Chromosome Biology ALL Flashcards
1
Q
DNA replication is
A
- Accurate + precise copying of DNA of genome
- Happens 1 per cell division cycle
- Each daughter cell inherits identical DNA
- Evidence
2
Q
Stages in DNA replication
A
- Initiation (origin recognised + opened)
- Elongation (DNA synthesised)
- Termination (stops polymerase)
3
Q
Organisation of chromosome
A
- Chromosomes = scaffolded by proteinaceous matrix
- 2nm diameter duplex DNA → wrapped around histone octamer → 30nm fibre → loops that condense in metaphase
4
Q
Chromosome banding
A
- Different points of chromosome replicated at different points in S phase
- Stain w/ Giemsa dye, gives G band (region w/o actively transcribed gene
- Oligomycin gives R bands that are GC rich
5
Q
Gene-rich DNA replicated early
A
- R bands = G rich, early
- G bands = gene poor, replicate later
- Micro-array analysis of replication origin
6
Q
Spatial localisation of replication in 3D
A
- Label cells w/ different times, take serial selections using 3D FISH
- Green (early-replicating) localised more to centre of nucleus
- Relate to chromatin in TAD (Repressed TAG = towards periphery)
7
Q
Comparison of prokaryotic + eukaryotic DNA replication
A
- Prokaryotes = 4.5x10^6 nt, single circular chromosome, 1 bi-directional origin
- Eukaryotes = 3x10^9 nt, many linear chromosomes, 2x10^4-5 origins
8
Q
Evidence
A
- Pulse-labelled cells
- EM = origins seems as open ‘bubbles’
- confocal microscopy (synchronise or arrest cells)
9
Q
Mapping origins of bi-directional replication
A
Shotgun cloning
- Extract genome, cut w/ RE, clone fragment into vector, see if grow on medium w/o His
- If grow, sequence acts as autonomously replicating sequence, supports replication of plasmid
10
Q
Mapping origins of bi-directional replication
E.g. S cerevisiae
A
- Consensus in ARS consensus in ACS box
- Recognised by ORC
- B domain = 3’ to T rich strand of ACS
- Str2 histone deacetylase silences some origins in yeast- epigenetic control
- In addition to AT rich seq, also have ORB + DUE
11
Q
Does the origin act in vivo?
A
- 2D gel mapping
- Yeast genome, cleave w/ RE, run down well, rotate 90o, apply ↑ V, transfer to nitrocellulose, probe against region of genome of interest, hybridise
- Bubble arch = if have origin
- If have passive replication by replication fork outside, give y arch
- If replication origin is to one side, start off with bubble arch then get y arch
- But messy
12
Q
Replication of eukaryotic DNA viruses
A
- like SV40
- Has small ds genome, requires viral protein for recognition of origin to let virus replicate independent of host
- Region that binds Tag (recognition origin protein)
13
Q
Simple origin
A
- Origin consists of core origin (CORE) that binds initiator flanked by DUE + auxiliary sequence that binds TF
- AT rich
14
Q
Defining proteins binding to ARS
A
Experiment (yeast replication origin in absence of protein, expose to DNAse1 → hypersensitive site where DNA slightly bent, footprint where DNA sat on DNA (x show fragment of DNA)
Mutate different regions of origin, mutated A = no protein footprint, mutate B1
15
Q
ORC
A
- 6 subunits, binds ACS
- Subunits are ↑ conserved
- Needs ATP to bind DNA
16
Q
ORC structure
A
- Winged helix domain, DNA binding HTH, B sheet wing, AAA+ ATPase
17
Q
ATP binding + hydrolysis by ORC
A
- ↑ ATP binding to complex in presence of origin DNA
- Mutate different subunits e.g. ORC5 mutant x bind ATP
- ORC binds ATP in presence of ARS, endogenous ATPase activity inhibited by ARS
18
Q
ORC binding to ARS
A
- DNAse I footprinting
- Mapped binding of individual subunits of ORC to ARS DNA
- ORC binds centrally within ARS consensus sequence
- ORC binds nucleosome deleted region
- ORC binding distorts ARS DNA helical axis by 35o
19
Q
S pome replication origin
A
- S pome ARS cloned w/ shotgun method
- Unlike S cerevisiae, x have ARS consensus sequence
- Also ATP rich, clusters of AT regions
- Also promoter but x need transcription initiation for firing
- Longer
- Specific origins of replication but x contain specific sequences
20
Q
How do ORC recognise origins of replication in S pome
A
- Recognise certain motifs
- Also hexameric recognised complex (4 in Pombe, bind specifically to origin via NTD)
- Orp4 has 9 repeats of AT hook DBD
- Spacing important to allow Orp to interact w/ DNA
- Quasi-random distribution
21
Q
Metazoan origin
A
- Lack consensus origin sequence
- No sequence specificity requirement for replication of exogenous DNA
- Indicates low origin specificity in early embryos
- Human = shotgun plasmid 2D mapping
22
Q
Origin plasticity related to transcription
A
- E.g. mammalian DHFR gene can amplify ↑ times by selecting methotrexate
- B globin is ↑ transcribed, v repetitive loci, replicate early
- non-B cells replicated passively through passage through a fork initiated at a ds origin
- Pre-B cells that are transcripting at that locus replicate entire locus early
- Xenopus have temporal developmental pattern
- In early embryo x transcription
23
Q
Mammalian origins are found mainly in promoters
A
- 46% replication origins in Ch3
- Generally promoters have ↑ controlled chromatin organisation, open + accessible
24
Q
Metazoan origins
A
- Can identify by sequence or individual origins
- Chromatin state = important
- Histone acetylation can promote initiation
- ORC can be recruited at distinct sites but ORC-interacting factors
- MCM coats chromatin
- Chromatin structure may contain DNA loop, inter-origin distance
- Replication factories
25
Replicon model
- Initiator ORC binds cis acting replicator
- If take all yeast + look at ORC + MCM bs = ↑ sites
- ↑ replication origins
