Hu - HR Flashcards
1
Q
What can break ds DNA?
A
- gamma radiation
2
Q
How are DNA DSBs gen?
A
- when 2 complementary strands of DNA double helix are broken simultaneously at adj sites –> not at exactly the same bp
3
Q
What happens after DSB gen?
A
- 2 DNA fragments gen become physically dissoc from each other
- these ssDNAs can be degraded in cells
- formation of DSB likely cause bp deletion
4
Q
What happens if DSB occurs w/in gene?
A
- prob compromises function of prot encoded or lead to ORF shift
5
Q
What can happen if DSBs not repaired correctly?
A
- can cause various genomic rearrangements, eg. DNA deletion, translocations and fusion –> common in cancer cells
6
Q
What are the 2 main sources of DNA damage?
A
- random DNA damage events and programmed ds break formation
7
Q
How can random DNA damage occur?
A
- ionising radiation and radio-mimetic chemicals (eg. sulphur mustard) –> can break dsDNA and gen DSBs
8
Q
How can mechanical stress lead to DNA damage?
A
- mechanical stress on chroms also leads to DSBs
- eg. if not properly condensed during mitosis lagging chrom will be cut into 2 by cytokinesis
9
Q
How can other types of DNA damage be transformed into DSBs?
A
- eg. ss break, once rep fork catches site, rep of undamaged strand completed as normal, but for nicked strand rep stops at nick site and forms DSB
- DIAG*
10
Q
How does programmed DSB formation occur?
A
- can occur as intermediates in biological events, eg. DSB induces V(D)J recomb in dev lymphoid cells (essential in prod of highly diverse pool of Abs)
- eg. during meiosis cells form DSBs to initiate HR
11
Q
What are the 2 options for repair of DSBs?
A
- NHEJ
- HR
12
Q
What does NHEJ involve?
A
- direct ligation of 2 DNA fragments w/o need for homologous template
13
Q
What does HR involve?
A
- damaged chrom uses homologous seqs from undamaged DNA mol as template to repair break
- error-free (ie. high fidelity)
14
Q
Is HR conserved?
A
- highly conserved from bacteria to humans
15
Q
What process is HR similar to?
A
- DNA rep of leading strand in certain scenarios
16
Q
How doe DNA rep of leading strand occur, and how is this similar to HR?
A
- 5’ to 3’ end
- pairing of ssDNA w/ template DNA req for ssDNA extension
- HR is the same –> 1st requirement is to have this ssDNA
17
Q
What are the diff steps of HR?
A
1) DNA end resection
2) strand invasion
3) strand extension and 2nd end capture
4) DNA ligation and formation of double Holliday junction
5) resolve holliday junction
6) resolve double holliday junction
18
Q
What occurs during DNA end resection (1)?
A
- prod 3’-ssDNA tail from DSB → vital to allow pairing of damaged DNA w/ template DNA
- formation of 3’-ssDNA req nucleolytic degrad of 5’ terminated strands = 5’-3’ end resection
19
Q
What occurs during strand invasion (2)?
A
- gen 3’-ssDNA searched homologous region in template DNA through bping
- displaces other strand from template DNA and forms bps w/ its complement
- this forms a D-loop structure –> similar to rep fork but lack lagging strand
- then damaged DNA extended through DNA synthesis
- therefore role of strand invasion is to establish rep fork like structure and prepare it for DNA synthesis
20
Q
What occurs during strand extension and 2nd end capture (3)?
A
- extension of invading strand in D loop = 1st end synthesis (similar to leading strand extension in DNA rep)
- other end will pair w/ displaced strand and also extended by DNA synthesis
21
Q
What occurs during DNA ligation and formation of double Holliday junction (4)?
A
- once damaged DNA sequences filled, 2 DNA fragments ligated by DNA ligase
- strands of rep DNA will still pair w/ template DNA
- at pairing transition sites, forms 2 X shaped structures = double Holliday junction
- damaged DNA seq repaired, but 2 DNA mols intertwined
22
Q
What is a Holliday junction?
A
- branched nucleic acid structure that contains 4 double stranded arms joined together
23
Q
What occurs during resolution of holliday junction (5)?
A
- opp 2 strands are cut or nicked to sep 2 double helixes and nicks ligated
- ligation results in 2 sep molecules
- either cut horizontally or vertically w/ 50/50 chance
24
Q
What occurs during resolution of double holliday junction (6)?
