DNA and Nuclear Architechture Flashcards
(33 cards)
Nucleic acid structure
Eukaryotic DNA viruses visualised by EM, relaxed or supercoiled (topoisomerases relax by transient breaking and resealing). Supercoiled circle helix coiled around itself. Intense DNase I digestion (micrococcal nuclease also) of core particles-> regular periodic pattern of 10-12bp (each turn), indicating that DNA wrapped around surface of histone core octamer. High-salt denaturing can separate DNA+ histones. DNA coiling-> 1 constrained supercoil per nucleosome/146bp (against calibration curve from gel ladder). X-ray nucleosome structure shows central globular histone domains in centre w/ N/C terminal tails extending out. In vivo, nucleosome assembly mediated by histone chaperones+ chromatin assembly factors. Form from histone subcomplexes (H3/H4 tetramer+H2A+H2B dimers). Linker length varies, H1/linker association helps form higher order structures.
Finding histone sizes with gels
Histone sizes can be found by boiling in amphipathic detergent (SDS)+ running on polyacrylamide gel (3D “fishing net”, proteins fall through pores)- stacking gel concentrates samples/aligns proteins before run to prevent any proteins getting a “head start”-> streaky gel.
Use SDS-PAGE buffer system w/ Tris-Gly pH8.3 in sample+ top solution, tris-HCl pH 6.8 in stacking gel, and tris-HCl 8.8 in main gel and in solution at the bottom, so HCl pulls proteins down w/H+ and Gly pushes protein down. Find that Hs relatively small, H1»H3>H2A>H2B>H4.
Histone modifications: overview
Tails acted on by protein modifying enzymes. Specific aas often post-translationally modified, e.g., Lys acetylated/ mono/di/tri methylated- other mods include Pi+ ubiquitylation. Epigenetic info, heritable+ reversible (dynamic for response to env+ to differentiate), read by proteins that bind specifically to modified histone tails. E.g., Lys 9 of H3 acetylation (recruits chromatin remodelling enzymes) -> active (enable transcriptional machinery access) and trimethylated (bound by HP1 heterochromatin protein)-> inactive, attracting different proteins. Immunostaining with fluorophores and antibodies for established heterochromatin markers and for a certain histone modification can show mods co-localising to heterochromatin, IDing mod’s effect on packing. Methyl lys can be ID’d by mass spec (tricky)/ immunostain w/ fluorophores (easier)- show it underlies heterochromatin.
Activating histone modifications
Activating mods: Lys (sometimes Arg) acetylation recognised by bromodomain containing proteins (HATs acetylate neighbouring histones, spreading- +ve feedback). H3 Lys4 trimethylation deposited methyltransferase proteins associated with active gene. Monomethylation usually effects enhancers. Proteins binding K4 Me3 can interact TFs+PolII
Repressive histone modifications
Repressive mods: Methylation H3 Lys27- mono/di/tri- tri specifically associated with gene silencing PRC2 (polycomb repressor complex). H2B Lys119 ubiquitination by PRC1-> silencing. K9 3xMe associated w/ heterochromatin. K27 Me3 can block acetylation, bring in proteins that block TFs/PolII binding.
Other mods: citrullination, Pi.
Condensation of chromatin
Nucleosome subunits-> Hierarchy of folding. Dynamic accessibility-> exp and rep.
Most condensed in metaphase- higher order packing involves loop formation, attachment of chromatin fibres to underlying matrix/scaffold structure. Proteins involved in packing explored by random mutagenesis (surviving cells that can’t make chromosomes sequenced to find relevant proteins/loci).
Condensation regulated by condensins, belonging to structural maintenance of chromosomes proteins (SMCs). Complexes of SMC2+4 w/ kleisin proteins (CAPs) can clamp chromatin fibres, forming horseshoe structure closed+ joined by kleisin-> mediate condensation.