- e.g. S cerevisiae has 2000kb stretch, 14 Ars, only 5 act as OBR so context important
26
Why replicate once
- If fails to prevent, get 4 copies of genome not 2
| - Oncogenes can be over-copied
27
How to regulate replication = only once
- Experiment w/ hola cells at diff stage of cell cycle, G2 nucleus x replicate
- Experiment 2 w/ Xenopus cell extract, analysed DNA On CsCl gradient, DNA undergo 1 round of replication (1 heavy, 1 light chain)
28
Replication licensing factor model
- Gained access to nuclei only when envelope breaks down
- Once binds chromatin = stable
- So, in G1 nuclei have this factor bound to chromosomes that entered during M
- Licenses DNA for replication
- Factor destroyed by DNA replication in S phase
- In G2, x active licensing factor
29
Regulating firing of the replication origin
- Ensures DNA replication = only once per cycle
- Involves:
1. Binding of initiators (ORC) to replicators (ARS/OBRs)
2. Licensing DNA for replication (assembly of pre-replicative complexes at origins
3. Assembly of pre-initiation complex (helicase, Mcm2-7)
30
CDK + DDk
- At M/A transition, x cyclins as degraded by APC
- Make pre-RC here
- In late M/early G1, cdc6 made, binds ORC in presence of ATP
- Helps recruit mcm2-7 by ATP hydrolysis, Cdt1 released leaving ORC-CDC6-MCM
- Need another MCM
31
ATP binding to ORC
- If block ATP binding, prevents loading
| - Important cancer recognition target
32
Initiation complex forming + firing
- Initiation complex involves loading of replisome components (Cdc25, GINS, cdc6) + firing factors (Skl2, Sld3)
- Firing of replication origin (load DNA pol + other replisome factors, activate IC by phosph)
- Cdc45 recruited to early origins in G1 + late in S phase
33
Firing factors
- Act w/ Dbp11
- If all mutated = lethal so essential
- S-CDK phosph Sld2/3, promotes assoc w/ Dbp11, bind Mcm
34
Switch from initiation to elongation
- 2 forks move away bi-directionally
- Leading strand is cont. w/ PolE, lagging = discontinuous
- CDC45-MCM-GINS stabilises replisome
35
Early + late firing origins
- Context determines if early or late
- E.g. moving efficient early-firing origin to sub-telomeric region where DNA is replicated late confers late replication
36
Affinity for firing factors
- Early origins have ↑ affinity for firing factors, shortage
- Origins in middle of S have ↓ affinity + late S have lowest
- Once fired, firing factors released
- Then bind region of next highest affinity
37
Factors determining origin firing
- Slowing replication speed recruits latest origin
- Licensing factors loaded only in late M/G1, firing factors can be loaded at diff time
- Late origins can be made to fire early (overexposes, ↑ histone acetylation)
38
Late firing origins are actively suppressed
- Late origins x fire if forks stalled/blocks
- Intra-S checkpoint
- Yeast w/ mutated rad53 or ori2-1
- ATM/ATR are kinases
- Prevent replication w/ DNA damage (in yeast, cells treated w/ DNA damaging agent suppress firing, WT cells stop when exposed to y-irradiation, once repaired resume)
- Rad53 inhibits Sld3 by phosph, inhibits DDK
39
Preventing re-replication
- Control MCM loading
- Cdc6 required in late M/early G1
- MCM chaperone cdt1 normally sequestered by geminin
- At M, Gemini is ubiq through APC + degraded → cdt1 chaperone MCM in G1
- Release of cdt1 at loading, PCNA (PIP box)
40
Overexpression of licensing + firing factor (cancer)
- ORC factors ↑ in human cancer, cdc6 particularly ↑ in breast cancer
- MCM dpf4 + cdc7 important in cancer
- MCM staining = better diagnostic for cervical cancer (MCM is expressed in whole epithelial)
41
Oscillation in CDK activity couples mitosis + S phase
- M/A destruction of cyclins → inactivation of Cdk1
- G1/S = Clb kinases active, inactive cdc6, cdc45 loaded onto chromatin in cyclin-cdk dependent reaction, MCM activates
- S phase checkpoint (S,G2, early M)
42
Coordinating progress through S
- Some origins fire early (R bands, exon dense), some late
- Some x act as origins in vivo but replicated passively
- Yeast mutant Clb5/6 = in M assoc w/ Cdk1, M/A transition degrade cyclin Clb + -ve regulatory phosph of Cdk1, G1 = make cyclin but -ve reg. on phosph on Cdk , G1/S -ve re removed by phosphatase
43
Genetic recombination
- Process where DNA is broken + rejoined into new segments
- Mechanisms of recomb. conserved
- 4 types: homologous recomb, site-specific recom, transposition + illegitimate recombination
44
Genomic alteration associated w/ HR
1. Gene conversion (both sister chromatids genetically identical so not issue, but 2 homologs can carry different alleles → gene conversion)
2. Cross over (exchange of chromosomal segments, often btw DNA repeats)
45
Role of HR
1. DNA repair (uses undamaged sister chromatid as a template, loss of heterozyg)
2. DNA replication (collapsed + broken replication forks, RDR)
3. Meiosis (programmed ds breaks by spoII, can lead to crossover)
4. Mating type switching in yeast (a or alpha type, haploid switch mating type through recombination at MAT locus, HO endonuclease)
5. Antigenic variation by African trypanosome (VSG, different copies produced)
46
RDR in fission yeast
- Collapsed fork undergoes reversal
- 4 way DNA junction structure
- Exonucleases resect, ss DNA bound by RPA, Rad52, Rad51, D loop structure, MCM2-7
- D loop is unstable
47
Meiosis vs Mitosis
- In 2 phase, homologs are replicated → 2 pairs of sister chromatid
- In meiosis, diploid chromosome → haploid
- Programmed ds also made, reciprocal exchange of DNA occurs, tension sensing mechanism
48
Holliday model
- 1 strand of DNA nicked in both molecules, nicked strand re-anneals to paired DNA, 4 way junction, further unwinding + re-annealing, HJ moves
49
Neuospora crassa lifecycle