A
- if have DNA mol carrying alleles A and B, and other carries a and b
- HR takes place in between and forms double Holliday junction
- during resolution there are 2 cuts at 2 Holliday junctions
- if both cuts on same strands it is straightforward and get repair w/o CO –> after 2 DNA mols sep and ligated, A/B and a/b still linked, so no exchange between 2 mols
- if 2 cuts on diff strands then get repair w/ CO
- -> to sep 2 DNA mols junction has to be turned 180°
- -> after ligation A linked w/ b in 1 DNA mol and a linked w/ B in other mol
- -> so damaged chrom and template chrom exchange genetic material = chromosomal CO
25
What are the diff sources of homologous templates?
- sister chroms
- homologous chroms
- homologous seqs from non homologous chrom
26
How can sister chroms be used as a source of homologous template, and when are they used?
- identical copies formed by DNA rep, exchange between them wont alter genetic composition (as exactly the same)
- used high freq in mitosis, low freq in meiosis
- CO has no effect
27
How can homologous chroms be used as a source of homologous template, and when are they used?
- chrom pairs of same length, centromere position and staining pattern, w/ genes for same prots at corresponding loci, but can be diff alleles
- high freq in meiosis (as need to break gene linkage and create genetic variation), low freq in mitosis
- CO has effect
28
How can homologous seqs from non-homologous chrom be used as a source of homologous template, and when are they used?
- repetitive elements make up 1/3 human genome, if DSB occurs here then other repetitive seqs in non-homologous chroms can be used as repair template, leading to chromosomal translocation
- low freq in mitosis/meiosis, as rare type of repair
- CO has effect
29
What are the consequences of COs in meiosis?
- CO between homologous chroms breaks linkage in meiosis
| - CO formation during meiosis prod 50% germ cells which have recomb genotypes
30
What are the consequences of a CO in mitosis?
- if have heterozygosity between 2 homologous chroms, then DSB between this locus and centromere, repaired using homologous chrom as template, CO formation causes DNA exchange
- this pair of sister chroms sep into 2 daughter cells
- homologous chroms randomly sorted into 2 daughter cells
- so 50% chance that daughter cells contain homozygous alleles = loss of heterozygosity --> can have serious consequences
31
How is heterozygosity defined?
- homologous chroms have 2 diff alleles at locus in diploid cell
32
How can LOH have such serious consequences?
- most mutations recessive, heterozygosity provides genetic buffering of toxic mutations
- LOH common genetic event in cancer dev and involved w/ loss of WT tumour suppressor gene in many inherited cancer syndromes
33
What is the genetic consequences of CO using diff homologous templates?
- sister chroms = no genetic change as identical
- homologous chroms
= in meiosis can break genetic linkage and lead to recomb in meiosis (important for creating genetic variation)
= in mitosis could cause LOH (may increase cancer risk)
- homologous seqs from diff chroms = chrom translocation, can cause many diseases, eg. cancer, infertility, Down Syndrome
34
Why is end resection 5’ to 3’?
- if had 3’-5’ resection, would prod 5’-ssDNA, could invade DNA by pairing w/ complementary strand of template DNA, would create D loop
- but would have 5’-ssDNA so DNA synthesis can’t extend it
35
What is nucleolytic degrad?
- hydrolytic cleavage of phosphodiester bond linking 2 nts in DNA backbone, by a water mol, breaks DNA chain, carried out by DNAse
36
What does DNA resection involve?
- formation of 3’-ssDNA req nucleolytic degrad of 5’ terminated strands
37
What are the 2 types of DNAses, and where do they cleave?
- endonuclease --> cleaves phosphodiester bond w/in DNA chain
- exonuclease --> cleaves nts 1 at a time from 3’ or 5’ end, have polarity of digestion (either 3’-5’ or 5’-3’)
38
If looking for key prot(s) for initiation of DNA resection, what characteristics are expected?
- role in DNA damage repair --> so mutation makes cells sensitive to DNA damage
- binds DNA damage sites at early stage (as resection is 1st step in repair)
- nuclease activity
- mutation defects gen of 3’-ssDNA during DSB repair
- based on this prot Mre11 found as the nuclease initiating resection
39
What evidence is there for the fact that Mre11 is req for DNA damage repair?
- budding yeast cells treated w/ 2 chemicals to induce DSBs
- -> HU (hydroxyurea) = decreases dNTP prod by inhibiting ribonucleotide reductase, causing stalked DNA rep forks, eventually leads to DSBs
- -> CPT (camptothecin) = a TOPI inhibitor, stab TOPI-DNA covalent adducts and induces SSBs, which eventually leads to DSBs
- in cells w/ WT Mre11, DSB can be repaired and cells able to grow w/ media containing HU and CPT
- if Δmre11, failure to repair DSBs, inhibits growth under DNA damage conditions
40
What is ribonucleotide reductase req for?
- biosynthesis of dNTPs
41
What evidence is there that Mre11p recruited to DNA damage sites at early stages of DNA damage repair?
