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NUCLEUS AND DNA ORGANIZATION, GENETIC PROCESSES - Coggle Diagram

- - - - INNER MEMBRANE
        
        associated with nuclear lamina
        
        lamina is connected to chromatin
        
        key in MECHANOTRASDUCTION
      - OUTER MEMBRANE
        
        continuous with the ER - surrounded by ribosomes
        
        this facilitates transition form transcription to translation
      - the two membranes are similar but not identical
        
        the envelope breaks down when triggered by the disassembly of the laminate in mitosis
        
        evolution hypothesis: invagination of the plasma membrane
    - - each pore is occupied by a large protein assembly: NUCLEAR PORE COMPLEX NPC
        
        mediates transport
        
        passive form small solutes and ions
        
        active and selective for bigger molecules (RNA, proteins, ribonulceoproteins)
        
        can occur against gradient
        
        requires energy
        
        depends on signal sequence = NLS or NES
        
        NLS: nuclear localisation signal = basic residues ex KKKRK and can be both mono partite or bipartite
        NES: nuclear export signal = hydrophobic residues (most L - like LXXXLXXLXL)
        
        signals are sufficient for transport but not for retention
        
        they are not cleaved = can be reused
        
        architecture
        
        charachteristic octagonal radial ring symmetry
        
        filaments for structural support
        
        nuclear basket
        
        cytoplasmic fibrils
        
        made of proteins called NUPs: nucleoporins
        
        one of the most important NUP group is the FG-nucleoporins
        
        they create and dynamic, disordered meshwork that regulates and facilitates passages for the correct molecules (= if they are correctly signaled) ==> selective barrier
        
        NPC proteins aren't just inert channels --> they also contribute to chromatin organisation/gene regulation/cell cycle control
        
        several NUPs during mitosis are recruited to the kinetochore and participate in the mitosis spindle assembly checkpoint
        
        ex RAE1 (RNA export 1) is part of mRNA export during interphase but binds to microtubules and kinetochores during cell division
      - discontinuities of about 80-100 nm
        
        number is variable and reflects the necessity of bidirectional communication between cytoplasm and nucleus
        
        key gateway for bidirectional traffic
      - TRANSPORT
        
        IMPORT
        
        NLS is recognised by an importin (alpha+beta complex or only beta)
        
        cargo-importing complex diffuses through the NPR translocating through the FG mesh
        
        Ran-GTP binds the complex (on the importin) causing a conformational change that causes cargo release
        
        the resulting importin-Ran-GTP complex exits the nucleus back to the cytoplasm following the RanGTP gradient
        
        in the cytoplasm Ran-GAP converts RanGTP to RanGDP allowing dissociation with the importin
        
        the importin is now free and the RanGDP returns to the nucleus where it will be converted back to RanGTP by Ran-GEF
        
        EXPORT
        
        when RNA has to exit is is often packed into a RNP (ribonucleoprotein) complex
        
        rRNA is associated with ribosomal proteins to form ribosomal subunits
        
        mRNA is associated with multiple hnRNP (heterogeneous nuclear ribonucleoproteins) that help in the export process and usually dissociate in the cytoplasm
        
        the cargo (protein or RNP) with NES is recognised by an exportin
        
        Ran-GTP also binds forming a ternary complex: cargo+expo+RanGTP
        
        the complex bins FG-NUPs and translocated through the mesh work to the cytoplasm
        
        in the cytoplasm Ran-GAP stimulated hydrolysis
        
        GTP hydrolysis triggers conformational change that causes dissociation of the exportin from its cargo which is now free
        
        exportin and RanGDP return to the nucleus where Ran-GEF D-->T
        
        very selective
        
        proteins can be transported in a fully folded state
        
        importins and exportins are part of the karyopherin family
        
        small GTPase Ran: Ran-GTP and Ran-GDP create the gradient and ensure cargo release and correct placement of importins/exportins
        
        Ran acts as a molecular switch:
        
        GTP bound = active form and in nucleus
        
        GDP bound = inactive form and in cytoplasm
        
        Ran-GEF (guanine exchange factor) D-->T (nucleus)
        Ran-GAP (GTPase activating protein) T-->D (cytoplasm)
        ==> they maintain the correct placement of RanGTP/GDP in order to keep the correct gradient/concentration
    - - thin dense meshwork made up of intermediate filaments that lies under the INM
        
        tightly anchored to the INM through various integral membrane proteins: like LBR and emerin
      - it's composed of nuclear laminas: fibrous proteins of the intermediate filament family
        
        in humans the main ones are:
        