Nuclear matrix/scaffold
Detergents/salts release nuclei/chromosomes, liberate DNA from underlying protein structure (matrix), which DNA attached to in loops ~60kbp. DNA attached= MAR/SAR (matrix/scaffold associated regions)- isolated after digesting. Can study this by prepping nuclei, extracting histones, cleaving with restriction nucleases, separate out scaffold w/ still bound DNA, remove and sequence- knowing seq of MARs helps discover how they regulate expression by altering coiling. To verify this, DNA fragments could instead be added to scaffold to see what “sticks”. MARs generally v. AT rich w/ weak “consensus” sites for topoisomerase II (suggesting it may be involved in controlling DNA loop coiling). Constituents of matrix still ill-defined but scaffold attachment factor (SAF) A (can directly bind MAR elements) + few other factors characterised.
Interphase chromosomes
Can sometimes be seen under light microscope, e.g.: lampbrush chromosomes of newt oocytes partially condensed in early meiotic prophase for months; very transcriptionally active, densely packed nascent RNP (ribonuclein- RNA and RNA-binding proteins (RBPs) particles coat loops so they can be seen- hybridisation of DNA probes to chromosome preparations confirm organisation is strictly seq-specific+ individual condensed loops correspond to specific active genes.
Interphase nuclei show both hetero+ euchromatin, formation of which involves histone mods. Formation due to post-translational histone tail mods.
Hybridisation experiments with interphase chromosomes
Hybridisation experiments w/ probes for specific chromosomes (FISH) show discrete chromosome territories w/ dynamic locations in interphase nucleus. High throughput DNA seq map 3D DNA-DNA interactions: Hi-C (high res chr conformation capture) based on ligation of proximal DNA segments+ sequencing these junctions: DNA crosslinked+ cut, ends marked w/biotin+ ligated, DNA purified+ sheared+ biotin-marked paired ends sequences-> plot interactions/ proximal sequences. Model interactions within chromosome territory (+ between chromosomes) by IDing topologically associated domains (TADs)- megabase resolution.
Many activities in the interphase nucleus occur in discrete foci…
Many activities in interphase nucleus occur in discrete foci, e.g., clusters of DNA replication forks/ RNA synthesis+ machinery. These locations are dynamic+ associated w/nuclear matrix. Nucleolus specifically separated by region of dense heterochromatin- rDNA transcribed to pre-rRNA by RNAPI, then processed to mature rRNAs+ assembled w/ imported ribosomal proteins, then ribosomal units exported into cytoplasm to be assembled into ribosomes.
Nuclear envelope and pores
Envelope= 2 membranes+ underlying lamina (2D meshwork of Lamins A, B, C, intermediate filament proteins). Lamina directly contacts chromatin+ inner nuclear membrane, some evidence that fibres extend into nuclear lumen, possibly contributing to matrix. Inner membrane has receptors that bind lamina, outer membrane continuous w/ RER+ contains ribosomes. Both membranes encapsulate perinuclear space, continuous w/ ER lumen. Both perforated by nuclear pore complexes.
Nuclear pores: macromolecular complexes ~150MDa made of nucleoporins, form aqueous channels. Main f(x)= regulated transport of macromolecules (but also small molecule diffusion)
Nuclear transport: Nuclear assembly in mitosis- prophase and anaphase
Nuclear assembly in mitosis: Prophase: disassemble (nuclear env breakdown due to cyclin B-CDK1 phosphorylating proteins). Lamina depolymerise-> soluble lamin A/C+ membrane-associated lamin B. NPCs-> soluble nucleoporin subcomplexes. Chromatin directly accessible to cytoplasm.
Anaphase: Inactivate CDK, fall in kinase activity, lamins+ NPCs dephosphorylated.
Telophase: chromatid separation, nuclei reassemble (phenomenological inversion of disassembly).
