (Dam)
  • sequence specific DNA modifying enzyme
  • Scans DNA for GATC
  • When found, Adenine is methylated GA*TC which indicates it is the old strand
  • sequences remain unmethylated for about 10 mins after replication meaning it has 10 minutes to find any misincorportated nucleotides before it loses ability to tell old strand apart from new strand
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14
Q

What happens to GATC sequences?

A
  • GATC sequences occur every 256 bp on average
  • recruited Mut H scans for GATC, cuts unmethylated strand 5’ to the G to make single nick in backbone
  • Uvr D (weakly processive 3’ to 5’ helicase) will initiate DNA unwinding at the nick and move towards the Mut S/L complex towards the mismatch
  • Exonucleases (3’ to 5’ or 5’ to 3’) remove strip of ssDNA that contains the mismatch
  • replication machinery fills in the gap, will repair up to 256 nt just to fix the one mistake
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15
Q

What are the equivalent proteins in human mismatch repair?

A
  • MSH is human version of Mut S
  • MLH is human version of Mut L
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16
Q

Structure and function of MSH

A

Heterodimer used for mismatch detection
- MSHα has affinity for mismatches and small (~2nt) loops
- MSHβ has affinity for small to large (~13nt) loops

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17
Q

Structure and function of MLH

A

Heterodimers used for recruitment
- possible has weak endonuclease activity
- disturbs the PCNA sliding clamp (which allows for pol delta and epsilon to have high processivity) which stops reaction
- recruits exonucleases

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18
Q

4 Steps of mismatch repair in Humans

A

occurs during replication
1. heterodimers of MSH’s follow pol to detect problems
2. if a mismatch is detected, heterodimer of MLH is bound which finds/releases the PCNA clamp
3. without PCNA pol delta/epsilon loses processivity and is released
4. MSH/MLH recruit helicases, replicases, and exonucleases to degrade the newly synthesized DNA

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19
Q

What are some errors and problems with human mismatch repair

A
  • inherited defects occur more because mismatch repair rates are 100-1000x lower
  • most are caused by mutations in MSH2 and MLH 1 which causes a dominant phenotype of heritable cancer risk (HNPCC = hereditary non-polyposis colon cancer)
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20
Q

How likely is HNPCC?

A
  • 80% of people with one of the mutations (MSH2 or MLH1) will get colon cancer and/or endometrial cancer in women
  • otherwise risk is only 4%
  • average age of onset is 44
  • direct testing can be antibody stain of tissues to look for missing proteins
  • indirect testing can be DNA sequencing to look for varying length/unstable repeats
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21
Q

Microsatellite testing for HNPCC

A
  • determines if individual has a mismatch repair defect
  • can’t determine which MMR gene is mutated, just that there is a mutation
  • uses normal tissue and polyp tissue from an individual
  • microsatellite is stable in cells without defect (mostly the same length)
  • microsatellite is unstable in defect cells (varying lengths)
22
Q

Antibody-labelling for HNPCC

A

normal MSH2 is brown, mutated MSH2 has no brown because there is no MSH2

23
Q

What is a non-replicative mutation?

A

-more problematic because mutation of DNA occurs before replication so there is no way to tell which strand is old and which is new
-extra UV/mutagens can make mutation rates higher than the cell’s ability to repair

24
Q

what are the 5 main types of non-replicative mutations?

A
  1. base loss
  2. cytosine deamination
  3. strand breakage
  4. alkylation
  5. UV dimerization
25
Q

describe base loss

A
  • when a base breaks off
  • depurination (A/G) occurs at 580/hour and the repair rate is too fast to measure (TFTM)
  • depyrimidation (C/T) occurs at 29/hour (less frequent) and the repair rate is TFTM
26
Q

describe cytosine deamination

A
  • cytosine deaminates and becomes uracil
  • becomes problematic with more replications where eventually C-G becomes T-A (C-G → U-G (mismatch) →U-A →T-A)
  • occurs at 8/hour and repair rate is too TFTM
27
Q

describe strand breakage

A
  • occurs at 2300/hr, repair rate is 20000/hour
  • could get saturated and not all breaks can be fixed
28
Q

describe alkylation

A
  • example is mustargen
  • occurs at 130/hour, repair rate is 50000/hour
29
Q

describe UV dimerization

A
  • responsible for highest mutagen rate (50000/hour) but has an equal repair rate (50000/hour)
    -5% of UV dimers are still present after 3 weeks
  • can cause the system to get flooded and lose ability to repair all mistakes
30
Q

what is a consequence of UV damage?