        Lamin A and C: derive form alternative splicing of a single gene LNMA on chromosome 1
        
        Lamin B: its isoforms are encoded by two separate genes: LMNB1 (chromosomes5) and LMNB2 (chromosome19)
        
        structurally they are similar to cytoplasmic intermediate filaments but they have specific additional domains:
        
        alpha helical rod domain
        
        C-terminal domain: NLS + imunoglobin-like fold + CAAX box (this box will undergo farnesylation in PTM)
        
        lamin assembly occur by general polymerization:
        
        dimer formation: parallel head-to-tail coiled-coil dimers
        
        dimers associate in polar head-to-tail polymers (there is a small staggered overlap)
        
        two polymers associate anti-parallel = protofilaments
        
        3/4 protofilaments associate laterally = intermediate filament
      - function
        
        gene regulation: bridge between the envelope and chromatin (through LBR and emerin) --> helps organize the 3D genome, influences chromatin structure and contributes to gene silencing
        
        mechanotransduction: it participates to long-range mechanics interaction and signalling connection between nucleus and cytoskeleton
        
        for this function the LINC complex is key
        
        Linker of Nucleoskeleton and Cytoskeleton
        
        physical linker, continuous bridge
        
        made form two main protein families
        
        SUN proteins: embedded in the INM and bind laminas and chromatin
        
        Nesprins: they are embedded in the ONM
        
        KASH domain: C-terminal that interacts with SUN
        
        N-terminal domain projects into the cytoplasm
        
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        essential for:
        
        correct positioning and shaping of the nucleus
        
        important during cell migration through confined environments (ex diapedesis)
        
        mechanical flexibility off the nuclear envelope
        
        role in chromosome dynamics: tether telomeres of homologous chromosomes connecting them to cytoskeletal motors through nesprins
        
        mechanotransduction
        
        mechanics: cytoskeletal support for the nucleus --> strengthens the envelope making it resistant to deformation
        
        mitosis: drives the disassembly and reformation of the nucleus
        
        cyclin-dependant kinases (Cdk) phosphorylate laminas (especially A and C) causing depolymerisation ==> lamina disassembly is a major driving force for nuclear envelope breakdown
        
        lamins A and C become soluble while laminating B remains attached to membrane fragments (thanks to the farnesyl group and interaction with receptor LBR)
        
        in telophase phosphatases remove phosphate groups from laminas allowing them to repolymerize --> lamin B serves as a scaffold
        
        architecture: helps define the overall shape and size of the nucleus and contributes the the distribution of pores
        
        provides a platform for assembly of protein complexes
- - - - non-histone proteins
        
        functions
        
        functional/regulatory roles: transcription factors, co-activators, co-repressors, chromatin remodelling complexes
        
        structural roles: cohesins, condensins, scaffold
        
        cell-type specific and condition dependant
        
        recognises specific DNA sequences
      - histone proteins
        
        they bind the negatively charged DNA backbone and organize it into higher-order structures
        
        they are sequence independent but have sequence preference: certain dinucleotides (AA TT AT) bend more easily toward the minor groove making those regions more favourable for nucleosome positioning
        
        five gene families/classes: H1, H2A, H2B, H3, H4
        
        highly conserved in evolution in almost all eukaryotes (and also present in archea)
        
        genes are often present in multiple copies in the genome
        
        similar general organisation
        
        histone fold domain (globular region) composed of several alpha helices ==> mediates interaction with other histones and DNA
        
        the fold domain allow histones to interact via a characteristic handshake interaction
        
        N-terminal tail that protrudes out of the nucleosome ==> rich in basic residues and main site of PTMs
        
        interaction with: DNA, other nucleosomes
        
        covalent modification of histone tails
        
        methylation - often associates with repressed chromatin
        
        phosphorylation - involved in process such as: mitosis, DNA damage response, remodelling
        
        acetylation - neutralizes = creates more permissive DNA
        
        ubiquination and SUMOylation - signalling and regulation
        
        some classes also have a C terminal main with basic residues (H1, H2A, H2B)
        
        useful way to describe structure:
        
        variable region V rich in positively charged residues (tail)
        
        constant region C the globular core which is structurally conserved
        
        they assemble into a NUCLEOSOME
        
        two H3+H4 dimers and two H2A+H2B dimers assemble into a histone octamer (first to form and to bind DNA = H3-H4 tetramer)
        