- 2 mating types
- Meiosis → 8 spores in same ascus
- Recombination = 2:2:2:2 or 2:4:2
- Ratio of 6:2 or 2:6 also well documented (non-reciprocal exchange of info btw chromatids)
- 4:4 ascus (2 of paired products segregate as post-meiotic division)
- 5:3 or 3:5 spore ratio ascus = asymmetric exchange of a ss from 1 chromosome to another
50
Meselson + Radding model
- Endonuc. makes a ss cut in 1 chromatid
- DNA synthesis → displaced nicked strand → invades 2nd chromatid → loop excised → free ends ligated to form HJ
- Junction can migrate to 2 heteroduplexes
- Resolved by opposite strands cleaving + rejoining along horizontal or vertical line
- Asymmetric duplex made
51
1983 ds break model
- Hotspot for meiotic recomb. correlates w/ site of ds break
- Recombination initiated from ds break
- 2 ends resected to expose 3' ended DNA region
- 1 ss end invades homologous DNA → D loop (x degraded)
- DNA synthesis extends from D loop
- Resolution
- GOOD EVIDENCE
52
Other DSB pathways
1. SSA (ds break flanked by directly repeated sequences, compl sequences anneal, 3' flaps cut away, nicks sealed
2. BIR (2nd end of ds break x anneal to D loop, DNA synthesis continues until end of chromosome)
3. SDSA (extended D loop unwound, released strand anneals to other end of break)
53
3 stages of recombination
1. Initiation
2. Synapsis (homologous strand paired + strand exchange occurs btw them)
3. Post-synapsis (migration + resolution or dissolution)
54
Identification of recombination genes
| 1. Mutants w/ altered capacity for recomb
- 2 types of mutants predicted: x produced recomb in a cross or ↑ exchange btw genetic marker
- Cross used Her strain of E coli
- Hfr- (sensitive to streptomycin) w/ F- strain (Str resistant) ↑ mutation needed
- Recomb btw tDNA + F- cell → recomb F- resistant to Str that grow w/o Pro
- If mutant in F- cell ↓ efficiency of recomb, ↓ recombinants recovered
- Found ↓ recombination frequency than WT parent
- Repeat w/ different donor
55
Identification of recombination genes
| 2. Mutants defective in DNA repair
- Mutants that inactivate recomb genes make cell ↑ sensitive to killing effect of DNA damage
- RecA mutant = hypersistive to UV light
- RecB + C identified
- Double mutant, suppressor (mutations at new locus or mutations in 2 new loci)
56
Identification of recombination genes
| 3. Screen for relevant biochemical activities in fractionated cells
- Used an assay
- Run products on native + denaturing gels using radioactive labels + detect w/ autoradiography
- HJ resolves makes pair of symmetrical inversions close to junction
57
Identification of recombination genes
| 4. Identify similar structures to E already known
- Proteins that are well conserved = SbccD or Mre11-Rad50
58
Biochemistry of recombination
| Initiation of HR
- ds break = resected, RecB,C,D
- RecB(3'-5'), RecD(5'-3')
- Rapid degradation of 3' end, less 5'
- At Chi, slows down (1000bp/s→150bp/s), conf change in RecB where switch to 5' end degradation more
- Makes 3'ss tails w/ Chi site, RecA can bind + invade using 3' end
59
Biochemistry of recombination
| Homologous pairing + strand exchange
- RecA (e coli), Rad51 (eukaryotes)
- During HR, ssDNA protected by SSB in E coli/ RPA in humans
- RecA displaces + searches for homologous sequence
60
RecA structure
- Core domain w/ walker A+B
- ATP lies btw adjacent monomers, ATP bound form has ↑ affinity for DNA
- RecA-DNA needs to bind ATP to search for homologous DNA
61
Rad51 filament formation single molecule analysis
- DNA stained w/ YOYO1 dye
- Bead moved into buffer that stretches out DNA, then to another channel w/ fluorescently labelled Rad51, pulled back to buffer channel for visualisation
- After 18 mins, several Rad51 nucleation events
- 4/5 monomers of Rad51/RecA = nucleation unit
- Competition w/ SSB prevents unwanted recombination
- Dimer of RecA is minimal oligomer needed
62
RecA/Rad51 mediators
- RecA poorly competes w/ SSB
- Helped by RecF, RecO + RecR (E coli)
- RecFOR promotes recA nucleation on SSB coated ssDNA
- RecOR aids nucleation at other sites
- RecO traps DNA transiently released by ssDNA
- In yeast, mediators = Rad52 + Rad55-Ra57
- In humans, BRCA2 mediates filament formation, inhibits Rad51 ATPase
63
Strand exchange
- ATP binding needed for both
- Thought find homologous DNA by simple collision
- Filament has a weaker 2o DNA bs next to ssDNA, DNA randomly sampled for homology
- Minimum 8bp needed
64
Experiment
| Strand exchange
- Circular ssdNA incubated w/ SSB, RecA + buffer
| - RecA exchanges complementary DNA strand from the linear duplex to circular + ss linear molecule
65
3D homology search
- fluroescently labelled RecA interacts w/ bacteriophage ds DNA
- This DNA is dipped into reservoir containing ssDNA filaments then → observational chamber
- Can manipulate ds DNA
- Pairing of RecA to dsDNA = ↑ efficient when DNA is in tangled state
- Intersegmental contact sampling to search for homologous DNA
66
Biochemistry of recombination
| Holliday junction branch migration + resolution
- D loop = resolved by cross over or non crossover by HJ resolvases like RuvC or Gen1
- Also can be processed w/ dissolution
- HJ move towards each other until collapse into hemicatenane
- Cleavage of 1 strand enables uncleared strand to pass through nick
- Nick resected by ligase
- RuvB anchored on DNA, translocates along DNA pumping out DNA across surface of RuvA (ATP hydrolysis)
67
Site specific recombination (SSR) overview
- Protein binds recognition site + catalyses exchange w/ another site that is recognised by the same protein
- Basis of recognition = protein DNA + protein-protein interaction
68
SSR
- Used by bacteriophage lamda
