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MBB313 - Genome Stability and Genetic Change > Barnes - MMR, DNA helicases, NHEJ, VDJ > Flashcards

Barnes - MMR, DNA helicases, NHEJ, VDJ Flashcards

1
Q

In summary what happens in MMR?

A
  • recognition and repair of single bp mismatches and small insertions/dels
  • monitors rep
  • monitors recomb between homeologous
  • v important for genome integrity
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2
Q

What do inherited mutations in MMR lead to?

A
  • HNPCC (Hereditary Nonpolyposis Colorectal Cancer)
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3
Q

What are homeologous seqs?

A
  • seqs that are not quite homologous, but nearly
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4
Q

What types of mismatches are recognised by MMR?

A
  • non watson crick bps

- short insertion/del loops of typically 1-3nt (could be a few more)

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5
Q

What can’t MMR recognise, and what does?

A
  • bulky structures –> adducts etc.

- worked on by other systems (nucleotide excision repair)

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6
Q

What are non Watson Crick bps, and what is the result of them?

A
  • simply incorrect addition of bases

- half cells will have mistake conserved

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7
Q

When do short insertion/del loops typically occur?

A
  • if have microsatellite and pol slips when passing through, then when starts rep again might not start in exactly the right place (wrong repeat)
  • if starts too early get extra microsatellite
  • or too late and get less copies of microsatellite in daughter cell
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8
Q

What % of colorectal cancers are due to inherited mutations, and MMR mutations?

A
  • 30% due to inherited mutation

- and 10% of those due to MMR mutations (2-4% of all colorectal cancer cases)

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9
Q

What are MMR deficient cells esp prone to?

A
  • microsatellite instability –> hallmark of this type of cancer
  • so increased risk of acquiring mutations that transform cells to be cancerous
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10
Q

Is MMR proofreading, why?

A
  • no, only recognising mismatches that have escaped proofreading by pols
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11
Q

What happens when pol recognises a mismatch?

A
  • stall, sometimes fall off and other enzs come and remove mismatched strand
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12
Q

Do pols have proofreading activity, if so how?

A
  • Most have intrinsic proofreading activity –> exonuclease activity to rewind back and synthesise the DNA region again
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13
Q

Does pol proofreading and MMR work well in WT cell?

A
  • work together v well

- estimate only 1 error every 250 gens in S. cerevisiae

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14
Q

Is it ideal to have no mistakes made by pols/MMR?

A
  • don’t want no mistakes, as need some for evo to act on for selection
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15
Q

What is the E. coli paradigm of MMR?

A
  • rep error causes mismatch
  • MutS recognises mismatch
  • MutS attracts MutL and MutH
  • MutH nicks newly synthesised strand
  • exonucleolytic degrad passed the mismatch
  • resynthesis
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16
Q

What would happen w/o MutS?

A
  • get more digested DNA, as recognises mismatches for repair
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17
Q

What does undigested DNA look like on a gel?

A
  • largest band at top of gel
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18
Q

What would happen if no MutS in DNA w/o any mismatches?

A
  • wouldn’t matter
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19
Q

Does MutS bind homoduplex or heteroduplex DNA?

A
  • binds mismatches (heteroduplex), but doesn’t bind at all to perfectly matched DNA (homoduplex)
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20
Q

What is the structure of MutS?

A
  • homodimer = ring around DNA
  • mismatch recognition dom –> creates kink at mismatched site in the DNA (only 1 subunit actually binds mismatch = asymmetry)
  • ATPase dom –> binds ATP once mismatch identified (important for next stages of MMR
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21
Q

What does MutS recognise, and how?

A
  • heteroduplex DNA
  • mismatches sensed due to changes in thermal stability (ie. interactions between mismatches bps are weaker)
  • diff efficiency for recognising diff types of mismatch
  • mechanism:
  • -> MutS binds nonspecifically to DNA
  • -> creates bend in DNA duplex to test thermal stability
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22
Q

What is hemimethylation?

A
  • half DNA meth
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23
Q

Why do E. coli have hemimeth?

A
  • tell MMR which strand to repair
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24
Q

How do E. coli use hemimeth to aid MMR?

A
  • DNA of some bacteria methylated at GATC sites by Dam methylase –> adds methyl group to A of GATC
  • Dam methylase about 2 mins behind rep fork (lag), so for a while DNA hemimeth
  • therefore in mismatched DNA, it is the newly unmeth strand that needs to be nicked and removed
  • GATC sites fairly rare so MMR machinery needs to diffuse away from mismatch in order to find one and work out which strand to repair
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25
Q

How does MutS change conform after mismatch recognition, and why does it change?

A
  • “sliding clamp”
  • forms ring around DNA and can move along
  • 600x more stable on DNA than when MutS searching for mismatches
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26
Q

How does MutS find GATC sites=?

