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
2
Q
What do inherited mutations in MMR lead to?
A
- HNPCC (Hereditary Nonpolyposis Colorectal Cancer)
3
Q
What are homeologous seqs?
A
- seqs that are not quite homologous, but nearly
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)
5
Q
What can’t MMR recognise, and what does?
A
- bulky structures –> adducts etc.
- worked on by other systems (nucleotide excision repair)
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
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
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)
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
10
Q
Is MMR proofreading, why?
A
- no, only recognising mismatches that have escaped proofreading by pols
11
Q
What happens when pol recognises a mismatch?
A
- stall, sometimes fall off and other enzs come and remove mismatched strand
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
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
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
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
16
Q
What would happen w/o MutS?
A
- get more digested DNA, as recognises mismatches for repair
17
Q
What does undigested DNA look like on a gel?
A
- largest band at top of gel
18
Q
What would happen if no MutS in DNA w/o any mismatches?
A
- wouldn’t matter
19
Q
Does MutS bind homoduplex or heteroduplex DNA?
A
- binds mismatches (heteroduplex), but doesn’t bind at all to perfectly matched DNA (homoduplex)
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
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
22
Q
What is hemimethylation?
A
- half DNA meth
23
Q
Why do E. coli have hemimeth?
A
- tell MMR which strand to repair
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
25
How does MutS change conform after mismatch recognition, and why does it change?
- "sliding clamp”
- forms ring around DNA and can move along
- 600x more stable on DNA than when MutS searching for mismatches
26
How does MutS find GATC sites=?
- has to go maybe few 100 bp to find GATC site
27
How is ATP involved in MutS function?
- MutS has ATPase dom
- ATP hydrolysis needed for testing for mismatches, but is suppressed after mismatch recognition
ATP bound stably in sliding clamp mode
28
What is the role of MutL?
- the “matchmaker”
| - contacts w/ mismatch bound MutS
29
What is the structure of MutL?
- also forms homodimer ring around DNA
30
What does MutL interact w/, and what is the purpose of these interactions?
- MutH endonuclease, which nicks DNA
- UvrD helicase, which unwinds DNA from the nick
- exonucleases to remove the newly synthesised strand
31
What is the role of MutH endonuclease
- cleaves unmeth stand at hemimeth GATC site
32
What does MutH endonuclease req to carry out cleavage?
- metal binding (Mg2+)
- hemimeth GATC recognition
- interaction w/ MutL
33
What type of DNA does MutH cleave?
- only hemimeth
| - unmeth and fully meth isn't
34
What controversy is there around MutS, but what is known?
- 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
35
How might MutS interact w/ the 3 central MMR prots?
- 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)
36
What are the ds steps of MutS?
- 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
37
What pols carry out leading and lagging strand rep in euk MMR?
- leading strand rep by Polε
| - lagging strand rep by Polα and Polδ
38
Is there a euk MutH homolog?
- no, but variety of mechs have been proposed and prob work together to ensure strand discrimination
39
What are the effects of msh2/3/6 mutations on MMR?
- msh2 mutants have bigger defect in MMR
| - msh3 and 6 have defect but nowhere near as big
40
What is the effect of having double/triple mutations to msh2/3/6?
- 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
41
What can be concluded about the epistatic relationship between the 3 MutS homologs?
- most important prot is msh2
| - then mhs3 and 6, prob act in parallel pathway
42
In a gel shift experiment what are the diff bands?
- 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
43
In a gel shift experiment where is the naked DNA?
- at the bottom
44
Can msh2/3/6 bind DNA on their own?
- no
45
Can msh2/3/6 or any combos between them bind to homoduplex DNA?
- no
46
What is the euk homolog of MutS?
- msh prots
47
What does the fact that msh prots are heterodimers reflect?
- that bacterial homolog is an asymmetric homodimer
48
How does msh2 contact the mismatch?
- binds ATP
| - pairs with Msh3 or Msh6, which contact the mismatch
49
Which combos of msh prots bind to larger and smaller IDLs?
- Msh2 + Msh3 (= MutSβ) bind to larger IDLs
| - Msh2 + Msh6 (= MutSα) bind to bp-bp mismatches and 1 bp IDLs
50
What is the role of msh4 and msh5?
- meiosis specific homologs
51
What diff MLH prots are there?