- Mre11 labelled w/ fluorescent prot
- induced DSBs
- repair prots should be recruited to this region
- before damage Mre11 floating around nucleus, but 1 min after DSB induction, Mre11 recruited to damage sites and formed strip of fluorescence
- entire repair process between 30 mins and up to several hours, so 1 min is early stages
42
What evidence is there for exonuclease activity of Mre11?
- Mre11 has nuclease domain, but nuclease activity undetectable --> implies other prots might be req for its nuclease activity
- when Mre11 purified from cells, prots Rad50 and Nbs1 co-purified
- form MRN complex --> has nuclease activity
- these prots highly conserved in euks
43
What is the yeast ortholog of Nbs1 and the MRN complex?
- Nbs1 = Xrs2
| - MRN = MRX complex
44
What direction of exonuclease activity does MRN complex have, and how was this found out?
- would expect 5’ to 3’ in order to prod 3’-ssDNA
- to test this DNA mols labelled w/ radioactive P32 (at either 5’ or 3’ end), which prod autoradiographic signals
- after incubation w/ MRN complex, 3’-P32 removed, but not 5’ end --> revealed that MRN complex degraded 3’ end nts and has unexpected 3’ to 5’ exonuclease activity
45
Does the MRN complex have endonuclease activity, how was this shown?
- yes, shown by digestion of circular DNA (which resists exonuclease activity as no 3’ or 5’ end)
46
How does MRN complex prod 3’-ssDNA using its nuclease activity?
- both endo and exonuclease activities req for resection in sequential manner
- Mre11 nicks dsDNA by endonuclease activity
- then degrades DNA in 3’-5’ direction towards DSB end, by exonuclease activity
- thus prod 100-300nt 3’-ssDNA
47
How do cells reg endo and exonuclease activity of MRN complex?
- req Sae2 (yeast)
| - ortholog = CtIP
48
What is the effect of Sae2 on Mre11s nuclease activity, how was this determined experimentally?
- 50bp dsDNA used as substrate, 1 of 3’ ends labelled w/ P32
- DNA incubated w/ MRN complex or MRN complex + Sae2 for up to 1 hr
- dsDNA denatured to ssDNA
- size of the 1st product is 1nt, as prod by hydrolysing 1st nt at 3’ end, so represents exonuclease activity
- sizes of 2nd product vary, as prod from random cut in middle of DNA, represents endonuclease activity
- product 2 req Sae2, but has little effect on product 1 --> therefore Sae stimulates endonuclease activity, but has no effect on exonuclease activity
49
What is the result of CtIP mutations in humans?
- Seckel and Jawad syndrome
- belong to group of genome instability disorders, collectively referred to as DNA damage response (DDR) and repair defective syndrome
50
Why do cells use such a complicated pathway to initiate DNA resection, instead of just 5’ - 3’ digestion (straightforward and high efficiency)?
- complicated as time consuming, energy consuming and low efficiency
- 5’-3’ nucleolytic degrad req accessible 5’ end for exonuclease, but often accessibility blocked
- -> by covalently bound prots at 5’ end (attached by eg. DSBs prod by TOPII in cancer treatment, or Spo11 during meiosis)
- -> or 2° structure at DSB site can prevent 5’-3- digestion, eg. when inverted repeat seqs form cruciform DNA structure through self folding, structure cut and transformed to DSB, ends capped by hairpin structure, but no free 5’ ends for exonuclease
* DIAG*
51
What is the main function of Mre11 in DNA resection?
- to initiate DNA resection by cleaning DSB ends
52
How does Mre11 perform its role in DNA resection?
- MRN complex cuts at internal site, 100-300bp away from DSB ends, and degrades DNA towards the ends
- so can release covalently bound prots from DSB sites or prod 5’ end in the hairpin capped ends
53
Why is DNA end resection by MRN complex and Sae2 not efficient for HR, and what is the result of this?
- slow and limited to only 100‐300 nt
- resection can be processed up to 3.5 kb in human cells --> suggests there's other resection pathways which are more efficient
54
What redundant pathways can take over DNA end resection form MRN complex and Sae2?
- Exo1 pathway
| - Sgs1/Dna2 pathway
55
What is Exo1?
- a 5'-3' exonuclease
56
Is Exo1 more efficient than MRN complex?
- carries out resection faster --> more efficient and can prod 5-10kb ssDNA
57
How was it experimentally proven that dsDNA w/ short 3’-ssDNA gen by Mre11 is preferential substrate for Exo1?
- 3 types of dsDNA used as substrates for Exo1
- 1st w/ 4 extra nts at 3’ ends, mimicking Mre11 resection DNA
- showed resection of dsDNA w/ 3’-ssDNA by Exo1 most efficient and those w/ 5’-ssDNA the least efficient, thus indicating Mre11 resected DNA is preferred substrate of Exo1
58
How does binding of RPA w/ ssDNA prevent over resection by Exo1?