A
  • has to go maybe few 100 bp to find GATC site
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27
Q

How is ATP involved in MutS function?

A
  • MutS has ATPase dom
  • ATP hydrolysis needed for testing for mismatches, but is suppressed after mismatch recognition
    ATP bound stably in sliding clamp mode
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28
Q

What is the role of MutL?

A
  • the “matchmaker”

- contacts w/ mismatch bound MutS

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29
Q

What is the structure of MutL?

A
  • also forms homodimer ring around DNA
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30
Q

What does MutL interact w/, and what is the purpose of these interactions?

A
  • MutH endonuclease, which nicks DNA
  • UvrD helicase, which unwinds DNA from the nick
  • exonucleases to remove the newly synthesised strand
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31
Q

What is the role of MutH endonuclease

A
  • cleaves unmeth stand at hemimeth GATC site
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32
Q

What does MutH endonuclease req to carry out cleavage?

A
  • metal binding (Mg2+)
  • hemimeth GATC recognition
  • interaction w/ MutL
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33
Q

What type of DNA does MutH cleave?

A
  • only hemimeth

- unmeth and fully meth isn’t

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34
Q

What controversy is there around MutS, but what is known?

A
  • how diffusion away from mismatch and complex formation w/ other prots happens
  • but importantly there is a loading of a no. of these complexes onto the mismatched DNA
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35
Q

How might MutS interact w/ the 3 central MMR prots?

A
  • MutS recognises a mismatch
  • then recruits MutL, then MutH, the whole complex moves together
  • MutL and MutH can detach and move more quickly in search of hemimeth sites (= yoyo model)
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36
Q

What are the ds steps of MutS?

A
  • UvrD helicase unwinds DNA back towards mismatch
  • exonucleases: at least 4 diff ones –> both 5’-3’ and 3’-5’, so doesn’t matter which side nick is on
  • DNA pol synthesises strand again
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37
Q

What pols carry out leading and lagging strand rep in euk MMR?

A
  • leading strand rep by Polε

- lagging strand rep by Polα and Polδ

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38
Q

Is there a euk MutH homolog?

A
  • no, but variety of mechs have been proposed and prob work together to ensure strand discrimination
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39
Q

What are the effects of msh2/3/6 mutations on MMR?

A
  • msh2 mutants have bigger defect in MMR

- msh3 and 6 have defect but nowhere near as big

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40
Q

What is the effect of having double/triple mutations to msh2/3/6?

A
  • if mutate msh3 and msh6 together then nearly as bad as msh2
  • but if mutate msh2 w anything else then doesn’t make it any worse
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41
Q

What can be concluded about the epistatic relationship between the 3 MutS homologs?

A
  • most important prot is msh2

- then mhs3 and 6, prob act in parallel pathway

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42
Q

In a gel shift experiment what are the diff bands?

A
  • if prot binds, then moves slowly and get a band

- if bands all the way across, then just 2° structure from substrate and not relevant

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43
Q

In a gel shift experiment where is the naked DNA?

A
  • at the bottom
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44
Q

Can msh2/3/6 bind DNA on their own?

A
  • no
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45
Q

Can msh2/3/6 or any combos between them bind to homoduplex DNA?

A
  • no
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46
Q

What is the euk homolog of MutS?

A
  • msh prots
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47
Q

What does the fact that msh prots are heterodimers reflect?

A
  • that bacterial homolog is an asymmetric homodimer
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48
Q

How does msh2 contact the mismatch?

A
  • binds ATP

- pairs with Msh3 or Msh6, which contact the mismatch

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49
Q

Which combos of msh prots bind to larger and smaller IDLs?

A
  • Msh2 + Msh3 (= MutSβ) bind to larger IDLs

- Msh2 + Msh6 (= MutSα) bind to bp-bp mismatches and 1 bp IDLs

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50
Q

What is the role of msh4 and msh5?

A
  • meiosis specific homologs
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51
Q

What diff MLH prots are there?

A
  • MutL homologs

- PMS (post meiotic seg) prots

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52
Q

What is MutLα in humans and yeast?

A
  • humans = MLH1-PMS2 dimer

- yeast = MLH1-PMS1 dimer

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53
Q

What is the role of MutLα?

A
  • mostly involved in post replicative MMR

- also is an endonuclease

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54
Q

What is MLH1-MLH3 heterodimer mostly used for?

A
  • reg of meiotic recomb
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55
Q

What additional role do many euk MutL homologs have, to matchmakers?

A
  • also have endonuclease activity
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56
Q

What is the role of MutL homologs?

A
  • take the role of E. coli MutH –> endonucleolytic cleavage of one strand in heteroduplex DNA
  • no seq specificity
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57
Q

What type of enz is exo1?

A
  • 5’-3’ exonuclease
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58
Q

What is the role of exo1?