- MutL homologs
| - PMS (post meiotic seg) prots
52
What is MutLα in humans and yeast?
- humans = MLH1-PMS2 dimer
| - yeast = MLH1-PMS1 dimer
53
What is the role of MutLα?
- mostly involved in post replicative MMR
| - also is an endonuclease
54
What is MLH1-MLH3 heterodimer mostly used for?
- reg of meiotic recomb
55
What additional role do many euk MutL homologs have, to matchmakers?
- also have endonuclease activity
56
What is the role of MutL homologs?
- take the role of E. coli MutH --> endonucleolytic cleavage of one strand in heteroduplex DNA
- no seq specificity
57
What type of enz is exo1?
- 5’-3’ exonuclease
58
What is the role of exo1?
- req for reconstituted eukaryotic MMR in vitro
| - has been shown to interact with MutS and MutL homologues
59
Why is exo1 not req in vivo?
- prob redundant systems that can take over if its absent
60
What is the probable mech of exo1 for strand discriminate?
- exo1 is loaded readily at the nicks in-between Okazaki fragments (lagging strand only)
61
What is PCNA?
- proliferating cell nuclear antigen
| - processivity factor for DNA polymerase
62
What are the roles of PCNA?
- enhances MutLα endonuclease activity (not essential)
| - interacts with exo1
63
How can interaction w/ PCNA result in strand discrimination in euk MMR?
- due to asymmetrical loading of PCNA onto nascent DNA
64
Do exo1 indep pathways exist?
- 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
65
What is the 2nd main job of MMR?
- monitoring HR
66
What is conjugation, and what can it measure?
- integration by recomb of DNA from 1 bacteria species into another
- way of measuring recomb between homeologous species
67
What were the findings of an experiment that looked at interspecies recomb and measured conjugation?
- 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
68
How does strand invasion result in heteroduplex, and what happens next?
- once occurred, then have mismatches in this region
69
What is the action of MMR machinery on heteroduplex DNA?
- acts to recognise heteroduplex DNA and process it
70
What is the general trend in recombination efficiency as sequence divergence increases?
- recomb decreases = loglinear relationship
71
What is the general effect of removing the MMR pathway (msh2 KO)?
- amount of recomb increases
72
What is the effect of very low levels of divergence on recomb efficiency?
- 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)
73
What is the effect of mismatches, and the MMR system, on homologous recombination?
- mismatches reduce efficiency of MMR
- mismatches reduce amount of recomb
- MMR limits recomb between homeologous DNA molecules
74
What is the purpose of MutS homologs used experimentally to recognise mismatches?
- to trap mismatches intermeds to mark them as a problem and stop them being resolved
75
What is heteroduplex rejection, and how does it occur?
- unwinding of mismatched DNA by helicases, not necessarily part of machinery for post replicative MMR = “anti-recombination”
- MutS homologs attract 3’-5’ helicases
76
How does heteroduplex rejection differ in E. coli and S. cerevisiae?
- 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
77
Where does MMR act in HR?
- at diff stages
78
What are MMR homologs important for in meiosis?
- promoting CO outcomes
79
What are msh4 and msh5 homologous to, and how do they differ to this?
- meiosis specific MutS homologs
- not involved in post replicative MMR or in mitotic HR
- homologous w/ other MutS homologs, but lacking mismatch recognition dom
80
What is the role of msh4-msh5 dimer?
- recognise and stab of strand invasion intermeds
81
What is the mlh1-mlh3 dimer, and what is its role?
- MutL heterodimer specialising in dHJ resolution
- endonuclease activity
- promotes CO, rather than non CO outcomes
82
What are DNA repair helicases involved in?
- many aspects of DNA repair, recomb and rep
83
What are the roles of some RecQ helicases?
- lots of interlinked roles, inc:
- -> BLM: promotes non CO outcomes in HR
- -> WRN: removes structures at telomeres so they can be rep
84
What are helicases, what do they function in?
- 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
85
How do helicases perform their role?
- through disruption of H bonds between DNA and/or RNA strands
- translocate along ssDNA, fuelled by hydrolysis of ATP
86
What does direction of helicases mean?
- describes direction of the translocation, NOT strand being displaced from the duplex
- *DIAG*
87
How do the 6 helicase superfam differ?
- 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
88
How many RecQ members are there in humans?
- 5 members
89
What are the important human SF2 DNA helicases?