        DNA wraps around this octamer approximately 1.7 turns (about 147 bp)
        
        histone H1 binds DNA at the entry and exit sites stabilising the wrap
        
        nucleosomes are separated by a linker DNA region of variable length (usually about 50 bp)
        
        histone chaperones and other assembly factor help coordinate
        
        they help in managing free histones and correct reassembly of nucleosomes during remodelling
        
        creation of nucleosomes creates the typical structure of the 10nm fibres: beads and string = most decondensed visible form
        
        form this the 30nm fiber can create: main form in physiological conditions = regular, highly ordered structure where histone H1 is crucial
        
        it has 2 models
        
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        they are not static: DNA continuously winds and unwinds around the core --> slides along in an ATP-dependant manner
        
        ATP-dependant chromatin remodelling complexes, they:
        
        side along DNA
        
        partially unwrap DNA
        
        remove and insert nucleosomes
        
        exchange histone dimers (with variants)
      - histones provide the basic conserved scaffold for DNA compaction, binding DNA mainly via charge and forming nucleosomes while non-histone proteins add specificity and flexibility, shaping higher order structures and finely tuning when and where genes are expressed
  - - - protective: defence form physical damage and nucleases
      - regulatory: the degree of local compaction establishes the ability of that portion of DNA to be transcribed
        
        chromatin isn't heterogeneous
        
        euchromatin
        
        transcriptionally active, more open form because genes are accessible (decondensed during interphase)
        
        heterochromatin
        
        transcriptionally inactive, tightly packed chromatin
        
        constitutive heterochromatin
        
        always present in a condensed state and typically contains DNA with structural functions often rich in ripetitive sequences
        
        facultative heterochromatin
        
        chromatin that can switch
      - steric: reduce volume
    - - naked B-form DNA (never exist freely in the nucleus
        
        nucleosomes
        
        chromatosomes = nucleosome + H1 + wrapped DNA
        
        10 nm fiber
        
        30 nm fiber = native interphase chromatin fiber
        
        DNA loops (50.000 - 200.000 bp) anchored at their base to a protein scaffold or nuclear matrix
        
        many loops in series create thick fibres
        
        looped organisation in present also in the interphase and it functional to structural compaction but also to the definition of functional domains
        
        protein scaffold = dense structure composed of non-histone proteins including:
        
        scaffold-specific proteins
        
        SMC proteins (structural maintenance of chromosomes) such as:
        
        cohesins
        
        typically built as heterotetramers they form a ring or hairpin like structure capable of embracing chromatin fibres
        
        mian functions
        
        regulatory factors
        
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        structural elements
        
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        condensins
        
        usually composed of 5 subunits and embrace and re-organize long stretches of chromatin loop
        
        typical roles
        
        driving chromatin companion in meiosis and mitosis
        
        creating and stabilising loop domains
        
        they are large and contain an ATPase domain since they work in a ATP dependant manner
        
        topoisomerase II: helps manage DNA topology (supercoiling and decatenation)
        
        only specific DNA regions can attach to the scaffold = non-coding AT-rich sequences = SAR/MARs (Scaffold Associated region or Matrix Attachment regions) ==> preferred docking site
        
        during metaphase it goes through further compaction
        
        it has an x shape which consists in two sister chromatids (each one containing a full and identical set of DNA molecule + histone/non proteins) held together at the centromere (primary constriction)
        
        centromeres are the sites where kinetochores assemble
        
        divide each chromatid in two arms
        
        q arm long
        
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        p arm short
        
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        telomere: terminal regions of constitutive heterochromatin with short repetitive sequences (in humans TTAGGG)
        
        they are crucial to maintain structural integrity and allow interaction with the nuclear lamina
        