- RPA has much higher affinity for ssDNA than dsDNA and isn’t directly involve w/ resection process
- w/ RPA resection stops at a certain point and prod uniform ssDNA (rather than cont until DNA completely degrad), as RPA binds ssDNA prod by Exo1 and prevents further resection
- in vivo RPA also protects 3’-ssDNA from other nucleases
59
What is the role of RPA?
- to protect ssDNA prod by resection
60
How was an experiment carried out to examine how Sgs1/Dna2 resects DNA, and what were the findings?
- dsDNAs incubated w/ either Sgs1, Dna2 or Sgs1+Dna2.
- heat treatment prod ssDNA, as breaks H bonds holding 2 strands together
- Sgs1 unwound 2 annealed strands of dsDNA and sep dsDNA into single strand = a DNA helicase
61
What is the role of Sgs1 in DNA resection?
- to unwind dsDNA to prod special type of DNA as Dna2’s substrate
62
What is the function of Dna2?
- has flap endonuclease activity
- substrate is dsDNA w/ 5’-ssDNA overhang
- Dna2 randomly cleaves w/in ss regions of substrate that form flaps, eg. 5’-ssDNA overhang
63
What is an overall summary of DNA resection?
- Mre11 complex binds DSB and Sae2 stim endonuclease activity to nick DNA
- Mre11 resects DNA by 3’-5’ exonuclease activity to prod short 3’-ssDNA tail
- RPA binds ssDNA to protect from degraf
- then 2 redundant pathways take over resection: Exo1 or Sgs1/Dna2 and prod 3’-ssDNAs up to 3.5kb
64
What is the purpose of resection?
- to prod a 3' ssDNA tail
65
What happens during strand invasion?
- 3’-ssDNA invades homologous region in repair template DNA by pairing w/ complementary strand
- as a result 3’-ssDNA tail displaces like strand of template and form D loop
- so damaged DNA establishes rep fork like structure and extended by DNA rep
- invasion by strand exchange
66
What are 3 key prots involved in the strand exchange reaction?
- RPA, Rad51, Rda52
67
Where is Rad51 highly exp in higher mammals?
- in organs expected to be undergoing recombs, eg. thymus and testis
68
What is Rad51?
- the recombinase in euks
69
How is it known Rad51 is essential in mammals?
- homozygous mutant mice didn’t survive past 4 cell stage and not poss to prod homozygous Rad51 mutant cell lines, whose lethality is due to chrom fragmentation (as DSBs can’t be repaired)
70
Does Rad51 interact w/ other prots?
- functions in complex w/ other prots, most of which belong to RAD52 epistasis group
- group also inc Mre11 and Xrs2 (in mammals p95 and Nbs1)
- suggests all work in same pathway, ie. HR
71
What else does Rad51 interact w/ in mammals?
- tumour suppressors
72
Does Rad51 have orthologs?
- in mammals there are many Rad51 orthologs (working on diff types of HR)
73
How does Rad51 promote strand invasion, experimental evidence?
- expected to have interaction w/ 3’-ssDNA tail by resection
- experiment showed Rad51 directly interacts w/ ssDNA:
- -> Rad51 incubated w/ circularised ssDNA and observed w/ electron microscopy
- -> results indicated that Rad51 polymerises along ssDNA to form active nucleoprot filament
74
How does strand exchange efficiency alt when DNA/Rad51 ratio decreases?
- when ratio decreases (while levels of Rad51 increase), strand exchange efficiency 1st increases, then decreases
- so excessive Rad51 inhibits strand exchange
75
How was Rad51s DNA binding property investigated?
- EMSA (electrophoretic mobility shift assay)
76
How is EMSA used for in vitro detection of DNA/prot interaction?
- principle based fact that DNA/prot complexes migrate slower (due to increased mass), than free DNA mols in native PAGE or agarose gel electrophoresis
- so if tested prots can bind DNA, DNA/prot complexes migrate slower
- if more prots binds to 1 DNA mol, migration even slower
77
Why is EMSA also referred to as gel shift/gel retardation assay?
- as rate of DNA migration shifted/retarded when bound to prots
78
Does Rad51 bind ss or ds DNA?
- both
| - but higher affinity for binding ssDNA
79
How was it discovered experimentally that Rad51 binds ssDNA and dsDNA, and how did?
- by incubating w/ either --> showed slower migration w/ both
- higher affinity for ssDNA
- therefore excessive Rad51 formed nucleoprot filaments w/ dsDNA and inhibited strand exchange reaction w/ ssDNA
80
What is it an essential step for Rad51 to assoc w/, and why is this a problem
- essential to assoc w/ 3’-ssDNA prod by DNA resection
| - but RPA preoccupies this 3’-ssDNA tail (after prod by DNA resection)
81
What is the effect of excess RPA, why?