- 2 main subgroups: RecQ and Fe-S
90
Are the members of RecQ fam structurally similar?
- conserved helicase dom and other doms conserved between some but all fam members
91
In what roles do SF2 helicases act?
- 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
What do RecQ fam helicases interact w/?
- lots of complicated interactions and overlapping effects
| - often interact w/ each other, but also w/ lots of other prots
93
What is the importance of E. coli RecQ?
- founding member of fam
94
What is the role of E. coli RecQ?
- 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
What is the S. cerevisiae homolog of RecQ?
- Sgs1
96
How has RecQ been assoc w/ genetic disorders?
- 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
What are the symptoms of Werner Syndrome?
- accelerated ageing
| - cardiovascular disease
98
What are the symptoms of Bloom Syndrome?
- growth retardation
| - diabetes prone
99
What are the symptoms of Rothmund-Thomson Syndrome?
- growth retardation
- light sensitivity
- cataracts
100
What type of helicase are the RecQ fam?
- 3’-5- helicases (translocation 3’-5’ along DNA and displacement of other strand 5’-3’)
101
How does sister chromatid exchange differ in cells deficient in BLM?
- increased
102
What is the consequence of the fact that there is increased sister chromatid exchange differ in cells deficient in BLM?
- 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
What are the diff roles of BLM at various stages of recomb, how do they affect recomb?
- 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
What is the balance of the diff roles of BLM reg by?
- by various PTMs
105
How is BLM important in unwinding D loops?
- 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
What are some eg.s of inapprop recomb events?
- chromosomal instability and sister chromatid exchange
107
What makes up the BLM dissolvasome?
- BLM + TOPOIIIα (+ RMI 1-2)
108
What is TOPOIIIα, and what is its role?
- type 1 tpm
- creates nick in 1 strand of DNA duplex
- important for cleavage of dHJ structures → specifically in non CO orientation
109
What is a model for the mech of TOPOIIIα?
- important for moving dHJ towards each other = branch migration
- then TOPOIIIα could come along and create nicks to undo them
110
Why is there increased sister chromatid exchange in BLM deficient cells?
- 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
What is the role of WRN in HR, and how does it differ from BLM?
- 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
When can rep fork stalling occur?
- eg. in presence of unusual DNA structures or adducts
113
What happens when a block is encountered by rep fork?
- 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
How are the RecQ helicases involved w/ telomeres?
- 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
What is the shelterin complex?
- prots that assoc w/ telomere for protection and to differentiate form ds break
- inc Trf1, Trf2, Pot1 etc.
116
How does shelterin complex form a loop structure?
- 3’ overhang of the telomere end invades the ds region
117
How is WRN involved in telomeres and D loop unwinding, why is this important?
- 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
What is a G quadruplex?
- 2° structure in DNA caused by Hoogsteen bonds between G bases
119
How does G quadruplex DNA affect rep?
- prevents transcrip or rep machinery from proceeding
120
Where is G-quadruplex DNA found, why?
- t/o genome
| - but pref found in G rich strand of telomeres
121
How is G-quadruplex linked to RecQ helicases?
- both WRN and BLM involved in unwinding and resolution of these structures, so that info not lost when telomeres replaced
122
How do BLM and WRN affect HR?
- BLM = increases, also increase in sister chromatid exchange
- WRN = decreases
123
How do BLM and WRN result in cancer?
- BLM = LOH
| - WRN = deficient recomb, chromosomal translocations and dels
124
How are BLM and WRN involved in telomeres?
- 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
Do BLM and WRN lead to premature ageing?
- BLM = no
| - WRN = yes
126
Where are Fe-S doms often found?
- in many prots
| - often redox reactions
127
What does reactive iron in Fe-S dom have a role in?
- DNA binding
128
What direction are Fe-S helicases?
- translocate in 5’-3’ direction
129
What do mutations in in XPD2 cause, why?
- cause disorders inc Xeroderma Pigmentosum = skin pigmentation disorder
- have in common an extreme sensitivity to sunlight
- because XPD is involved in NER
130
What is NER responsible for?
- repairing bulky adducts caused by UV damage of DNA --> these distort the helical structure of duplex DNA
131
What happens when UV reacts w/ thymine, why is this bad?
- linkage between 2 bases next to each other
| - bad for further rep/transcrip/doing anything else w/ that bit of DNA
132
What is XPD part of?