A
  • req for reconstituted eukaryotic MMR in vitro

- has been shown to interact with MutS and MutL homologues

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59
Q

Why is exo1 not req in vivo?

A
  • prob redundant systems that can take over if its absent
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60
Q

What is the probable mech of exo1 for strand discriminate?

A
  • exo1 is loaded readily at the nicks in-between Okazaki fragments (lagging strand only)
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61
Q

What is PCNA?

A
  • proliferating cell nuclear antigen

- processivity factor for DNA polymerase

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62
Q

What are the roles of PCNA?

A
  • enhances MutLα endonuclease activity (not essential)

- interacts with exo1

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63
Q

How can interaction w/ PCNA result in strand discrimination in euk MMR?

A
  • due to asymmetrical loading of PCNA onto nascent DNA
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64
Q

Do exo1 indep pathways exist?

A
  • yes
  • exo1 can remove the mismatched strand
  • but not essential for MMR in vivo (mild mutator phenotype)
  • so there is 2nd, Exo1-indep, pathway of eukaryotic MMR –> not clear what alt exonuclease is
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65
Q

What is the 2nd main job of MMR?

A
  • monitoring HR
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66
Q

What is conjugation, and what can it measure?

A
  • integration by recomb of DNA from 1 bacteria species into another
  • way of measuring recomb between homeologous species
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67
Q

What were the findings of an experiment that looked at interspecies recomb and measured conjugation?

A
  • looked at recomb between Hfr DNA from S. typhimurium and circular chrom of E. coli
  • 735x more recomb in absence of MutS prot
  • so MMR somehow blocking recomb between these divergent seqs
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68
Q

How does strand invasion result in heteroduplex, and what happens next?

A
  • once occurred, then have mismatches in this region
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69
Q

What is the action of MMR machinery on heteroduplex DNA?

A
  • acts to recognise heteroduplex DNA and process it
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70
Q

What is the general trend in recombination efficiency as sequence divergence increases?

A
  • recomb decreases = loglinear relationship
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71
Q

What is the general effect of removing the MMR pathway (msh2 KO)?

A
  • amount of recomb increases
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72
Q

What is the effect of very low levels of divergence on recomb efficiency?

A
  • if perfectly matched (0% divergence) then exactly the same for MMR KO and WT cells
  • but if look at even v low amounts of mismatches then see sharp reduction in WT cells comp to MMR mutants (single mismatch enough to reduce recomb efficiency in MMR+ cells)
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73
Q

What is the effect of mismatches, and the MMR system, on homologous recombination?

A
  • mismatches reduce efficiency of MMR
  • mismatches reduce amount of recomb
  • MMR limits recomb between homeologous DNA molecules
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74
Q

What is the purpose of MutS homologs used experimentally to recognise mismatches?

A
  • to trap mismatches intermeds to mark them as a problem and stop them being resolved
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75
Q

What is heteroduplex rejection, and how does it occur?

A
  • unwinding of mismatched DNA by helicases, not necessarily part of machinery for post replicative MMR = “anti-recombination”
  • MutS homologs attract 3’-5’ helicases
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76
Q

How does heteroduplex rejection differ in E. coli and S. cerevisiae?

A
  • in E. coli = UvrD (so is the same)

- in S. cerevisiae = inc Sgs1 and Srs2 (not involved in post replicative MMR but are important in other repair pathways

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77
Q

Where does MMR act in HR?

A
  • at diff stages
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78
Q

What are MMR homologs important for in meiosis?

A
  • promoting CO outcomes
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79
Q

What are msh4 and msh5 homologous to, and how do they differ to this?

A
  • meiosis specific MutS homologs
  • not involved in post replicative MMR or in mitotic HR
  • homologous w/ other MutS homologs, but lacking mismatch recognition dom
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80
Q

What is the role of msh4-msh5 dimer?

A
  • recognise and stab of strand invasion intermeds
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81
Q

What is the mlh1-mlh3 dimer, and what is its role?

A
  • MutL heterodimer specialising in dHJ resolution
  • endonuclease activity
  • promotes CO, rather than non CO outcomes
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82
Q

What are DNA repair helicases involved in?

A
  • many aspects of DNA repair, recomb and rep
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83
Q

What are the roles of some RecQ helicases?

A
  • lots of interlinked roles, inc:
  • -> BLM: promotes non CO outcomes in HR
  • -> WRN: removes structures at telomeres so they can be rep
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84
Q

What are helicases, what do they function in?

A
  • class of enzs that cat sep of duplex NA into single strand in ATP dep reaction
  • function in DNA mod processing, inc DNA rep, DNA repair, recomb, transcrip, translation and many other NA related processes
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85
Q

How do helicases perform their role?

A
  • through disruption of H bonds between DNA and/or RNA strands
  • translocate along ssDNA, fuelled by hydrolysis of ATP
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86
Q

What does direction of helicases mean?