- Minimum requirement = small core DNA site (inverted repeat of recombinase bs)
- Monomer of recombinase binds each of the repeats
69
Outcomes of SSR
- Depends on relative orientation of 2 core sites
| - Excision, integration or inversion
70
Roles in nature
1. Flp (maintaining copy no. of 2 micron plasmid in yeast, ssrecombinase encoded by 2 micron plasmid, recognises FRT, Flp ↑ copy no by recombining 2 FRT sites on plasmid, single origin of replication, recombine 1 newly replicated FRT, resolved)
2. Resolving chromosome dimers (circular chromosomes = hard to segregate chromosomes to daughter cell, bifunctional fork move away from OriC, resolution → crossover)
3. Integration + excision of bacteriophage (life cycle, integration + excision of DNA depends on lambda integration)
4. ss inversion system (on/off switch for gene expression, e.g. salmonella, Hin drives switch btw 2 different flagella filament proteins FljB/C)
5. Resolution of transposition intermediates (Tn3 transposon)
6. Acquisition of new drug resistance (e.g. integrons, incorporate exogenous ORF by SSR + convert to functional genes, intl + attl, array of gene
71
SS recombinase families
1. Tyr recombinase family
2. Ser " "
- Common = (inverted repeat bound by recombinase, 1 phosphodiesterase bond cleaved, SN2 reaction)
- Difference = (Tyr catalyses 2 pairs of strand cleavage, Ser = both simultaneously)
72
Tyr recombinase family
- Flp, Cre v similar = overall 2-fold symmetry
- Clamp around DNA substrate
- CTD a-helical + has AS residues, NTD ↑ varied
- Catalytic domain = 6 conserved residues
- 1 strand cleaved in each ds DNA molecule
- Each cleavage → phosphoTyr linkage btw recombinase 3' end of DNA
- Form HJ intermediate, resolved
- Synaptic complex
73
Ser recombinase family
- Conserved domain, 4 motifs w/ catalytic residues in A + C
- NTD = AS, CTD = DBD
- Asp + 2Arg crucial for catalysis
- 180o rotation of 2 subunits within complex
74
Controlling direction of reaction
- DNA must be bent so 2 monomers interact
- Tyr recombinase, synaptic complex has spacer antiparallel → predictive strand exchange
- Ser recomb also needs bp of comp 2 bp to properly align 3'OH w/ 5' phosphoser
- Sites = parallel, unproductive synaptic complex → all 4 strands cleavage + 180o rotation
75
Controlling direction of recombination
- Sites in direct repeat in same DNA combine → deletion
- Diffrent sites recombine → integration
- Many recomb systems flip balance using accessory proteins + DNA seq elements
E.g. = bacteriophage lamda from E coli
76
Filter to sense connectivity btw distant DNA sites
- Resolvases like Tn3 avoid catalysing inversion + intermolecular recom by only being active in a specific complex
- Different no. of DNA crossings needed
77
Transpositional recombination overview
- Similar to SSR as involves protein DNA + protein-protein interactions
- Only 1 of the recombining partners is specifically recognised by the protein that catalyses transposition
78
3 general mechanisms for genetic mobility
1. SSR
2. Transposition - transposes binds inverted DNA at end of element, each transposes cleaves ends of elements + transfers to new molecule
3. Target-primed reverse transcription - transposable element copied from its DNA by RNA pol + produces RNA copy of element → encodes reverse transcriptase that nicks target DNA + element copied in
79
Transposable elements
- Abundant, 12% of c elegans genome
- Most a silent, some are active
- Specific DNA/RNA elements can move
- Source of spontaneous mutation
- Can lead to gene rearrangements
80
Classification of eukaryotic transposable elements
1. Class I = retrotransposons, transpose via RNA int, use reverse transcriptase, split into LTR, non-LTR or DIRS
2. Class II - DNA transposons
81
LTR transposons
- Inc retroviruses like HIV
- Both use integrate
- Retroviruses can be distinguished from LTR transposons by presence of envelope gene located at 3'
82
Non-LTR transposons
- x have LTR
- Transposes via target-primed reverse transcriptase
- uses endonuclease to nick DNA (restriction-like or AP)
- e.g. = LINES/ SINES
83
DNA transposons
- Divided into autonomous elements + non-autonomous
84
Classification of prokaryotic transposable elements
1. Simple insertion sequences (800-1500bp, inverted terminal repeats flanking gene for transposase)
2. Compound transposons (have 2 insertion sequences which cooperate to transpose own DNA as well as DNA in btw)
3. Complex transposon (terminal inverted repeat flanking range of genes)
85
Transposable element abundance + activity
- Most common = Tc1/Marnier family
| - Only around 10 elements are active inc P elements in Drosphilia
86
Mechanism of transposition
- Liberation from donor DNA + insert/joining to target DNA
| - Can either paste in or copy w/ reverse transcriptase
87
Transposition types
Conservative vs replicative
| Both use endonucleolytic cleavage of phosphodiester bonds, transfer ends into target DNA
88
Conservative (cut + paste) transposition
- Tc1/Marnier family
- Typically these transposes = DBD, CT catalytic domain,
- Asp + Glu coordinate 2 Mg2+
- 2 transposes recognise inverted repeats, bind w/ HTH → 'single end' complex
- Cleave 5' of inverted repeat → 'paired end complex'
- 3' end cleaved + release 3'OH termini
- Bind target DNA
- 2 strand cleavages liberate from end of donor DNA
- Fill in w/ DAN pol
- Transposon footprint
89
Coordination of transposition w/ DNA replication
- Conservative = wasteful
- Overcome by coordinate
- Excision of transposon ensures ds break is repaired by HR
90
Replicative transposition
- Only 1 strand cleaved at each transposon end
- Strand transfer of cut 3' end into target DNA → Shapiro int.
