Chromosome Biology ALL Flashcards
DNA replication is
- Accurate + precise copying of DNA of genome
- Happens 1 per cell division cycle
- Each daughter cell inherits identical DNA
- Evidence
Stages in DNA replication
- Initiation (origin recognised + opened)
- Elongation (DNA synthesised)
- Termination (stops polymerase)
Organisation of chromosome
- Chromosomes = scaffolded by proteinaceous matrix
- 2nm diameter duplex DNA → wrapped around histone octamer → 30nm fibre → loops that condense in metaphase
Chromosome banding
- 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
Gene-rich DNA replicated early
- R bands = G rich, early
- G bands = gene poor, replicate later
- Micro-array analysis of replication origin
Spatial localisation of replication in 3D
- 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)
Comparison of prokaryotic + eukaryotic DNA replication
- Prokaryotes = 4.5x10^6 nt, single circular chromosome, 1 bi-directional origin
- Eukaryotes = 3x10^9 nt, many linear chromosomes, 2x10^4-5 origins
Evidence
- Pulse-labelled cells
- EM = origins seems as open ‘bubbles’
- confocal microscopy (synchronise or arrest cells)
Mapping origins of bi-directional replication
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
Mapping origins of bi-directional replication
E.g. S cerevisiae
- 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
Does the origin act in vivo?
- 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
Replication of eukaryotic DNA viruses
- 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)
Simple origin
- Origin consists of core origin (CORE) that binds initiator flanked by DUE + auxiliary sequence that binds TF
- AT rich
Defining proteins binding to ARS
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
ORC
- 6 subunits, binds ACS
- Subunits are ↑ conserved
- Needs ATP to bind DNA
ORC structure
- Winged helix domain, DNA binding HTH, B sheet wing, AAA+ ATPase
ATP binding + hydrolysis by ORC
- ↑ 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
ORC binding to ARS
- 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
S pome replication origin
- 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
How do ORC recognise origins of replication in S pome
- 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
Metazoan origin
- 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
Origin plasticity related to transcription
- 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
Mammalian origins are found mainly in promoters
- 46% replication origins in Ch3
- Generally promoters have ↑ controlled chromatin organisation, open + accessible
Metazoan origins
- 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
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
Why replicate once
- If fails to prevent, get 4 copies of genome not 2
- Oncogenes can be over-copied
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)
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
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)
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
ATP binding to ORC
- If block ATP binding, prevents loading
- Important cancer recognition target
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
Firing factors
- Act w/ Dbp11
- If all mutated = lethal so essential
- S-CDK phosph Sld2/3, promotes assoc w/ Dbp11, bind Mcm
Switch from initiation to elongation
- 2 forks move away bi-directionally
- Leading strand is cont. w/ PolE, lagging = discontinuous
- CDC45-MCM-GINS stabilises replisome
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
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
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)
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
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)
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)
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)
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
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
Genomic alteration associated w/ HR
- Gene conversion (both sister chromatids genetically identical so not issue, but 2 homologs can carry different alleles → gene conversion)
- Cross over (exchange of chromosomal segments, often btw DNA repeats)
Role of HR
- DNA repair (uses undamaged sister chromatid as a template, loss of heterozyg)
- DNA replication (collapsed + broken replication forks, RDR)
- Meiosis (programmed ds breaks by spoII, can lead to crossover)
- Mating type switching in yeast (a or alpha type, haploid switch mating type through recombination at MAT locus, HO endonuclease)
- Antigenic variation by African trypanosome (VSG, different copies produced)
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
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
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
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
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
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
Other DSB pathways
- SSA (ds break flanked by directly repeated sequences, compl sequences anneal, 3’ flaps cut away, nicks sealed
- BIR (2nd end of ds break x anneal to D loop, DNA synthesis continues until end of chromosome)
- SDSA (extended D loop unwound, released strand anneals to other end of break)
3 stages of recombination
- Initiation
- Synapsis (homologous strand paired + strand exchange occurs btw them)
- Post-synapsis (migration + resolution or dissolution)
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
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)
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
Identification of recombination genes
4. Identify similar structures to E already known
- Proteins that are well conserved = SbccD or Mre11-Rad50
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
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
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
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
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
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
Experiment
Strand exchange
- Circular ssdNA incubated w/ SSB, RecA + buffer
- RecA exchanges complementary DNA strand from the linear duplex to circular + ss linear molecule
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
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)
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
SSR
- Used by bacteriophage lamda
- Minimum requirement = small core DNA site (inverted repeat of recombinase bs)
- Monomer of recombinase binds each of the repeats