Interphase: Mlns of proteins+ RNAs cross envelope via NPCs
NPCs and nucleoporin lining central gate of the NPC
NPCs: Image reconstructions/isolated NPCs+ high-res studies (cryoelectron tomography)-> ring/ 8 subunits, fibrils project from both surfaces of NPC, those on inside organised as baskets. Top to bottom: Cytoplasmic filaments, cytoplasmic ring, luminal spoke ring, nuclear ring, nuclear basket, distal ring (“plug” sits in the middle).
Nucleoporin lining central gate- rich in FG (hydrophobic phenylalanine/gly) repeats interacting each other to form dynamic hydrogel model. Postulated models: virtual gate (noncohesive FG repeats- would bind translocating proteins+ retain them), selective phase models (nonsaturated or saturate hydrogel). Currently favour latter- cohesive FG-repeats form barrier against diffusion of general macromolecules+ solvent for translocating molecules. Exp: purify FG rich proteins, condense-> form hydrogel in vitro. Mutating out Fs-> droplet, not gel. (also can track import by phase contrast and confocal imaging. Add cargo to media, wait 30 min and see if moves in, adding importin beta to see it aids import)
Size limit of NPCs and nuclear localisation sognals
Size limit: passive diffusion of small molecules (effective pore diameter ~9nm= physical 60nm channel+ ctrl by physical properties like hydrogel).
Large proteins: microinjection into cytoplasm exp-> nuclear proteins re-accumulate by selective entry- can study import of nucleoplasmin and SV40 large T antigen.
Nuclear Localisation signals (NLS): small peptide motifs for import, can fuse to non-nuclear proteins (e.g., BSA)-> nuclear accumulation. Mutations in some Lys to Thr/Asp in nucleoplasmin/ SV40 T antigen-> abolish transport. Mostly Lys (+ Arg) essential. Inject radioactive nucleoplasmin (heads-> no uptake, tails-> uptake) or tail-coated colloidal gold into, e.g., Xenopus oocyte cytosol-> can track passage via NPC central channel by autoradiography/EM.
Nuclear transport process (import)
Transport: 1) rapid binding cargo-> cytoplasmic side of NPC, 2) slower, energy-dependent translocation. Need importin and Ran (GTPase).
Alpha subunit/importin binds NLS, beta subunit docks at NPC. After transport, nuclear ran-GTP-> dissociation of importin from NLS cargo. Alpha and beta of importin separately exported-> cytoplasm (ran-GTP dependent). Importin beta reversibly interacts FG repeats-> can cross hydrogel, mediate import. Ran+ importin cycles coupled for regulated import.
Ran cycle
Ran cycle: In cytosol, ran-specific GAP (GTPase activating protein) simulates endogenous GTPase of ran, converts ran GTP->GDP. In nucleus, chromatin-bound nt exchange factor RCC1 helps exchange GDP->GTP. Overall ran-GDP/GTP gradient across nuclear env. Ran GDP needed for importin/cargo binding (cytosol), Ran-GTP for cargo dissociation from importin+ cargo binding to exportins (nucleus).
Nuclear export
Export: RNA exported as protein complex (always fully processed before). Export signal-dependent, carrier mediated. Giant mRNA transcript (e.g., insect salivary glands) unfold to pass 5’ end first. Cytoplasmic proteins have nuclear export signals (some binding RNA) often involve amphipathic aas. NES recognised by exportins (related to importin beta), transport ran-dependent. Ran levels equilibrates across nuclear env by NTF2.
HIV unspliced transcripts bind Rev (interacts exportin, mediating transport). Small RNAs bind their exportins directly. tRNAs interact exportin-t. microRNA precursors/ other small non-coding RNAs-exportin 5.
Higher levels of transport in the nucleus
Higher levels of transport: Some proteins only enter when released from cytoplasmic anchors, e.g., some TFs-> gene regulation (anchor/release). Pi of NLS/NES currently investigated- e.g., cyclin B1 shuttles nucleus<->cytoplasm in S/G2, but Pi NES in early mitosis blocks export-> nuclear accumulation. (Unidirectional ctrl of shuttling or non-shuttling substrate by Pi). Also, Binary switch (e.g., Hog1p triggered to enter/leave nucleus by kinase/PPase respectively)
Eukaryotic chromosome replication (differences to prokaryotes)
Differs from Prok: initiate at many sites/chromosome, 1 round rep and cell division complete before next starts, DNA assembled to higher order structure after rep. Rep origins better understood in Prok- euk knowledge from biochem and genetic analysis of e.g., cases and high throughput DNA seq.