- 2 3' ends → replication of transposon
- Intermolecular replication transposition → co-integrate structures where donor + target replicon are joined but separated by directly repeated copy of element
- Resolution
91
Life cycle of DNA transposon
- Retrotransposons are transcribed
- Assoc. w/ mRNA
- In retroviruses, envelope gene means mRNA + proteins → infectious particles by bonding off
- In virus-like particles, pol derived protease cleaves protein
- RNA reverse transcribed to comp. DNA
92
Reverse transcription of retroviral DNA
- 3' end of host tRNA binds 5' of viral RNA
- Reverse transcriptase makes short segment of RNA
- RNase H exposes 3' end of - DNA
- tRNA mostly degraded
- Short stretches of RNA persist, primes synthesis of + DNA which proceeds to 5'
- 2nd template switch
93
Integrase integration
- Integrates into cDNA of host
| - Generates 2 base recessed 3' ends in LTR + staggered ends in target DNA
94
Strand cleavage
- Transposition requires assembly of synaptic complex
- Precise cleavage to liberate 3'OH
- Cleavage of compl strand sometimes occurs
- Hairpins hydrolysed → 3'OH
- 3' ends transferred into target DNA
95
Disadv of transposition
- Insertion near genes can Δ gene expression (can carry gene promoter)
- Indirectly harm by acting as sites for non-allelic HR
96
Transposable elements targeting specific sites
- E.g. in some yeast specifically target promoter regions or genes transcribed by RNA pol III
- E.g. TyI integrase in yeast has LTR that interacts w/ subunit of RNA Pol III
97
Regulation of transposable elements
- small RNAs silence
- KRAB domain (>400 genes), bind transposable elements in a sequence-specific manner + recruit KAP1 → H3 lys 9 methyl transferase recruited → epigenetic silencing
- De-reg = hallmark of cancer
98
Adv of transposition
- Antibiotic resistance
- VDJ recomb = antigen receptor diversification (relies on transposition-like reaction + NHEJ, RSS bound by VDJ recombinase which initiates recombination
99
Transposon as genetic tool
- Typical gene transfer = 2 plasmids
| - ↑ efficiency of transposons in catalysing genomic insertion
100
Genomic DNA is continuously degraded
| Types of DNA damage
- DNA damage = through endogenous (hydrolytic/oxidative) or environmental (physical & chemical)
- Different types of damage = ss or ds breaks in sugar phosphate backbone, dirty/clean, DNA mismatches, missing bases, altered bases
101
DNA mutations
- Deletions, insertions or substitutions
- Mutagenesis of single bases = transitions or transversions
- Large scale mutations have ↑ effects, loss of genetic info → loss of heterozygosity
102
Frequencies of DNA damage
- Depurination = 12,000 per day, ds = 9
- Chemical agents used in chemotherapy
- Mutation = 1.1-2.5x10^-8 (compared to 50,000ss = very little, good repair)
103
Strategies for DNA repair
1. Direct reversal of repair or damage
| 2. DNA damage tolerance + potential for mutagenesis
104
Types of DNA damage
1. Spontaneous de-amination of C→U (2 ways could occur, U bp w/ A not G)
2. De-amination of other bases (e.g. nitrous acid reacts w/ amino group → deamination, A→hypoxanthine
3. H atoms on bases change (tautomer, amino tautomerises to imino, keto to enol)
4. Alkylating agents (mono functional or bifunctional, often nucleophilic centres)
5. Loss of bases through deprivation/depyrimidination (cleavage of N-B-glycosyl bond → abasic site
105
Direct reversal of DNA damage
- UV radiation → CPD + 6-4PP
| - Both can be reversed w/ DNA photolyases (contain non-covalently bound chromophores)
106
Direct reversal of O6-G + O4-T alkylations
- O6-MGTI transfers methyl from O6 of G (DNA) to C (protein)
- 2 Cys acceptor receive methyl
- Once methylated, O6-MGT1 x be regenerated, finite damage that can be repaired
- Cont exposure to alkylating agents → mutations
- Methylated O6-MGTI = transcriptional activator, activates genes w/ Ada box (transcribes genes needed for BER)
- Humans = o6-AGT, also uses Cys
107
UV light + thymine dimers
- UV → photoproducts that are covalently linked btw adjacent pyrimidines (CPD)
- Issue as have covalent crosslinks that Δ ability to form bp + distort DNA ds helix
108
NER
- Recognise damage, incision of affected DNA either side, excise, fill in w/ polymerase + ligase
- GC-NER vs TC-NER
- UvrA,B,C (mutagenesis)
- UvrA + B = important for proximal DNA damage recognition + act as platform
- UvrC = nuclease that acts on either side of damaged DNA, cuts 3' to DNA
- UvrD = helicase that removes
- Eukaryotes = XPC + XPE, XPD (helicase that unwinds)
- In Tc-NER, recognition = DNA poly transcribing stalls, CSA + B associate = assembly point for factors
109
NER + disease
- XP have defects in NER
110
BER
- Similar to NER: recognise, remove, use undamaged strand as template
111
DNA glycosylases
- Remove effected base by cleaving N glycosidic bond → apurinic site
- Carry out both recognition + excision
- Monofunctional = remove bases only leaving AP site
- Bifunctional = additional lyase that cleaves 3' of basic site
- Recognise CHEMISTRY of base damage
112
Uracil DNA glycosylase
- Spontaneous deam. of C→U = repaired
- UDGs are specific for U in DNA
- Scan along flipping out bases to see if fit in AS
- Specificity = bs too small for A + G, T has bulky C5 methyl so x fit
113
AP endonuclease + DNA processing E
- Need 3' to use as a primer
- AP cut leaving 3'OH
- E.g. Hunan apex1 has AP endonuclease, 3' phosphatase and 3' diesterase activity
114
Different pathways
- Pathway II: DNA glycosylase cleaves N glycosidic bond → remove base → Apex1 cleaves 5' site revealing 5'P + 3'OH, DNA pol fills in, ligase seals
- Pathway I: Type II glycosylase removes base → opening of ribose sugar + cleaving of backbone (x use for synthesis, need processing e.g. w/ Apex1)
- Pathway III: type II glycosylase give 3'P + 5'P, need to restore 3'OH (w/ PNKP) then DNA synthesis
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Oxidative damage
- ROS e.g. H202
- 2 main ways attack DNA:
- Addition to double bonds of bases OR H abstraction from deoxyribose sugars