A
  • describes direction of the translocation, NOT strand being displaced from the duplex
  • DIAG
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87
Q

How do the 6 helicase superfam differ?

A
  • some, eg. RecQ and Fe-S helicases are monomeric and contain tandem repeat of RecA like motor core
  • some, eg. those involved in rep, are hexameric
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88
Q

How many RecQ members are there in humans?

A
  • 5 members
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89
Q

What are the important human SF2 DNA helicases?

A
  • 2 main subgroups: RecQ and Fe-S
90
Q

Are the members of RecQ fam structurally similar?

A
  • conserved helicase dom and other doms conserved between some but all fam members
91
Q

In what roles do SF2 helicases act?

A
  • act in many stages of rep and repair
  • inc HR repair, DNA end resection, nucleotide excision repair, fork regression, lagging strand processing, mt DNA rep etc.
92
Q

What do RecQ fam helicases interact w/?

A
  • lots of complicated interactions and overlapping effects

- often interact w/ each other, but also w/ lots of other prots

93
Q

What is the importance of E. coli RecQ?

A
  • founding member of fam
94
Q

What is the role of E. coli RecQ?

A
  • can bind and unwind no. of substrates in vitro
  • involved in no. pathways, inc. processing of ds breaks ot make 3’ overhangs and working w/ topIII to catenate and decatenate DNA
95
Q

What is the S. cerevisiae homolog of RecQ?

A
  • Sgs1
96
Q

How has RecQ been assoc w/ genetic disorders?

A
  • rare autosomal recessive disorders
  • WRN in Werner syndrome
  • BLM in Bloom syndrome
  • RECQ4 in Rothmund-Thomson Syndrome
  • all have cancer predisposition
  • shows members of fam have distinct roles and can’t sub for each other
  • lack of helicase function = chrom instability = cancer
97
Q

What are the symptoms of Werner Syndrome?

A
  • accelerated ageing

- cardiovascular disease

98
Q

What are the symptoms of Bloom Syndrome?

A
  • growth retardation

- diabetes prone

99
Q

What are the symptoms of Rothmund-Thomson Syndrome?

A
  • growth retardation
  • light sensitivity
  • cataracts
100
Q

What type of helicase are the RecQ fam?

A
  • 3’-5- helicases (translocation 3’-5’ along DNA and displacement of other strand 5’-3’)
101
Q

How does sister chromatid exchange differ in cells deficient in BLM?

A
  • increased
102
Q

What is the consequence of the fact that there is increased sister chromatid exchange differ in cells deficient in BLM?

A
  • isn’t inherently so bad, but:
  • -> is sign of genome fragility and somatic mutations
  • -> high levels of recomb can also lead to LOH, can cause cancer etc.
103
Q

What are the diff roles of BLM at various stages of recomb, how do they affect recomb?

A
  • promotes DSB processing by exo1 (pro recomb)
  • reg of Rad51 dep D loop formation (could be pro or against recomb)
  • promotion of synthesis dep strand annealing, avoids COs
  • promotion of non CO outcome (important in dissolution of Holliday junction structure)
104
Q

What is the balance of the diff roles of BLM reg by?

A
  • by various PTMs
105
Q

How is BLM important in unwinding D loops?

A
  • if D loop forms and not advantageous, perhaps if mismatches there, then BLM involved in removing Rad51 from invading DNA strand
  • important for suppression of mutagenic recomb events
106
Q

What are some eg.s of inapprop recomb events?

A
  • chromosomal instability and sister chromatid exchange
107
Q

What makes up the BLM dissolvasome?

A
  • BLM + TOPOIIIα (+ RMI 1-2)
108
Q

What is TOPOIIIα, and what is its role?

A
  • type 1 tpm
  • creates nick in 1 strand of DNA duplex
  • important for cleavage of dHJ structures → specifically in non CO orientation
109
Q

What is a model for the mech of TOPOIIIα?

A
  • important for moving dHJ towards each other = branch migration
  • then TOPOIIIα could come along and create nicks to undo them
110
Q

Why is there increased sister chromatid exchange in BLM deficient cells?

A
  • sister chromatid exchange happens when DNA breaks repaired via exchange of genetic material from diff parental strands
  • hyperrecomb in cells lacking BLM
  • also goes down diff pathway to that involving TOPOIIIα, therefore causing COs instead
111
Q

What is the role of WRN in HR, and how does it differ from BLM?

A
  • WRN deficient cells show defects in HR (the opp to bloom)
  • WRN helps stim resection in early stages of recomb
  • redundant w/ BLM at some of other stages too
  • also involved in NHEJ
  • promotes recomb –> needed for efficient recomb
112
Q

When can rep fork stalling occur?

A
  • eg. in presence of unusual DNA structures or adducts
113
Q

What happens when a block is encountered by rep fork?