A
  • stacked thymines (thymine dimers) and rarely T-C (cyclopyrimidine dimers) sequences can be fused together by UV light
  • thymine dimer (CPD) or 6-4 photoproduct creates a bulge in DNA because the rise between T-T is reduced and hydrogen bonds with A’s are displaced
31
Q

Describe direct thymine dimer repair

A
  • bacteria and most eukaryotes (but not humans) have photolyase with breaks apart T^T
  • repair (photoreactivation) occurs when the enzyme captures a photon of visible light and uses the energy to break the T^T bond
  • DNA does not have to be resynthesized
32
Q

What is another function of photolyases?

A

Evolved into blue light sensors for circadian rhythms in plants and animals, and as magnetoreception in birds

33
Q

How do humans repair thymine dimers?

A
  • evolution selected away from ability to produce photolyase
  • instead we use nucleotide excision repair (NER)
  • recognizes mutations by localized DNA shape changes
34
Q

describe the bacterial NER system

A
  1. UvrA (ultra violet repair A) recognizes dimers
  2. UvrB flexes/wraps DNA creation bubble at lesion to recruit UvrC
  3. UvrC is endonuclease that cuts 8 bp upstream and 4 bp downstream of lesion on the strand that has the bulge
  4. UvrD is helicase which removes the DNA between the cuts
  5. polymerase and ligase fill in the correct sequence
35
Q

What are the equivalent NER genes in humans?

A

called XP# or CS# (where # = A,B,C,D…) because they cause 2 disorders
-xeroderma pigmentosum (XP)
- cokayne’s syndrome (CS)

36
Q

How does human NER recognize lesions?

A

can be one of 2 pathways, repair after is the same
1. XPC/XPE (global genome repair): surveys every sequence all the time for UV damage
2. CSA/CSB (transcription coupled receptor): detects RNA pol that stalls because it can’t transcribe thymine dimers - immediate repair

37
Q

What is xeroderma pigmentosum?

A
  • condition where people have little/no ability to repair UV light damage
  • any exposure to sun causes severe burns (some don’t “burn”, they just mutagenize)
  • rate of skin cancer is thousands of times above normal
  • average age where patients get skin cancer is 10
  • 8 genes found to be mutated in disease, 7 are implicated in NER
  • autosomal recessive, 1 in 250 000 cases
38
Q

What is Cockayne’s syndrome?

A
  • not associated with cancer because global genome repair pathway is active which fixes damage but results in slower gene expression because transcription coupled pathway is faulty
  • phenotype includes dwarfism, deafness, microcephaly, mental defects, sun sensitivity, and degenerative blindness
  • progeria phenotype results in premature aging (live to 12 years) due to some CS proteins interacting with telomeres
    -autosomal recessive, 1 in 100 000
  • no accepted reason for these phenotypes associated with lack of transcription-coupled repair
39
Q

What is base excision repair?

A

used to repair improper bases that do not significantly distort the DNA structure, often detected by sensing the minor groove
1. a non-standard base is removed by a specific DNA glycosylase but the deoxyribose and phosphate stay intact (only the base removed, not the nucleotide)
2. an AP (apurinic/apyrimidinic) endonuclease cuts 5’ to the missing base
3. A dRpase (deoxyribose-5-phosohatase) removes the sugar and phosphate
4. Human polβ fills in and ligase seals

40
Q

what are 3 ways mutagens can react with bases causing modified/improper bases?

A
  1. Alkylating agents attach to bases and can methylate (methyl/ethyl methane sulfonate MMS and EMS are common chemicals used to mutagenize in the lab)
  2. deamination removes NH2 and replaces it with oxygen producing peroxides, nitrous acid, and formaldehyde
  3. oxidation replaces H’s with O’s or OH’s producing peroxides and other rare chemicals
41
Q

What is interesting about human glycosylases?