- Radical attack → sugar fragmentation/base loss/ strand break
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DNA damage by ionising radiation
- IR directly damages through ionisation of base/sugar
- Due to H20 in system, species formed through radiolysis of H20 = main source of IR damage as → ROS
- 2 ss on opposite strands → DSB
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ss break repair signalling
- PARP = add ADP ribose onto proteins
| - Secrete signals for repair proteins e.g. histones (opens structure)
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Sources of DSB
- O2 radicals
- Ionising radiation
- DNA cleaving agents e.g. bleomycin
- If replication fork encounters SSB → replication run-off
- 9 per day, most cytotoxic (illegitimate joining/breakdown by nucleases)
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Pros/Cons of DSB
GOOD
Generated under normal conditions e.g. meiotic recombination, V(D)J recombination
BAD
Crossing over → loss of heterozygosity, recomb. btw repetitive DNA → loss of genetic info, de-reg DSB repair → gross chromosomal rearrangement, aberrant ds break repair → loss of genome integrity
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HR in E coli
- Need homologous chromosome
- DS break w/ homologous sequence for repair, detect ds break, resect DNA ends → 3' overhand, RecA facilitates strand invasion, synthesis using 3'OH as primer, holiday junction resolution
- Conserved in vertebrates
- Resolving
1. Double Holliday junction cleaved w/ nuclease
2. Migration of both Holliday junction together → catenene, resolved w/ topoisomerase
3. SDSA = HJ migration that flips out ss structure making homologous region that can anneal, then fill in gap
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NHEJ overview
- Process of direct DNA end-end fusion that x use strand exchange or homologous DNA
- DSBs detected, bound by proteins that facilitate assembly of repair factors
- End of DSB = often 'dirty', need to process 3'P (can → loss of genetic info)
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VDJ recombination
- Variable region fused to joining region inaccurately to cause mutation to provide ↑ diversity
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Components of NHEJ pathway
- Ku70/80 heterodimer binds DNA
- Artemis = 5'-3' exonuclease
- MRN = 3'-5' exonuclease
- DNA ligase IV
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NHEJ pathway
- MRN + assoc. Ctip bind DNA damage in response to IR
- Mutation in Mre11 → genomic instability syndrome like ATLP
- MRX assoc transiently w/ regions proximal to DSB
- MRN polarity = wrong direction, only 100bp resected
- Use exo1 (5'-3' exonuclease) + Sgs1-DNA2
- 3' overhand facilitates Rad51 invasion
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HR vs NHEJ
- Regulation of DNA end resection = determinant
- HR x good in G1 as x have homologous chromosome
- CtIP needed to activate MRN/MRX
- CtIP phosph on Ser267 by Cdc28 which is needed for Clb/Cln activation
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Genome size + replication rate
- E coli = 4.7Mbp to replicate in 20-30 mins
- Human = 3000Mbp, ↑ DNA
- Issues w/ DNA replication = big genome, only want to replicate once, needs to be accurate, all links btw 2 strands need to be removed,
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MMR
- Repairs base-base mismatches
- Replication slippage = type of error
- x detect a chemical change, hard to detect
- Solution = identify newly synthesised strand
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Discriminating daughter + parental strand
| Prokaryotes
- Identification of mismatches = methylation status of new DNA at GATC
- After replication, window where parent = methyl, daughter x (hemi-methylated)
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Methyl-directed MMR
- At methylated GATC, MutH aspic
- MutS binds mismatch + communicates to MutH via MutL
- Activates endonuclease activity of MutH, cleaves daughter
- Nick unwound by UvrD
- DNA synthesis
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How is MutH activated
- Different models
1. translocation model = extrusion of DNA through MutS dimer pulls MutH towards it
2. Sliding clamp where MutS moves to MutH
3. Spooling at 1 side of MutS dimer to reel MutH close
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Discriminating btw daughter + parental strand
| Eukaryotes
- Resection = issue for lagging strand as x know directionality
- Achieved w/ PCNA (maintained on lagging strand)
- MutS, L translocate, if meet PCNA in good direct, resect towards mismatch
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Translesion synthesis
- Damaged lesion encountered by replication forks stalls
- Could use HR to restart replication, then repair w/ NEJ
- Replicate past lesion w/ Pol w/ ↓ fidelity so can replicate past lesions
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Polymerase switch
- Rad6/MMS2 + Ube13 = ubiquitin conjugating E
- Rad18/5 = ubiquitin ligase
- When a replication fork encounters damage, PCNA is mono-ubiq
- PCNA becomes poly ubiq, has ↑ affinity for PolN, Pol, allowing translation synthesis
- Synthesises past damage
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DNA interstrand crosslink repair
- Covalent bond btw 2 bases on opposite strand (x use helicase or recombination)
- Combination of pathways
- e.g. = NER to cut out damage, HR to restart replication
Prokaryotes
cross-link w/ UvrABC → unhook lesion to give gap + ssDNA → RecA to invade if homologous chromosome
Eukaryotes
FA pathway, FA core recruits key FA protein → assembly of HR/nucleases that process ICL → unhook ICL one lower strand of DNA, fill in w/ DNA replication + bypass w/ translesion synthesis
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ICL defective repair
- Can try using another pathway
- Experiment w/ WT or knock down FRANCD2
- If compromised NHEJ can rescue w/ FA