- excess RPA inhibits strand invasion, by competing w/ Rad51 for ssDNA binding
82
How was it shown that RPA stimulation of Rad51 strand invasion activity is dep on temp?
- if DNA pre-incubated at low temps (0° or 22°) strand invasion efficiency low and greatly improved by RPA
- but DNA pre-incubated at higher temp (37°) v capable of strand invasion and addition of RPA has little effect
- therefore pre-heating DNA has same effect as RPA
- suggests assoc of RPA w/ ssDNA prevents formation of 2° structures and facilitates strand invasion
83
How does heating affect DNA, to eg. 37°?
- can denature DNA
| - 37° wouldn’t melt dsDNA, but can eliminate ssDNA 2° structures
84
How does Rad51 replace RPA and specifically bind 3’-ssDNA?
- req Rad52 --> can bind RPA and Rad51
85
How was it shown that Rad52 also binds ssDNA much more efficiently than dsDNA?
- EMSA
- human Rad52 can form pro/DA complex w/ ssDNA, but not w/ dsDNA
- also true of yeast Rad52
86
What have recent experiments shown about Rad52 binding?
- binds to ends of broken DNA
87
How was a strand invasion experiment carried out into the role of Rad52 in HR?
- if strand invasion occurred, joint mol (JM) formed and ran slower than dsDNA
- JM formation in these conditions req hRad52 and more efficient if ssDNA 1st incubated w/ hRad52
- if the Rad51 and 52 incubated together before addition of ssDNA, no reaction --> suggesting interaction inhibits binding to DNA
- suggests Rad52 1st binds ssDNA, then recruits Rad51 to ssDNA through interaction w/ Rad51
- another function of Rad52 is to displace RPA from ssDNA
88
What is the model for Rad51 mediated strand invasion?
- RPA binds 3’ overhangs, after resection, to prevent self-folding of ssDNA --> 2° structure of ssDNA caused by self folding blocks assoc of DNA repair prots, eg. Rad52, Rad51
- Rad52 can bind RPA/ssDNA filament and displace RPA --> v important for binding of Rad51 to ssDNA
- Rad52 recruits Rad51 to 3’-ssDNA
- Rad51 triggers strand invasion
89
What are 2 further prots involved in human HR?
- BRCA1/2
90
What is the importance of BRCA1/2 genes in cancer?
- tumour suppressors
- mutations account for 5% US breast cancers
- BRCA2 also gives increased risk of male breast, pancreas and prostate cancers
- when 1 copy of BRCA1/2 mutated, LOH is a factor for cancer dev
- but mutations of single allele can be sufficient for increased risk of tumour formation
91
Is LOH seen in all cancer cells, what does this suggest?
- seen in some cancer cells, but not all
| - suggests may be a later event, rather than initiating event
92
What is the structure of BRCA1, inc its interaction w/ other prots?
- acts as scaffold for multi prot interactions
- huge prot that interacts w/ no. of other prots
- BARD1 forms core complex w/ BRCA1 through interaction w/ BRCA1 ring domain and stab BRCA1
- interacts with CtIP (human orthologue of Sae2), through BRCT repeats motif
93
What is the role of the core complex of BRCA1, and what might this be req for?
- has ubiquitin ligase activity
| - radiation resistance and tumour suppression
94
What are mutations to ring dom of BRCA1 assoc w/?
- cancer
95
What does the fact that BRCA1 interacts w/ CtIP imply?
- that BRCA1 has role in DNA resection
96
How was is shown experimentally that knockdown of BRCA1 reduces DNA damage induced ssDNA foci?
- cells treated w/ gamma-radiation = DSBs
- DNA resection prod ssDNA at 3’ end of DSBs
- anti-ssDNA antibody bound ssDNA and gen green signals, while anti-BRCA1 antibody gen red signals
- once BRCA1 knocked down, amount of ssDNA indicated by green foci greatly reduced
- w/o IR, v little ssDNA foci
- greatly induced ssDNA foci formation by irradiation
- -ve control = nonspecific knockdown --> had no effect
- however, 2 indep BRCA1 knockdowns signif decreased ssDNA foci
- revealed that BRCA1 binds CtIP to facilitate DNA resection
97
What is the role of PALB2?
- essential for assoc of Rad51 at DSB site in vivo
| - key prot for recruiting Rad51 to damage sites in human cells
98
How does BRCA1 interact w/ PALB2?
- through its coiled coil domain
99
What happens in PALB2 deficient cells?
- Rad51 fails to bind damage DNA
100
What happens when PALB2 is introd into PALB2 deficient cells, and what does this suggest?