A
  • either reversal of fork using BLM helicase activity (BLM activities that contrib here inc helicase, branch migration, annealing)
  • or conversion to recomb intermed and break induced rep = strand invasion, then rep to chrom end (BLM reg resection and strand invasion)
114
Q

How are the RecQ helicases involved w/ telomeres?

A
  • Werner syndrome patients have premature ageing and WS cells in culture have reduced replicative lifespan and enter replicative senescence prematurely
  • Bloom syndrome patients don’t have these phenotypes
  • however both interact w/ telomere structures in vitro
  • and are involved in alt lengthening of telomeres, telomerase indep pathway
115
Q

What is the shelterin complex?

A
  • prots that assoc w/ telomere for protection and to differentiate form ds break
  • inc Trf1, Trf2, Pot1 etc.
116
Q

How does shelterin complex form a loop structure?

A
  • 3’ overhang of the telomere end invades the ds region
117
Q

How is WRN involved in telomeres and D loop unwinding, why is this important?

A
  • WRN interacts w/ shelterin complex –> assoc w/ telomeres at S phase
  • WRN important for unwinding D loop at telomere, so rep can go all the way to the end
  • otherwise would get rep fork termination, would cause gradual telomere loss (faster than normal)
118
Q

What is a G quadruplex?

A
  • 2° structure in DNA caused by Hoogsteen bonds between G bases
119
Q

How does G quadruplex DNA affect rep?

A
  • prevents transcrip or rep machinery from proceeding
120
Q

Where is G-quadruplex DNA found, why?

A
  • t/o genome

- but pref found in G rich strand of telomeres

121
Q

How is G-quadruplex linked to RecQ helicases?

A
  • both WRN and BLM involved in unwinding and resolution of these structures, so that info not lost when telomeres replaced
122
Q

How do BLM and WRN affect HR?

A
  • BLM = increases, also increase in sister chromatid exchange
  • WRN = decreases
123
Q

How do BLM and WRN result in cancer?

A
  • BLM = LOH

- WRN = deficient recomb, chromosomal translocations and dels

124
Q

How are BLM and WRN involved in telomeres?

A
  • BLM = some evidence in telomere rep but no increase in replicative senescence
  • WRN = get shorter faster, genetic instability at telomeres because WRN important in dismantling D loop structures during telomere rep
125
Q

Do BLM and WRN lead to premature ageing?

A
  • BLM = no

- WRN = yes

126
Q

Where are Fe-S doms often found?

A
  • in many prots

- often redox reactions

127
Q

What does reactive iron in Fe-S dom have a role in?

A
  • DNA binding
128
Q

What direction are Fe-S helicases?

A
  • translocate in 5’-3’ direction
129
Q

What do mutations in in XPD2 cause, why?

A
  • cause disorders inc Xeroderma Pigmentosum = skin pigmentation disorder
  • have in common an extreme sensitivity to sunlight
  • because XPD is involved in NER
130
Q

What is NER responsible for?

A
  • repairing bulky adducts caused by UV damage of DNA –> these distort the helical structure of duplex DNA
131
Q

What happens when UV reacts w/ thymine, why is this bad?

A
  • linkage between 2 bases next to each other

- bad for further rep/transcrip/doing anything else w/ that bit of DNA

132
Q

What is XPD part of?

A
  • TFIIH, a basal TF
133
Q

What is TFIIH important for?

A
  • initiation of transcrip
134
Q

How is TFIIH linked to the cell cycle?

A
  • link to CAK = CDK activating kinase complex –> link to cell cycle, needed for basal transcrip
135
Q

What are 2 important helicases for NER, and do they req ATP?

A
  • XPB: 3’-5’ and XPD: 5’-3’

- both ATP dep

136
Q

How is damage recognised for repair by NER?

A
  • XPC = general pathway

- transcrip machinery = transcrip coupled pathway

137
Q

What happens in NER after damage recognised?

A
  • opening of DNA helix around lesion by these helicases
  • excision of a piece of the damaged strand and resynthesis –> by unwinding small piece of DNA around thymine dimer (24-32 nucleotides), then can be snipped away and replaced
138
Q

Does XPD have a role in transcrip?

A
  • important in transcrip but just acts as a scaffold
139
Q

What is the role of XPD arch dom?

A
  • involved in interaction w/ transcrip machinery → but mainly linked to cell cycle functions (CAK complex)
140
Q

Why are there some mutations that cause defects in NER, but not transcrip?

A
  • arch role in transcrip machinery, but not NER

- XPD helicase activity only req in NER

141
Q

How do mutations in XPD result in disease?

A
  • defective NER
  • cells can’t repair UV damage
  • hypersensitivity to sunlight, skin cancer
142
Q

What is the backup pathway to NHEJ?

A
  • microhomology mediated end joining
143
Q

When does NJEH predominate, comp to HR?