- TFIIH, a basal TF
133
What is TFIIH important for?
- initiation of transcrip
134
How is TFIIH linked to the cell cycle?
- link to CAK = CDK activating kinase complex --> link to cell cycle, needed for basal transcrip
135
What are 2 important helicases for NER, and do they req ATP?
- XPB: 3’-5’ and XPD: 5’-3’
| - both ATP dep
136
How is damage recognised for repair by NER?
- XPC = general pathway
| - transcrip machinery = transcrip coupled pathway
137
What happens in NER after damage recognised?
- 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
Does XPD have a role in transcrip?
- important in transcrip but just acts as a scaffold
139
What is the role of XPD arch dom?
- involved in interaction w/ transcrip machinery → but mainly linked to cell cycle functions (CAK complex)
140
Why are there some mutations that cause defects in NER, but not transcrip?
- arch role in transcrip machinery, but not NER
| - XPD helicase activity only req in NER
141
How do mutations in XPD result in disease?
- defective NER
- cells can’t repair UV damage
- hypersensitivity to sunlight, skin cancer
142
What is the backup pathway to NHEJ?
- microhomology mediated end joining
143
When does NJEH predominate, comp to HR?
- in G1 phase
| - HR normally preferred in S/G2 phase of the cell cycle
144
Why is NHEJ often mutagenic?
- doesn't use template
| - and processing of ends is diff
145
How NHEJ outcomes so variable, ie. what happens?
- 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
What pathways compete for repair of ds breaks in DNA?
- HR and NHEJ
147
What does HR involve, and what does it req?
- 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
What does NHEJ involve, and when might this be adv over HR?
- v short resection, only tiny regions of homology used
- template indep so always poss
- prob going to lose a bit of seq (mutagenic)
149
Is NHEJ conserved?
- 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
What is the NHEJ system made up of in bacteria?
- 2 component minimal system, 1 prot to recognise DNA ends, 1 ligase
151
What occurs during general genome NHEJ in vertebrates?
- 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
Are Ku prots well conserved?
- homology between them t/o several conserved domains --> diffs at C-ter
153
What is Ku70-80 made up of?
- heterodimer of 70kD prot and 86kD prot in humans (name based on mw)
154
How does a footprint analysis work?
- 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
How does Ku70-80 bind DNA?
- 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
What is the role of Ku70-80, once it has bound DNA?
- 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
What is the role of Ku w/ telomeres?
- 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
What does Ku cause to happen at dysfunctional telomeres lacking Shelterin?
- promotes telomere fusion --> better than genetic material being lost completely
159
What complex is Ku a part of in vertebrates?
- DNA-PK complex
160
How was it shown that Ku is a part of the DNA-PK complex?
- 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
What does DNA-PKcs req to function?
- DNA dep prot kinase
| - no kinase activity w/o Ku and DNA
162
How is DNA-PKcs activity controlled by phos at 2 diff types of sites?
- 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
How is synapsis of DNA ends carried out by DNA-PK?
- 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
How have the slightly differing complexes in synapsis of DNA ends been demonstrated?
- w/ fluorophores, if complexes close enough together will glow
165
When can ligases simply be used to stick ends back together?
- in the case of a neat blunt ended ligation, or complementary overhangs this is easy to repair
166
What are important ligases?
- DNA ligase IV and XRCC4 (X-ray repair cross complementing prot 4)
- XLF (esp important for blunt end ligation)
167
Where does cooperative binding occur, in terms of ligation?
- big complex w/ ligases and upstream factors at the DNA ends
168
Can blunt end ligation be carried out most of the time?
- no, most ends need to be processed
169
Why do most ends req processing?
- DNA ends may have been nucleolytically degrad or otherwise mod
170
How are ends processed for ligation?
- tidying up DNA ends for ligation may req nucleases and/or pols
- removal of block ends groups by PNKP (polynucleotide kinase/phosphatase)
171
How is artemis nuclease recruited and activated?
- recruited to DNA ends by Ku
| - activated by DNA-PKcs
172
What is the role of artemis nuclease, and how is this carried out?
- 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
What other nuclease works in a similar way to artemis?
- 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
What are the pols in humans?
- pol mu and pol lambda
175
What is the role of pols?
- nt addition at the DNA junction, in template dep or indep manner
176
Why do pols have diverse outcomes and effects on cells?