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SOS response (E coli)
- Surveillance mechanisms that monitor structure of chromosome
- Coord LexA transcriptional repressor + DNA repair protein RecA
- ssdNA damage repaired w/ HR, recognised by RecA which initiates strand invasion
- RecA interacts w/ LexA by reliving repressor activity of LexA → transcribe SOS box genes
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DNA damage cell cycle signalling (All)
- Experiment show deletion of checkpoint genes → loss of cell cycle arrest
- Checkpoints ensure DNA replicated only once
- Chk1 (ATR) + Chk2 (ATM)
- Activated Chk2 phosph Cdc25 which degrades Cdk2/cyclinE via p53 via p21
- Chk1 phosph + inhibits Cdc7 needed for replication origin firing, also inhibits activation of Cdc25
- AT = chromosome instability, ATM kinase,
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MRN as sensor of DNA damage
- MRN assoc w/ DNA damage
- 3'-5' exonuclease property
- Mutations → ATLD/NBS, patients are sensitive to IR
- Nbs1 reacts w/ ATM through conserved CTD, Nbs1 binds ds break
- Active ATM = auto-phosph + forms dimer
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ATR activation
- ssDNA = trigger
- Experiment = look at RPA + ATR nuclei foci when DNA damage
- Knockdown
140
911 complex
- Identified in genetic screen yeast
- Similar to PCNA, ring-like structure
- Rad17 + 911 thought to load DNA damage sensing complex onto chromatin
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Tobp1
- ATR + Rad17/911 needed for Chk1 phosph but recruited independently of each other to ssDNA
- Reduction of TOPB1 in mammals → ↓ phosph of Chk1 + other ATR substrates
- 8 BRCT phospho-reogn motifs
- TOBP1 = ATR activation domain, recruited by 911
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Chromatin structure
- Chromatin → nucleosome → fibre
- Nucleosome = H2A,B,H3,H4
- N terminal tails protrude out = target for PTM which Δ chromatin structure
- Acetylation = -vely charged PTM, relax DNA (also =ve)
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Phosphorylation of histone variant H2AX
- Variant of H2A that has 15aa CT extension inc SQE motif
- H2AX phosph in response to DSB by ATM, ATR or DNA-Pk
- Staining pattern in nuclei
- After repair, H2AX is dephosph by PP2A
- Fast vs slow kinetics
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ATM-dependent phosphorylation of KAP-1
- Heterochromatin → repressors bind + recruit co-repressors like KAP-1
- In turn recruits chromatin modifying E like methyl transferases
- Knockout KAP-1
- ATM inhibits KAP-1 by phosph, can facilitate repair by relieving heterochromatin formation
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Genome instability syndromes
- XP, AT
- Null mutations in subset of DDR genes → embryonic lethality
- DDR critical as defects x compatible w/ cell survival
- Several diseases = assoc. w/ ↑ cancer risk
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Tumour progressoin
- 5-10% of cases= inherited, familiar cancer syndrome e.g. AT
- e.g. = Retinoblastoma = children inherit 1 WT + 1 mutant, loose 2nd → loss of heterozygosity
- Spontaneous mutation e.g. Burkitt's lymphoma = translation of c-myc to chromosome 14
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Defective DNA repair
- Leads to micro-satellite instability
- HNPCC
- Gross chromosomal rearrangements, DSB
- Illegitimate room of repetitive DNA elements in HR → translocations or deletions
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Sub-optimal repair mechanism
- Defective DNA damage also channels DNA damage through sub-optimal repair mechanisms → genome instability
- e.g. w/o FA use NEHJ
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Telomeres, DDR + senescence
- Replicative senesce = spontaneous proliferative arrest of untransformed cells after a certain no of cell divisions due to shortened telomeres
- Telomerase makes telomeres
- At some point, x form T loop + have exposed ds break → DDR active → senescence
- Experiment
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OIS
- Limit where tumerous cell stops growing - OIS
- Call can determine they're goring uncontrollably
- Experiment = DDR marker
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Synthetic lethality
- Malignant cells often loose DDR
- 2 pathways, if take away 1 survive, if takeaway both x
- E.g. exo1 + Sgs1 compensate for long range resection, 1 mutant = viable, both = not
- Chemotherapy using DSB, normal cells use NHEJ/HR, cancer cells x use HR, use NHEJ inhibitor so have no pathways
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PARP inhibitors
- PARPi = x do ss break repair, trap PARP at ss break
- Normally use HR + BRCA2
- BRCA2 mutated in breast cancer
- Give ParpI + can't repair
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Prokaryote DNA replication regulation
- Replicate as fast as possible
- Circular, single origin of replication
- 9 bp where DnaA binds, DnaB/C then bind w/ PolIII
- Key regulation = before helicase loads
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Prokaryotic helicase loading control
1. Levels + activity of DnaA
- Inactivation of DnaA (RIDA) (complex of clamp, ADP-Hda promotes hydrolysis of DnaA bound ATP → inactive DnaA)
2. DnaA binding to origin
- DatA locus (by OriC, hierarchy of affinity for DnaA, datA has highest binding (8x more), as is replicated ↓ [DnaA}
- OriC methylation (DNA transiently hemimethyl, SeqA binds + sequesters OriC, DnaA promotes initiation of 'old' methylated origins
3. Conformation of DNA (DnaA binding to datA stimulated by IHF + datA-IHF stimulates hydrolysis of DnaA (DDAH), DDAH is reg by supercoiling
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Prokaryotic DNA replication termination
- Occurs at Ter sites, trap 2 forks to meet each other
- Have polarity so stop replication in 1 direction
- Coord removal of 1 fork or 2 complexes (allow another Pol to be recruited)