A
  • in G1 phase

- HR normally preferred in S/G2 phase of the cell cycle

144
Q

Why is NHEJ often mutagenic?

A
  • doesn’t use template

- and processing of ends is diff

145
Q

How NHEJ outcomes so variable, ie. what happens?

A
  • ds break often processed in diff ways, eg. by exonucleases in cell, so often broken in various ways
  • this is diff everytime, processed diff, so what happens to breaks is quite random
  • break bound by Ku, attracts all other important prots (inc pols, nucleases for further processing and then specialised ligases for joining)
  • then specialised ligases to do joining
  • can involve deletion or addition of bases, or even both
146
Q

What pathways compete for repair of ds breaks in DNA?

A
  • HR and NHEJ
147
Q

What does HR involve, and what does it req?

A
  • resection of long sections of DNA near break, so can get extended region of homology (by creating long 3’ overhang of 100s of bps)
  • need homologous mol nearby to guide repair
  • in theory should be repaired perfectly (but LOH)
148
Q

What does NHEJ involve, and when might this be adv over HR?

A
  • v short resection, only tiny regions of homology used
  • template indep so always poss
  • prob going to lose a bit of seq (mutagenic)
149
Q

Is NHEJ conserved?

A
  • not much enz seq conservation, except some between KU enzs

- seems to be mostly convergent evo between species, both evolved sep to do similar thing

150
Q

What is the NHEJ system made up of in bacteria?

A
  • 2 component minimal system, 1 prot to recognise DNA ends, 1 ligase
151
Q

What occurs during general genome NHEJ in vertebrates?

A
  • induction of DSB
  • recognition of DSB by Ku heterodimer (once Ku bound, everything comes along at once and binds)
  • assembly and stab of NHEJ complex at the DSB
  • bridging of DNA ends
  • activation of DNA-PKcs kinase activity
  • DNA end processing (if req)
  • ligation
  • dissolution of NHEJ complex and repair is complete
152
Q

Are Ku prots well conserved?

A
  • homology between them t/o several conserved domains –> diffs at C-ter
153
Q

What is Ku70-80 made up of?

A
  • heterodimer of 70kD prot and 86kD prot in humans (name based on mw)
154
Q

How does a footprint analysis work?

A
  • low conc DNAse, so cuts every piece of DNA once at a random place, therefore creating ladder of fragments
  • if something bound to mismatch then protects it and can’t cut here
155
Q

How does Ku70-80 bind DNA?

A
  • binds ends of DNA
  • fits into minor and major groove contours, wraps around DNA
  • can bind even on DNA assoc w/ nucleosomes
  • forms ring around DNA so can only get on when there is a break
156
Q

What is the role of Ku70-80, once it has bound DNA?

A
  • protects DNA ends from degrad (from nucleases etc.)
  • holds 2 ends close together (helps when stick them back together)
  • acts as a ‘tool-belt’ –> other NHEJ prots interact w/ Ku w/ various reqs
157
Q

What is the role of Ku w/ telomeres?

A
  • chrom ends look a lot like DNA ds breaks and also need protection
  • Ku binds capped telomeres and protects them from recomb and degrad (together w/ shelterin complex)
  • binding also important for silencing of gene exp in telomeric regions
158
Q

What does Ku cause to happen at dysfunctional telomeres lacking Shelterin?

A
  • promotes telomere fusion –> better than genetic material being lost completely
159
Q

What complex is Ku a part of in vertebrates?

A
  • DNA-PK complex
160
Q

How was it shown that Ku is a part of the DNA-PK complex?

A
  • binding of Ku to DNA, then recruitment for DNA-PKcs (but all w/in a few secs of break forming)
  • this experiment showed addition of anti-Ku Abs prevents DNA-PKcs from binding to DNA
161
Q

What does DNA-PKcs req to function?

A
  • DNA dep prot kinase

- no kinase activity w/o Ku and DNA

162
Q

How is DNA-PKcs activity controlled by phos at 2 diff types of sites?

A
  • autophosphorylation: req for end-ligation as it moves DNA-PK out of the way so its not blocking DNA ends anymore
  • transphosphorylation: generally mediated by ATM, needed for recruitment of Artemis and therefore processing the ends
163
Q

How is synapsis of DNA ends carried out by DNA-PK?

A
  • synaptic complex holding broken DNA ends together
  • 1st long range complex w/ ends tethered but far apart, req DNA-PKcs but not catalytic activity
  • then brought closer together, this DOES req kinase activity
  • ligases and other factors from downstream in pathway also needed for close-range complex to form
164
Q

How have the slightly differing complexes in synapsis of DNA ends been demonstrated?

A
  • w/ fluorophores, if complexes close enough together will glow
165
Q

When can ligases simply be used to stick ends back together?

A
  • in the case of a neat blunt ended ligation, or complementary overhangs this is easy to repair
166
Q

What are important ligases?