A
  • each one has evolved to be highly specific to detect and remove a certain subset of bases
  • this is because there are several modifications that can be done to bases to result in non standard base pairing
42
Q

What is Translesion synthesis?

A
  • cellular life hacks
  • if DNA damage can’t be repaired by mismatch repair, base excision repair, or nucleotide excision repair, it may be necessary for cell survival to copy the DNA anyway
  • bacterial pol I/III and eukaryotic pol δ/ε are only able to catalyze DNA extension when proper base pairing is achieved so if this is not possible due to mutagenized template, bacteria and eukaryotes have loose/error-prone polymerases that can attempt to synthesize past an error
  • NOT repair, just damage tolerance
43
Q

What are key aspects of translesion synthesis (TLS) polymerases?

A
  • have very low processivity and synthesize only one or two bases before a better polymerase takes over
  • often very error prone with 1 in 20 errors on standard templates
  • tightly controlled and are induced by DNA damage by UV light
44
Q

what are some human examples of TLS polymerases?

A
  • Xi: incorporates bases randomly, effective for abasic sites
  • Eta: puts 2 A’s in opposite to an unfixed thymine dimer which allows synthesis of what should be correct accross a thymine dimer
  • Iota: synthesizes past 6-4 photoproducts and abasic sites by checking pairing along the side-face of the major groove (Hoogsteen)
  • Kappa: synthesize past G-G interstrand crosslinks (mustargen)
  • Zeta: extends synthesis correctly from mismatched bases
45
Q

Why are these TLS pols important for cancer research?

A

Inhibitors of the polymerases are potential chemotherapy drugs
The cells themselves normally help cancer survive other chemo drugs

46
Q

What are unintentional causes for double strand breaks (DSB)?

A
  • ionizing radiation and oxygen free radicals (environment)
  • replication of nicked NDA (nicked strand floats away because once helicase reaches it it’s no longer attached to the rest of the molecule
  • topoisomerase failure if it is blocked before it can reattach chromosomes
  • mechanical stress in mitosis such as if a microtubule is attached to both poles but the same chromosome, chromosome is ripped
  • accidental endonuclease action if a nick is created close by on opposite strand
47
Q

What are causes of intentional DSBs

A
  • V(D)J recombination (antibodies in B cells)
  • class or type switching such as mating type in yeast or antibody type switching
48
Q

what are the two possible mechanisms prokaryotes and eukaryotes may use to repair DSBs?

A
  • Homologous recombination: extremely accurate at finding broken sequence somewhere else and using it as template to attach things back together
  • non-homologous end joining: not as accurate, but has minimal sequence lost as it joins two sequences together
49
Q

when might there be a homologous sequence available for DSB repair?

A
  • in haploid and bacterial cells, may occur after DNA replication and before cell division
  • in sister chromatids because they are physically joined to one side of the break
  • in diploid/polyploid cells, may come from homologous chromosome and lead to crossover events
50
Q

5 steps of simplified homologous recombination

A
  1. DSB occurs (can be blunt ends or have overhang)
  2. 5’ ends are trimmed back with 5’ exonuclease which makes sure there is a single-stranded 3’ end that can be extended off by polymerase
  3. scanning proteins bind 3’ ends and search for homology (recA in prokaryotes, Rad51 in eukaryotes)
  4. Synthesis from homolog template (allow extension past where the break was previously, only need the homolog to synthesize past the break then we can put the pieces back together
  5. resolving the linked dsDNA molecules (resolvases cut them apart and ligate to 2 continuous dsDNAs which may cause recombination events because of cruciform structure)
51
Q

When is non-homologous end joining (NHEJ) used?

A
  • must be used when there’s no homolog, may be used when homolog can’t be found quickly
  • competes with homologous replication but is slower
52
Q

what initiates NHEJ?

A

the Ku protein complex (both prok/euk)
- has high affinity for DNA ends (5’, 3’, blunt, loops, ssDNA - all ends)
- in humans there are approx. 400 000 Ku complexes in every cell waiting to be deployed (often waiting at telomeres)

BMSC320 (9 decks)

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