- when introd RAD51 recruited to damage sites
| - implies BRCA1 has role in the recruitment of Rad51 to DNA damage sites
101
What are the roles of BRCA1 in DNA damage repair?
- v important to genome stability
- req for normal levels of HR in DNA damaged challenged cells
- activity could inc recruiting or helping resection prots
- interacts w/ prots that help Rad51 dep recomb
102
What are the roles of BRCA2 in DNA damage repair?
- essential gene req for cell prolif
- interactions w/ PALB2, Rad51 and Dmc1 (meiotic version of Rad1) imply key role related to Rad51
- studies in mice showed interaction w/ Rad51 and showed v similar exp pattern
- KO mouse embryos (from hetero cross) are radiation sensitive
- has OB fold motif → known as a ssDNA binding motif, suggests can directly bind ssDNA
103
How was BRCA1 binding to ssDNA showed experimentally, and what did the results suggest?
- EMSA showed it could specifically bind 3’ ssDNA
- BRCA2 slowed migration of ssDNA, but not dsDNA
- confirmed by EM
- suggests BRCA2 may play role in recruitment of Rad51 to ssDNA since Rad51 has no selective binding of ssDNA and dsDNA
104
What is the effect of BRCA2 on Rad51 binding to DNA?
- Rad51 binds ss and dsDNA (although pref ssDNA)
- BRCA2 greatly increased Rad51/ssDNA binding affinity and inhibited Rad51 binding to dsDNA
- likely that BRCA2 facilitated strand invasion by recruiting Rad51 to 3’-ssDNA tails prod by resection
105
How does BRCA2 also increase ability of Rad51 to cat strand exchange, experimental evidence?
- if strand invasion occurred, ssDNA would displace like strand and form JM w/ complementary strand
- BRCA2 increased JM
- the more BRCA2 prots, the more JMs prod
- suggests BRCA2 acts by targeting RAD51 to ssDNA over dsDNA --> enabling RAD51 to trigger strand invasion
106
In what way does Rad52 have same function as BRCA2?
- in human cells, BRCA2 is major recruiter of Rad51 --> KO of Rad52 in human cells has little effect, but if BRCA2 deficient, then Rad52 function becomes important
107
How can Holliday junctions migrate?
- branch in Holliday junction can migrate in either direction, by breaking old bps and forming new ones = process called branch migration
- migration process not spont --> req DNA unwinding, which req helicase activity and energy from ATP hydrolysis
108
How can Holliday junctions be resolved by HJ resolvases?
- HJ resolved by HJ resolvases through 2 steps:
- -> cut 4 way DNA branch at opp position and sep JMs
- -> nick ligated by DNA ligase
109
What are HJ resolvases, and what are their roles?
- group of structure selective endonucleases
- pref for binding and cleaving 4 way DNA junctions
- cleave HJ to gen unconnected DNA duplexes
- dimeric enzs and use twin active sites to cat 2 coord incisions w/in single prot HJ complex
110
In what organisms are HJ resolvases found?
- bacteria, bacteriophages, archaea, viruses, yeast, plants, mammals
111
What is RuvABC?
- HJ resolvase found in E. coli (and other proks)
112
What is the structure of RuvABC, and the role of each subunit?
- RuvA = specifically binds 4 way DNA junctions and recruits RuvC to HJ
- RuvB = DNA helicase and interacts with RuvA
- RuvA and RuvB promote branch migration
- RuvA and RuvB recruits RuvC to Holliday junction --> RuvC might displace RuvA to gain access to the junction
- RuvC = resolves (ie. cuts) junction to form 2 duplex products
113
What is a model for RuvC and the HJ complex?
- RuvC forms dimer to bind 4 way DNA junction and cut DNA junctions at opp positions
114
What is the resolvase identified in humans?
- hGEN1
115
What is the yeast equivalent of GEN1?
- YEN1
116
Does del of YEN1 affect cell survival under DNA damaging stress?
- no effect
117
Why did it take a long time for Holliday resolvases to be discovered?
- as not the major pathway for DNA damage repair in euks
118
What evidence is there for the fact that Holliday resolvases are not the major pathway for DNA damage repair in euks?
- deletion of YEN1 has no effect on cell survival under DNA damage stress
- treatment w/ several DNA damage reagents (HU, MMS, CPT) almost stopped growth of Rad52 deletion cells (as essential for strand invasion), but these effects not seen w/ YEN1 deletion cells
119
What diff strategies can cells undertake during DNA damage repair to avoid CO (as can lead to LOH)?
- use sister chroms instead of homologous chroms as repair templates
- or use diff pathways to resolve JMs in mitosis
120
What does the fact that many studies have revealed CO rarely assoc w/ mitotic DNA damage repair suggest?