        sister chromatids will separate during anaphase
        
        NOR: secondary constriction (nucleolar organiser region) containing rRNA gene clusters
  - - - combinatorial
      - interpreted by proteins
      - contributes to epigenetic memory
  - - - monosomy
      - trisomy
      - nullisomy
- - - - rRNA synthesis (by RNAP I and III)
      - processing and modification of RNA
      - assembly of ribosomal subunits
    - - after mitosis (starts in telophase) 5 chromosomes (13,14,15, 21, 22) carry rRNA genes on NOR and each NOR can start to form a small nucleolus that can then fuse into one/few
    - - enzymatic machineries for rRNA synthesis and maturation
      - ribosomal proteins synthesised in cytoplasm and brought back
      - rRNA transcript and processing intermediates
      - genes for some tRNAs (processing begins in the nucleolus)
      - multicopy genes coding for rRNA (located in NORs)
      - multiple RNPs and the telomerase complex
    - - heterogenous with distinct morphological regions
      - FC - fibrillar center: contains rDNA in a less condensed form - associated with transcription units of rRNAs
      - DFC - dense fibrillary component: contains actively transcribed rRNA genes, RNAP I and transcription factors
      - GC - granular component: contains rRNA undergoing later processing and pre-ribosomal particles (progressive maturation before export)
  - - - 1/few per nucleus defined by the presence of a specific marker protein: coilin
        
        sites where snRNAs and snoRNAs are processed and assembled with proteins into snRNPs (involved in splicing) and snoRNPs (involved in rRNA modification)
        
        involved in telomerase maintenance
        
        linked to DNA repair, telomer dysfunction, cancer and premature aging
      - GEMS (gemini of Cajal bodies)
        
        similar to cajal bodies but lack coilin
    - - typically found near gene-dense regions and telomers
        
        functions
        
        regulation of transcription
        
        participation in DNA repair
    - - where pre-mRNA splicing factors are enriched
        
        they are correlated with transcription activity
        
        in active cells splicing factors shuttle between speckles and transcription sites but when transcription is inhibited splicing factor accumulate in the speckles
        
        they don't contain RNAP II or pre-mRNAs
    - - characteristic circular appearance
      - built along an non-coding RNA = NEAT1 (acts as scaffold for multiple proteins)
      - function: retention and storage of specific nuclear RNAs + regulation of: circadian rhythms, cell differentiation and stress response
- - - - come form a nucleated precursor
      - before losing the nucleus the accumulate proteins, RNA and metabolites
      - they are much less flexible (less possibility to adapt) since the cannot synthesize new proteins and lifespan is limited
      - absence of nucleus frees up space improving efficiency
- - - - within territories
        
        telomere tend to localise near the periphery, near the lamina
        
        active regions may loop out toward the interior or towards interchromosomal domains
        
        INTERCHROMOSOMAL DOMAINS
        
        are enriched in pre-mRNAs and mRNAs in maturation and in transcription and processing machines
        
        act as transcription factories
        
        heterochromatin is near the lamina
    - - the genome, beyond the level of chromosomes, is partitioned in TADs: region of the genome that interact more frequently with themselves --> they can include part of one or more chromosomes that create a 3D pattern
      - they tend to segregate into active A compartments and inactive B compartments
- - - - primer removal and gap filling
        
        PRO
        
        RNAse H degrades the RNA part
        
        DNA Pol I removes the remaining ribonucleotides and fills the gap
        
        EUK
        
        FEN1 (flap endonuclease) cleaves the RNA/DNA flap
        
        DNA pol fills the gap
        
        RNAse H degrades RNA part
    - - in the lagging strand there are multiple
    - - after the bubble the two strands keep dividing
        
        the bubble creates two replication forks in opposite directions
        
        each forks creates two types of strands (forks are asymmetric)
        
        leading strand: will be synthesised continuously since the DNAP will move towards the opening
        
        lagging strand: the DNAP moves opposite form the direction in which the strands open so replication will be fragmented = Okazaki fragments
    - - replication starts at a specific site: origin of replication which is recognised by initiator proteins that bind to it creating a replication bubble
        
        the region of DNA replicated from a single origin of replication is a replicon
        
        PRO only have one replicon, EUK with linear DNA have multiple
  - - - extends primers and later fills gaps
        
        adds nucleotides only in 5-->3 direction (so reads the strand in 3-->5 direction)
        
        that's what there is a leading and a lagging strand
        
        chemical reaction
        
        the 3'-OH of the primer attacks the free alpha-phosphate of the incoming dNTP creating a new phosphodiester bond and releasing pyrophosphate (PPi)
        