Animal virus origins of replication
Animal virus Origins of rep: SV40+ polyoma virus- minichromosomes, only have ctrl elements for own rep- ori seq and initiator protein- other factors from host. 1st useful model sys for euk rep. Large T antigen (initiator protein) bind specific seq between 65bp ctrl region w/27bp inverted repeat (potential hairpin)+ conserved A/T rich element (not valid model for euk ori- virus replicates many times per cell cycle, defies cellular ctrls).
Autonomously replicating sequences (ARS) in yeast
Autonomously replicating seqs (ARS) in yeast: yeast plasmids= episomal minichromosomes, require ARS for rep- act as ori. Isolated from random genomic DNA by ability to allow origin-less circular plasmid to replicate as autonomous chr when ligated in (+ plasmid used to transform yeast).
Used deletion and point mutants to define ARS consensus. Mutations in flanking B element seq.s reduce efficiency. >200 ARS in budding yeast genomic DNA, act as ori here too, but rep not always from every possible site.
In vivo nuclease digestion-> ARS+ B elements protected. Oris recognised seq-specifically by origin recognition complex (ORC)- initiator protein binding ARS w/ ATP. Cdc6, Cdt1, MCM complex added stepwise, occupy B elements+ other positions.
IDing higher eukaryote replication origins
Higher Euk rep oris: several thousand/chromosome (colinear replication bubbles @ 30-300kb intervals). Site-specific ori mapped by transition point between leading+ lagging strand synth. High-throughput DNA seq+ computational analysis-> tens of Ks potential oris.
* SNS-seq: Small nascent DNA strands isolated from replicating cells, seq (activated origin denatured, ssDNA w/RNA primers at start isolated+ size-fractionated, unprimed DNA denatured with lambda nuclease, RNA primers removed, DNA seq’d)- largest amount of seq.s next to ori site.
* Initiation site seq: sync cells in G1, initiate rep, rep for short time, labeling DNA w/ modified nt- initiation-site associated DNA isolated, seq’d (Ini-seq)
* Okazaki fragment seq (OK seq): seq all Ok fragments: Peaks when just from left (Watson) strand to right (Crick) show transition from rightward to leftward rep (i.e., ori)
* For all 3, inefficient oris produce less sharp peaks. Could also isolate small rep bubbles. Complementary datasets suggest several oris aggregate to initiation “zone”.
Euk origin of replication features
Oris often correlate w/ GC rich regions, incl CpG islands, which may for G quadruplex, often have modified bases. Prominent oris often @/upstream of transcription start sites- when rep initiates at/near active gene promoters, rep and transcription elongation complexes go the same direction, preventing head-on collisions.
Genetic and epigenetic factors in ori specification unclear. No strict consensus motifs known, likely candidates incl. short GC-rich motifs (may-> unusual DNA structures, include specific DNA/histone mods).
Centromeres
Centromeres attach chromosomes to spindles: plasmids replicating by ARS lost in yeast without selective pressure, can be stabilised by CEN (centromere seq) of 3 conserved DNA elements (I+III consensuses (found by yeast mutant screens for non-segregation), and II AT rich 88bp element), microtubule attachment mostly at consensuses. Yeast centromeres= attachment sites for centromeric tubules, spindle MTs (part of kinetochore complex), when chromatids attached to mitotic spindle. Higher euk centromeric DNA has alpha-satellite DNA elements, assembled into specific centromeric heterochromatin (spreads further into chromosomes), contains centromere-specific H3 variant+ sections of normal H3 with dimethylated Lys4.