- diff nuclease activities (gen 0-14bp lost) and diff addition of nts → lots of heterogeneity at the joining site
177
Which occurs 1st nuclease or pol activities?
- no particular order for nuclease and pol activities (can occur either way round)
178
How can terminal microhomology mediate joining?
- base pairing between ends (before or after some processing) can bring the ends together and promote ligation
179
What is the fave substrate for NHEJ in vitro?
- 4bp region in overhang, preferred to a blunt end ligation
180
What do defects in vertebrate NHEJ cause?
- give phenotypes you’d expect --> eg. premature ageing, cancer
- but also immunodeficiency --> due to problems w/ V(D)J recomb
181
What is the essential decision point for NHEJ or HR, why?
- 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
How is Mre11 involved in decision between NHEJ and HR?
- Mre11 endo and exo activity channels the DSB into the HR pathway
183
How does alt-NHEJ differ to C-NHEJ?
- 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
How does alt-NHEJ occur?
- 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
When is alt-NHEJ used as a back up pathway?
- once resection begun but HR not poss
186
What are the effects of the cell cycle stage on decision between NHEJ and HR?
- 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
How important is cell cycle in making the decision between NHEJ and HR?
- most important in determining what happens at ds break
188
What is the effect of cell cycle stage on bacterial NHEJ?
- NHEJ non essential when cells growing healthily, as rep is constant, so duplicate genome is present to provide a template for HR
189
What is the effect of phosphorylation on the decision between NHEJ and HR?
- 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
Why is V(D)J recomb needed?
- responsible for creating enormous diversity of Abs and lymphocyte receptors in jawed vertebrates
191
What creates the breaks in V(D)J recomb, and how are they repaired?
- RAG prots
| - classical NHEJ
192
What is site specific recomb?
- 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
What are some other eg.s of site specific recomb?
- Cre/lox
| - other tyr recombinases
194
What is the role of IgG?
- binding and agglutinating pathogens
- targeting invaders for phagocytosis
- activating complement pathway
195
How is diversity of T cells gen?
- in a similar way to B cells
196
What is the structure of IgG?
- 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
What part of IgG is highly variable, why?
- 2 identical antigen binding sites
| - as has to recognise diff antigens
198
What part of IgG is conserved, why?
- constant region is conserved
| - this is part that interacts w/ other parts of IS
199
How are IgG genes assembled?
- somatic assembly from gene fragments during lymphoid cell dev
200
Where does diversity of IgG come from?
- random selection of gene fragments during dev
201
How many diff human IgG types are poss?
- 2.5 x 10^7
202
What makes up a H chain V region?
- 1 V (variable), 1 D (diversity) and 1 J (joining) segment
203
What makes up a L chain V region?
- 1 V (variable) and 1 J (joining) segment
204
How big is the V region, which segment is largest?
- all together around 100 AAs
- V is biggest
- D and J much smaller (around a dozen AAs each)
205
Overall, how is reorganisation achieved by V(D)J recomb?
- 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
What is SCID caused by?
- mutations in RAG1, RAG2 and various NHEJ components (amongst others)
207
What is SCID?
- severe combined immunodeficiency)
| - rare inherited disorder
208
What does SCID result in?
- defects in B and T lymphocytes, as genetic arrangements not able to take place
209
How can SCID be treated?
- sometimes w/ bone marrow transplants
210
What are recomb signal seqs (RSS) and where are they found?
- 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
How is this recomb restricted to B and T cell precursors?
- lymphoid specific exp of RAG1/2
212
In what way is exp of RAG2 limited, why?
- exp limits it to G2 --> means breaks will be repaired by NHEJ rather than HR
213
How do RAG prots make a hairpin?
- 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
What is the 12/23 rule?
- RAG prots only act on 1 12RSS and 1 23RSS of a locus
215
Why is the 12/23 rule important?
- 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
What is the role of HMGB1 in V(D)J recomb?
- partly controls 12/23 rule
| - by forming specific complex w/ RAG and 12RSS, but not 23RSS
217
What is "beyond 12/23 restriction", why is it needed?
- 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
How are hairpins handled by the NHEJ machinery?
- 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
What is TdT?
- V(D)J specific NHEJ factor
220
What is the role of TdT, how does it perform this?
- 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
Where is TdT exp?
- only in early lymphoid cells where V(D)J recomb occurring