        PPi is then hydrolysed to make the reaction energetically favourable
        
        it can be visualised as a right hand
        
        palm domain
        
        catalytic site where the bond is formed
        
        finger domain
        
        bends the template of about 90deg in order to correct the right base - helps in accuracy
        
        thumb domain
        
        holds the DNA-primer dupplex in place + stabilised the DNA-enzyme interaction = increase in processivity and efficacy
        
        there are different types
        
        PRO
        
        I and II are gap filling and repair, III is the main one
        
        EUK
        
        delta does lagging and eta does leading
        
        gamma is the main for mito and chloro
        
        beta is for repair and alpha for primer synthesis and partial elongation
        
        is a self-correcting enzyme
        
        if the growing strand is mismatched is can activate 3'-->5' exonuclease activity to remove the wrong base
        
        does exonucleatic proofreading
    - - solves the issue of supercoiling/unwinding problem acting as a reversible nuclease: creates a transient bond with DNA breaking the phosphodiester backbone, allowing the strand to pass/rotate and then reseals the break
        
        topo I: breaks one strand topo II: breaks 2 and consumes ATP
    - - RNAP that synthesizes RNA primers
    - - (PCNA x euk) ring shaped protein complex that encircles DNA increasing processivity (how many nucleotides are added before the polymerase falls off)
        
        the clamp loader is an ATP-dependant complex that opens the clamp and loads it onto the DNA
    - - bind cooperatively to the SSDNA maintaining the denatured state, avoiding the formation of secondary structures
        NB they don't cover the bases - they are still accessible to the DNA polymerase
    - - seals remaining nicks using ATP
    - - unwinds the DNA double helix at the fork using ATP hydrolysis to move along
    - - being the euk DNA linear after the removal of the last RNA primer there is no upper free 3'-OH to attach to fill the gap
        
        this issue would mean a continuous and quick shortening of DNA = lesion of genes
        
        telomeres solve this issue ==> they are repeating non-coding sequences that are repeated many times
      - they are active in: gremlin cells, some stem cells and some WBC
        
        in somatic cells they activity is very low or completely absent so the get shorter after every replication process
        
        when the cell reaches the Hayflick limit it stops dividing
        
        this concept links to cellular senescence and cancer
  - - - to prevent this cells combine a highly accurate polymerisation to various DNA repair systems
        
        polymerization
        
        error rate is reduced in three steps
        
        proofreading
        
        when a mismatch is incorporated despite the controls DNAP can switch to a 3-->5 exonuclease activity to cleve the incorrect base and proceed with a correct polymerisation
        = self correction machine
        
        MMR
        
        residual mismatches bend the helix strand and can be corrected by strand-directed mismatch repair
        
        in bacteria
        
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        accurate polymerization
        
        DNA polymerase is shaped like a right hand, when the correct base is incorporated the finger drain rotates of about 40deg into a closed conformation and specific AA residues (K, Y, R) bind with the incoming base while the palm domain forms Hbonds with the base pairs in the minor groove
        
        if these bond don't form correctly the enzymes enters a non optimal conformation and synthesis is slowed down
        
        this involves misincorporation errors
        
        DNA repair
        
        there are other types of errors that are not directly linked to mis incorporation soother are direct repair systems
        
        NER nucleotide excision repair
        
        targets bulkier regions that cause significant distortions where the bases may be correct but abnormally linked
        
        damage is recognised by proteins that sense distortions in the helix --> when it finds one it binds and and excision nuclease cuts the damaged strand on both sides
        
        a DNA helices helps detach the patch and a DNAP and ligase finish the work
        
        SSB
        
        they are repaired quickly and accurately
        
        specialised proteins like PARP1 (poly-ADP-ribose polymerase 1) detect the break
        
        PARP1 becomes activated and synthesised branched chains of ADP-ribose on itself and nearby proteins
        
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        BER base excision repair
        
        for small specific lesions or individual bases where the backbone is intact but the base is chemically abnormal
        
        DNA glycosylase - enzyme specialised to recognise a particular type of damaged base - identifies the error and flips the damage into the active site cleaving the glycosidic bond creating an AP site
        
        an AP endonuclease recognises the atypical sugar and cuts the DNA backbone
        
        a phosphodiesterase removes the remaining sugar creating a clean gap
        
        a repair type polymerase fills and a ligase seals
        
        DSB
        
        there are two types of systems and the choice of use depends on the stage of the life cycle of the cell
        
        in G1
        
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        after S
        
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- - - - translation: in the cytoplasm
      - DNA repair
      - transcription: in the nucleus
      - recombination: homologous or site specific
      - replication: during the S phase in the nucleus
      - they are all tightly connected to the cell cycle and cell's necessities
      - exceptions to the dogma: reverse transcriptase and prions