A
  • DNA ligase IV and XRCC4 (X-ray repair cross complementing prot 4)
  • XLF (esp important for blunt end ligation)
167
Q

Where does cooperative binding occur, in terms of ligation?

A
  • big complex w/ ligases and upstream factors at the DNA ends
168
Q

Can blunt end ligation be carried out most of the time?

A
  • no, most ends need to be processed
169
Q

Why do most ends req processing?

A
  • DNA ends may have been nucleolytically degrad or otherwise mod
170
Q

How are ends processed for ligation?

A
  • tidying up DNA ends for ligation may req nucleases and/or pols
  • removal of block ends groups by PNKP (polynucleotide kinase/phosphatase)
171
Q

How is artemis nuclease recruited and activated?

A
  • recruited to DNA ends by Ku

- activated by DNA-PKcs

172
Q

What is the role of artemis nuclease, and how is this carried out?

A
  • cuts many DNA substrates at the boundaries between ss and ds DNA
  • intrinsic 5’ exonuclease activity on ssDNA
  • in complex w/ DNA PKcs has endonuclease activity of 5’-3- overhangs and on DNA hairpins
  • really important part of VDJ recomb
173
Q

What other nuclease works in a similar way to artemis?

A
  • WRN has 3’-5’ exonuclease activity that is stim by binding to Ku and phos by DNA-PKcs
  • only human RecQ helicase w/ this type of activity
174
Q

What are the pols in humans?

A
  • pol mu and pol lambda
175
Q

What is the role of pols?

A
  • nt addition at the DNA junction, in template dep or indep manner
176
Q

Why do pols have diverse outcomes and effects on cells?

A
  • diff nuclease activities (gen 0-14bp lost) and diff addition of nts → lots of heterogeneity at the joining site
177
Q

Which occurs 1st nuclease or pol activities?

A
  • no particular order for nuclease and pol activities (can occur either way round)
178
Q

How can terminal microhomology mediate joining?

A
  • base pairing between ends (before or after some processing) can bring the ends together and promote ligation
179
Q

What is the fave substrate for NHEJ in vitro?

A
  • 4bp region in overhang, preferred to a blunt end ligation
180
Q

What do defects in vertebrate NHEJ cause?

A
  • give phenotypes you’d expect –> eg. premature ageing, cancer
  • but also immunodeficiency –> due to problems w/ V(D)J recomb
181
Q

What is the essential decision point for NHEJ or HR, why?

A
  • resection of ends is the essential decision point
  • -> NHEJ has basically no resection at end (usually less than 20 nt)
  • -> HR req lots of resection at end to create longer overhang
182
Q

How is Mre11 involved in decision between NHEJ and HR?

A
  • Mre11 endo and exo activity channels the DSB into the HR pathway
183
Q

How does alt-NHEJ differ to C-NHEJ?

A
  • alt, error prone pathway, w/ more extensive resection of DNA ends and use of small regions of homology uncovered to pair DNA strands (10 or 20 nts of homology, a bit more than in classical NHEJ)
  • more mutagenic
184
Q

How does alt-NHEJ occur?

A
  • PARP rather than Ku involved in binding the ends
  • end resection dep on nicking by Mre11 and exonuclease activities of
  • Mre11 and Exo1
  • annealing of microhomologies
  • flap removal –> poss as DNA around not necessarily homologous so can just remove
  • fill in synthesis by error prone pols
  • ligation
185
Q

When is alt-NHEJ used as a back up pathway?

A
  • once resection begun but HR not poss
186
Q

What are the effects of the cell cycle stage on decision between NHEJ and HR?

A
  • important to consider whether there will be a template available for HR
  • -> so HR favoured at S and G2 phase, and NHEJ at G1 phase
  • main role of 53BP1 is to stop end resection from happening in G1
187
Q

How important is cell cycle in making the decision between NHEJ and HR?

A
  • most important in determining what happens at ds break
188
Q

What is the effect of cell cycle stage on bacterial NHEJ?

A
  • NHEJ non essential when cells growing healthily, as rep is constant, so duplicate genome is present to provide a template for HR
189
Q

What is the effect of phosphorylation on the decision between NHEJ and HR?

A
  • complicated interactions of diff kinases
  • ATM = master regulator DNA damage response, promotes the HR pathway
  • ATM and ATR are in the same kinase fam as DNA-PKcs
  • DNA-PKcs also -vely reg ATM through phosphorylation
190
Q

Why is V(D)J recomb needed?

A
  • responsible for creating enormous diversity of Abs and lymphocyte receptors in jawed vertebrates
191
Q

What creates the breaks in V(D)J recomb, and how are they repaired?

A
  • RAG prots

- classical NHEJ

192
Q

What is site specific recomb?