- suggests resolvase not main pathway
- must be other pathways for HR in euks
- this is result of natural selection
121
What is HO system and how can it be used to measure CO rate?
- inducible DSB repair system
- gene encoding HO site specific endonuclease controlled by inducible promoter
- HO cuts both sister chroms at MATa site in ch5, so cells can’t use sister chroms as repair templates
- yeast strain also haploid and has no homologous chroms, but repair template seq MATa-inc (= MATa inactivation, can’t be cut by HO) inserted in ch3
- forces cells to use MATa-inc homologous seq to repair MATa DSB
- repair can prod either non CO or CO products
- after HR, restriction digest of non CO and CO by EcoR1 prod DNA fragments w/ diff sizes, distinguished by SB
122
What is the typical result of using a HO system as an assay to measure CO rate?
- before inducing HO endonuclease EcoR1 digest prod 2 parental DNAs
- after 1 hr induction of HO, HO cut DNA gen
- then HO exp switched off and cells started HR, and Ho cut DNAs disappeared
- repair prod far less CO products then non CO (5:95)
- therefore some prots must suppress CO formation
123
How does overexp of RAd51 affect HR?
- inhibits NHEJ and pushes cells to repair DSB using HR
124
How does del of Srs and/or Sgs1 affect no. of COs, what does this suggest?
- del of Srs2 mildly increases CO product
- del of Sgs1 greatly increases CO
- double del increases CO even more --> so effects of Srs2 and Sgs1 are additive, therefore suggests they work indep, likely they trigger alt pathways to resolve JMs after strand invasion
125
What is Srs2 and what is its role?
- DNA helicase and DNA dep ATPase
- involved in DNA repair and checkpoint recovery
- disassembles Rad51 nucleoprot filaments during recomb
- can unwind invading ssDNA/template DNA and disassemble D loop
126
What is Srs2 homologous to?
- functional human homolog, RTEL1
127
How does Srs2 activity repair DSB?
synthesis dep strand annealing
- after strand invasion and extension, instead of forming double HJ, Srs2 unwinds invading strand and disassembles D loops
- extended seqs of 2 ssDNAs, complementary to each other are annealed
- after gap filling by further DNA synthesis and ligation, DSB is repaired
- this pathway doesn’t nick template DNA
- no exchange of genetic materials between damaged DNA and template DNA --> therefore no COs
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What fam is Sgs1 a part of?
- RecQ family: the DNA helicase
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What is the role of Sgs1?
- Sgs1 eq for maintenance of genome stability in orgs from bacteria to human
- Sgs1 interacts with Top3 and stims its activity
- suppresses illegitimate recomb
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How are human homologs of Sgs1 and Top3 implicated in human disease?
- BLM and WRN can complement yeast Sgs1 mutant
| - implicated in Bloom and Werner syndrome
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What kind of enz is Top3?
- DNA topoisomerase III
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What is the role of Top3?
- Works on single DNA strand
- resolves DNA hemi-catenation, in which 1 single strand from 1 dsDNA interlaces w/ another single strand from another dsDNA
- suppresses illegitimate recomb
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How are JMs resolved by Sgs1/Top3?
- Sgs1 can move 2 branches of double Holliday junction towards each other
- once branches meet, tension caused by DNA right hand double helix will transform this part as hemi-catenation, which can then be resolved by Top3
- doesn’t involve nicking of template DNA, so no COs
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How does the purpose of HR differ in mitosis and meiosis?
- mitosis: repair spont DSBs, maintain genome stability
| - meiosis: create genetic variation by exchanging genetic material between homologous chroms
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How does the freq of HR differ in mitosis and meiosis?
- mitosis: about 1 per chrom
| - meiosis: 5-10 per chrom
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How does the source of HR differ in mitosis and meiosis?
- mitosis: spont
| - meiosis: programmed
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How does the outcome of HR differ in mitosis and meiosis?
- mitosis: non CO
| - meiosis: CO
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How does the repair template of HR differ in mitosis and meiosis?
- mitosis: sister chrom
| - meiosis: homologous chrom
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How does the recombinase used in HR differ in mitosis and meiosis?
- mitosis: Rad51
| - meiosis: Dmc1
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How do the main repair pathways used in HR differ in mitosis and meiosis?
- mitosis: Srs2 or Sgs1/Top3
| - meiosis: Mus81 or hGEN1
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What happens during meiosis?
- DNA rep
- synapsis forms by pairing homologous chroms --> brings the homologous chrom together and facilitates DSB repair using homologous chroms as template DNA
- DSBs are induced and repaired to form COs
- 2 rounds of chrom segregations prod 4 gametes, 50% have recomb genotype
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What are chiasmata?