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Eukaryotic DNA replication regulation
- DNA linear molecules, ↑ in size, need to be coordinated, early, late + dormant origins
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Eukaryotic DNA replication origin firing
1. loading helicase in inactive state
(ORC binds origin, Cdt1 recruited + cdc6 then MCM2-7, regulated by CDK, regulated in after to prevent re-replication)
2. Origin firing
(S phase, DDK recogn. NTD of MCM + phosph → bs for Sld3, Cdc45 binds, Dbp11 recruits GINS + polE, regulated)
3. Regulating epigenetic code
(Recycling or de novo assembly, histone chaperones e.g. CAF-1 by pCNA, 1/2 normal no., histone PTM diluted = regulatory marker, HTA1-H3/H4, HIR represses histone gene expression)
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Eukaryotic DNA replication termination
- Thought forks converge
- Converging CMGs bypass each other + translocate until reach Okazaki fragment
- Leading strand extends to Okazaki fragment + processed by recruitment of Pol gamma
- Removal of CMG
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Recognition of DNA damage
MMR
( base mismatches/smaller insertions/deletions, identify template strand, prokaryotes = hemimethyl (MutH nicks unmeth strand, eukaryotes = PCNA (RFC loads w/ defined orientation, when MutLa interacts in this orientation ensures cleavage = new strand, HMSH2/6 heterodimer, bends to find mismatch)
NER
(detects bulky lesions, GGR vs TCR, XPC recog, + binds strand opposite, Rad4 = recognised through DBD at ss DNA)
BER
( recognises chemical features of DNA bases, DNA glycosylases, specific, bind minor groove, kink DNA + flip bases out, mono functional vs bifunctional)
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Verification of DNA damage
MMR
(overlaying 2 crystal structures → MutS binds mismatch w/ Phe-X-Glu, heteroduplex kinked → URC (unbent), The disrupts base, Glu recogn misfired bases
NER
(TFIIH = key, XPB opens helix + anchors proteins, XPD has iron-sulfur cluster, forms tunnel through ssDNA fits through)
BER
(bases fit into glycosylase e.g. uracil glycosylase, A+G too big, T has bulky C5 methyl)
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DNA damage removal
MMR
(coordinate nicked DNA to mutS, MutS similar to Smc thought extrude DNA through loop, after MutH activated, helicase (UvrD) unwinds, MutLa nicks DNA in euk, exo1)
NER
(2 incisions made, XPG 3' end, XPF 5' after ERCC1-XPF joins)
BER
(short vs long-patch BER, mono functional vs bifunctional glycosylases)
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Re-synthesis
- Relies of DNA pol, RFC, PCNA
| - Pol uses undamaged ssDNA as template
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Repair of ds break overview
- DSB arise from sources like ROS, when DNA replication fork encounters DNA ss break
- Danger of DSB:
- If x detect, damaged cells x die + → progeny that have genomic instability
- DNA breaks → chromosome breakage 0> chromosome fragments unequally distributed → genomic heterogeneity
- Chromsome translocation (promoter of other gene)
- Telomerase makes telomere from DSB
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Repair of ds break
| NHEJ
- G1 where x homologous chromosome
- DSB recog. by Ku70/80, stops floating away, DNA-PKC recruited, Artemis phosph (exonuc), joined by PolX + ligase IV
- 'Microhomology' (just XRCC4 + ligase)
- Insertions, deletions + erroneous joining of DSBs → translocation, loss of chromosome material
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Repair of ds break
| HR
- Uses homologous sequences
- Broken ends resected (MRX/MRN then exo1, RPA coats ssDNA, Rad51 replaces + invades, min 8 bp, D loop, capture 2nd DSB → 2HJ, RuvC resolvase, dissolution)
SSA
(resection reveals compl. strands that recomb like NHEJ, ERCC1 assoc w/ XPF removes sequences btw direct repeats)
SDSA
(Invading strand displaced + anneals w/ 2nd resected DSB, x HJ formed)
BIR
(during S phase at telomeres or broken forks, 3' end of D loop extended, x have 2nd end to anneal w/ extended invading strand)
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Repair of ds break
| HR vs NHEJ
- HR ↑ efficient, NHEJ always on
- Resection = 1st major difference, Ct1p interacts w/ MRN, activated by cdc28 during S phase
Sister chromatid?
(epigenetic code btw new vs old, H4K20 methyl, H2AK15 recruits machinery to look for H4K20, TONSL-MMS22L reads H4KM20 in HR via ARD)
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Is ds DNA most dangerous damage?
- Purposefully used in VDJ recomb + meiosis
- Controlled e.g. meiotic hotspot
- 50,000 ss dna a day, mismatches likely to go un-noticed
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DDR
1. cell cycle control
(time for lesions to be removed, prevent firing of late origin, ssDNA → MRN → ATR, dsDNA → MRX → ATM, H2AX(MRN) phosph, RPA-coated ssDNA involves loading 911 + TOBP (ATR), ATR recruits Chk1, phosph Cdc7, ATR recruits Chk2, activate p53, keep pRb inactive, G1/S paused)
(ATM mutations → AT, Chk2 → LFS, p53 → Li-Fraumeni
2. Apoptosis
(damage x be repaired, p53 affinity for promoter ↑ in genes w/ cell cycle, ↓ apoptosis, transcriptional activator for Bax, binds Bcl2/Xl + frees Bax)
3.Senescence
(Permanent withdrawal from cell cycle, senescent cells release SASP, impact neighbours, maintains structure, OIS)
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ssDNA mutation
- XPG → XP (C to T mutations found via UV) (NER)
- BER = mut in 30% polB + DNA glycosylase → colon cancer
- MMR HNPCC = mutations in MSH2/MLH1
170
dsDNA mutation
HR: Brca1/2 → breast cancer, RecQ helicase mutations
NHEJ: Ku70/80 expression ↓ colon cancer
Genomic instability, loss of heterozygosity
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Synthetic lethality
- Defect in 1 gene = survive, 2 = die
- BER + NER Overlap, NRE components repair BER + NER can be backup for BER
- If HR defective, NHEJ can be used
- PARPi = knocks out 2nd pathway in cancer cells so x use