A
  • recomb that happens at breaks created “on purpose” at specific DNA seqs (rather than at non seq specific breaks –> either accidental or made by Spo11)
193
Q

What are some other eg.s of site specific recomb?

A
  • Cre/lox

- other tyr recombinases

194
Q

What is the role of IgG?

A
  • binding and agglutinating pathogens
  • targeting invaders for phagocytosis
  • activating complement pathway
195
Q

How is diversity of T cells gen?

A
  • in a similar way to B cells
196
Q

What is the structure of IgG?

A
  • Y shaped prot mol
  • 2 H chains = 3x C regions and 1x V region, joined by disulphide bonds
  • 2 L chains = 1x C region and 1x V region
  • 2 identical antigen binding sites
197
Q

What part of IgG is highly variable, why?

A
  • 2 identical antigen binding sites

- as has to recognise diff antigens

198
Q

What part of IgG is conserved, why?

A
  • constant region is conserved

- this is part that interacts w/ other parts of IS

199
Q

How are IgG genes assembled?

A
  • somatic assembly from gene fragments during lymphoid cell dev
200
Q

Where does diversity of IgG come from?

A
  • random selection of gene fragments during dev
201
Q

How many diff human IgG types are poss?

A
  • 2.5 x 10^7
202
Q

What makes up a H chain V region?

A
  • 1 V (variable), 1 D (diversity) and 1 J (joining) segment
203
Q

What makes up a L chain V region?

A
  • 1 V (variable) and 1 J (joining) segment
204
Q

How big is the V region, which segment is largest?

A
  • all together around 100 AAs
  • V is biggest
  • D and J much smaller (around a dozen AAs each)
205
Q

Overall, how is reorganisation achieved by V(D)J recomb?

A
  • DNA cleavage by RAG1 and RAG2
  • hairpin formation
  • DNA break repair by NHEJ
  • DNA ligase joins nicked and repaired hairpins to form coding joint and blunt ends ligated to form signal joint (excised)
206
Q

What is SCID caused by?

A
  • mutations in RAG1, RAG2 and various NHEJ components (amongst others)
207
Q

What is SCID?

A
  • severe combined immunodeficiency)

- rare inherited disorder

208
Q

What does SCID result in?

A
  • defects in B and T lymphocytes, as genetic arrangements not able to take place
209
Q

How can SCID be treated?

A
  • sometimes w/ bone marrow transplants
210
Q

What are recomb signal seqs (RSS) and where are they found?

A
  • found flanking each of these gene fragments
  • RSS has 2 conserved seqs: heptamer and nonamer –> conserved, but not esp tightly
  • sep by non conserved spacer of either 12 or 23bp
211
Q

How is this recomb restricted to B and T cell precursors?

A
  • lymphoid specific exp of RAG1/2
212
Q

In what way is exp of RAG2 limited, why?

A
  • exp limits it to G2 –> means breaks will be repaired by NHEJ rather than HR
213
Q

How do RAG prots make a hairpin?

A
  • nick at border between signal seq and adj coding seq, cut between heptamer and coding seq
  • free hydroxyl group at 3’ end and free phosphate at 5’ end
  • OH attacks phosphodiester bond of the bottom strand –> hairpin at “coding” end, DSB at “signal” end
214
Q

What is the 12/23 rule?

A
  • RAG prots only act on 1 12RSS and 1 23RSS of a locus
215
Q

Why is the 12/23 rule important?

A
  • makes sure the rearrangements happen in the right combos –> ensures only 1 V, (1 D if H chain) and 1 J, so recomb not poss between eg. 2 J segments
216
Q

What is the role of HMGB1 in V(D)J recomb?

A
  • partly controls 12/23 rule

- by forming specific complex w/ RAG and 12RSS, but not 23RSS

217
Q

What is “beyond 12/23 restriction”, why is it needed?

A
  • makes sure that 1 segment of each type is inc
  • need to ensure inc D in H chain, as poss to get just V and J as this fits the 12/23 rule (this mech not well understood)
218
Q

How are hairpins handled by the NHEJ machinery?

A
  • Artemis endonuclease action opens the hairpins at the coding joint
  • req autophosphorylated DNA-PKcs for endonuclease activity (on its own can only process exonucleolytically)
  • MRN can act as a backup
  • doesn’t matter if all components there at once, but that is the most efficient (shown experimentally)
219
Q

What is TdT?

A
  • V(D)J specific NHEJ factor
220
Q

What is the role of TdT, how does it perform this?

A
  • adds nts in template indep manner at coding joint
  • additions create additional diversity –> can go wrong, eg. often cause the prot to go out of frame and undergo premature termination of translation
  • signal joints on the other hand are generally fused precisely, generating a circular mol
221
Q

Where is TdT exp?

A
  • only in early lymphoid cells where V(D)J recomb occurring

MBB313 - Genome Stability and Genetic Change (3 decks)

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