- site of CO
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What is the other role of COs?
- ensure sister kinetochores pulled in same direction and homologous kinetochores pulled in opp directions
144
How are sister chromatids held together?
- sister chromatid cohesion
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What 3 types of genetic maps of human chroms are there, and what did they reveal?
- physical maps
- linkage based genetic map
- chiasma base genetic map
- 3 maps largely matched, revealed chiasmata are physical basis of genetic recomb
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What are physical maps based on?
- seq of nts
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What are linkage based maps based on?
- actual recomb freq
| - 1cM is distance between loci w/ 1% recomb freq
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What are chiasma based genetic maps based on?
- expected recomb freq calc from chiasma freq
- so if on av chiasma freq between 2 loci was 1, then expected recomb freq between them would be 50%, so genetic distance would by definition be 50cM
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What is the role synapsis and the synaptonemal complex?
- after DNA rep, synapsis occurs to pair homologous chroms w/ their counterparts
- the homologous chrom don’t align side by side, but on top of each other w/ synaptonemal complex holding them together
- this process brings homologous chroms together and facilitates repair using homologous chrom as the template DNA
150
How was it seen that DNA DSBs appear then disappear during meiosis?
- to initiate HR during meiosis, programmed DSBs induced after DNA rep and homologous chrom pairing
- experimentally, fragmented chrom appear at 2 hours after cells enter meiosis, run as distinguishable bands --> suggests that DSBs take place at hotspots (not randomly)
- intensities of these fragmented chroms reach peak at 4 hours, then disappear --> breaks repaired
151
Why is the mobility of DSB meiotic DNA normally slower on agarose gels relative to prediction from mol size?
- found that a prot covalently bound at the ends of meiotic DSBs
- mitotic DNA fragment is produced by BspHI digestion and not affected by pronase (a proteinase)
- but meiotic DNA runs faster after pronase digestion
- suggests meiotic DNA ends covalently bound to prot, which slows migration.
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Which prot can cut dsDNA and covalently bind DNA?
- topoisomerase
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How does Spo11 cleave DNA and form DNA/prot products?
- top like prot
- covalently binds meiotic DSBs
- binds and cleaves dsDNA
- after cleavage tyr in Spo11 forms covalent bond w/ 5’ end phosphate group of DNA
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How was it found that Spo11 can be released from chroms w/ covalently bound 20bp DNA?
- strain exp HA tagged Spo11 induced to enter meiosis
- denaturing extracts prep and then Spo11-HA immunoprecipitated w/ covalently bound DNA
- Spo11 digested by addition of pronase
- treated w/ 32P labelled nt and TdT, which added 32P labelled nt to 3’ ends
- after digestion Spo11 removed and DNA size around 20bps
- DNA not digested by any REs, so during meiosis Spo11 can be naturally released from DSB sites, w/ covalently bound 20bp DNA fragment
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How was is demonstrated experimentally that Spo11 is released from DSB sites by Mre11/Sae2 complex mediated resection?
- WB revealed immunoprecipitated Spo11 has several forms --> normal and DNA bound Spo11 (runs slowly, as MW slightly bigger)
- DNA bound Spo11 can’t cut DNA
- no Spo11/DNA products, as in these mutants DSBs form normally and Spo11 can bind DSB 5’ ends, but can’t be released from DNA
156
What is Dmc1 and its role?
- meiosis specific recombinase req for strand invasion during meiosis
- promotes inter-homolog repair, but mech remains unclear
157
How is Dmc1 similar to Rad51?
- similar biochem properties - binds ssDNA and dsDNA, forms nucleoprot filaments
158
How is Rad51 important to the role of Dmc1 in strand invasion in meiosis?
- Rad51 req for Dmc1 to bind DNA, but it's strand invasion activity is inhibited during meiosis
159
Why is the Sgs1/Top3 pathway inhibited in meiosis?
- to increase CO freq
160
What occurs during the Mus81/Eme1 pathway (during meiosis)?
- Mus81/Eme1 is endonuclease which cleaves branched DNA, can cut unligated HJ, leading to 100% CO products
- following strand invasion, Mus81/Eme1 nicks 1 of template strands, turning the HJ by 180°
- after 2nd capture and extension, nicks template strand again and sep JM
- after ligation, repair completed and prod all products w/ CO
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What are the diff pathways for meiotic DSB repair, in what proportions are they used and how many products w/ COs do they prod?
- 40% by Mus81/Eme1 --> 100% products w/ CO
- 40% by HJ resolvase YEN1 --> 50% products w/ CO
- 20% by Srs2 mediated synthesis dep strand annealing --> no products w/ COs
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Overall what % of DSBs in yeast will lead to CO (based on all 3 pathways)?
- about 60%
