Struktura a funkce buněčného jádraStruktura a funkce buněčného jádra

Josef SrovnalJosef Srovnal

LaboratoLaboratořř experimentální medicíny experimentální medicíny DK DK LF UP a FN Olomouc LF UP a FN Olomouc

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• Seznámit posluchače se strukturou Seznámit posluchače se strukturou buněčného jádra a chromozómůbuněčného jádra a chromozómů

• Popsat funkce buněčného jádra – replikace, Popsat funkce buněčného jádra – replikace, transkripcetranskripce

• Poukázat na možnosti léčebného ovlivnění Poukázat na možnosti léčebného ovlivnění procesů na úrovni buněčného jádraprocesů na úrovni buněčného jádra

Proč buněčné jádro?Proč buněčné jádro?

DNADNA - protein- protein

– – struktura a funkce struktura a funkce – – řízení řízení

– – přežití přežití – – množení množení

– – předání předání DNADNA

Buněčné jádroBuněčné jádro

Jádro lidské buňky:

• 5-8 mm v průměru

• 10% objemu buňky

• DNA 3x109 bp (haploid) ( 1 milion stran textu)

• celková délka DNA 2 m

• 2 x 23 chromosomů (od matky a otce)

• 23 tisíc genů

Hlavní metabolické procesy v jádře

• replikace DNA ____________________________________________

• transkripce

• processing RNA, splicing

• tvorba a transport ribosomálních podjednotek

• transport mRNA do cytosolu

SAMOSTUDIUM?, DALŠÍ PŘEDNÁŠKA? ____________________________________________

Struktura jádra

• jaderný obal

• jaderné membrány

• jaderné póry

• jaderná matrix

• jadérko

• filamenta

• chromatin

Jaderný obal

Prokaryota vs. eukaryota

Proč obal?

• Ochrana DNA při aktivitě cytoskeletonu

• Transkripce – splicing - translace

Jaderné póry

Protein synthesis in eucaryotes

• Eucaryotic cells have evolved numerous membranebounded compartments that segregate their various chemical reactions so as to make them more efficient, and the nucleus is one such compartment.

• The nuclear envelope keeps functional ribosomes out of the nucleus, preventing RNA transcripts from being translated into protein until they have been extensively processed (spliced) and transported out of the nucleus into the cytosol.

• Thus RNA splicing and transport steps are interposed between DNA transcription and RNA translation.

Nucleolus

This highly schematic view of a nucleolus in a human cell shows the contributions of loops of chromatin containing rRNA genes from 10 separate chromosomes.

The function of the nucleolus in ribosome synthesis

• The 45S rRNA transcript is packaged in a large ribonucleoprotein particle containing many ribosomal proteins imported from the cytoplasm. While this particle remains in the nucleolus, selected pieces are discarded as it is processed into immature large and small ribosomal subunits.

• These two subunits are thought to attain their final functional form only as each is individually transported through the nuclear pores into the cytoplasm.

Changes in the appearance of the nucleolus in a human cell during

the cell cycle

• Only the cell nucleus is represented in this diagram.

• In most eucaryotic cells the nuclear membrane breaks down during mitosis, as indicated by the dashed circles.

Jaderná matrix

• Eukaryontní chromosom – chromatin = komplex DNA + proteinůBakteriální chromosom – DNA

• Heterochromatin - trvale v kondenzovaném stavu• Euchromatin - v interfázi dekondenzován, v mitóze

kondenzován

• Nukleoskelet - komplexní struktura analogická cytoskeletu složená z jaderná laminy a několika typů filament nutných pro průběh replikace DNA, vazbu chromatinu a integritu jádra

Eukaryontní DNA je uspořádána do chromosomů.

Chromosom - dlouhá lineární DNA sbalená pomocí proteinů do složitějších struktur umožňujících snadné rozbalení a sbalení, čili úžasnou archivaci a zároveň rychlé čtení (milion stran textu v každé buňce).Struktura chromosomu se mění během buněčného cyklu (M-fáze – kondenzovaný, neaktivní, interfáze – dekondenzován, aktivní, transkripce).

Chromosomy

The functions of the three DNA sequence elements needed to produce a stable linear

eucaryotic chromosome.

• Each chromosome has many origins of replication, one centromere, and two telomeres.

• The centromere serves to hold the two copies of the duplicated chromosome together and to attach them, via a protein complex called a kinetochore, to the mitotic spindle in such a way that one copy is distributed to each daughter cell at mitosis.

The organization of genes on a typical vertebrate chromosome.

• Proteins that bind to the DNA in regulatory regions determine whether a gene is transcribed; although often located on the 5' side of a gene, as shown here, regulatory regions can also be located in introns, in exons, or on the 3' side of a gene.

• Intron sequences are removed from primary RNA transcripts to produce messenger RNA (mRNA) molecules.

• p- a q- raménko

The nature of the nucleosome

• (A) depicts two views of the three-dimensional structure of the histone octamer; the general path of the DNA wrapped around it is indicated by a coiled tube ( top) and a series of parallel lines ( bottom). Two H2A-H2B dimers ( blue) flank an H3-H4 tetramer. The histone octamer is thus composed of two each of histones H2A, H2B, H3, and H4, with a total mass of about 100,000 daltons.

• (B) The nucleosome consists of two full turns of DNA (83 nucleotide pairs per turn) wound around an octameric histone core, plus the adjacent "linker DNA." The part of the nucleosome referred to here as the "nucleosome bead" is released from chromatin by digestion of the DNA with micrococcal nuclease.

Histones

• Principal Structural Proteins of Eucaryotic Chromosomes

• Histones are relatively small proteins

• They are present in such enormous quantities (about 60 million molecules of each type per cell) that their total mass in chromatin is about equal to that of the DNA.

• Most highly conserved of all known proteins

Nucleosomes as seen in the electron microscope

• These electron micrographs show chromatin strands before and after treatments that unpack, or "decondense," the native structure to produce the "beads-on-a-string" form.

• The native structure, known as the 30-nm fiber, is shown in (A).

• The decondensed, "beads-on-a-string" form of chromatin is shown at the same magnification in (B).

The bending of DNA in a nucleosome.

• The DNA helix makes two tight turns around the histone octamer. This diagram is drawn approximately to scale to illustrate how the minor groove is compressed on the inside of the turn. Due to certain structural features of the DNA molecule, A-T base pairs are preferentially accommodated in a narrow minor groove.

The way histone H1 is thought to help pack adjacent nucleosomes

together.

• The globular core of H1 binds to each nucleosome near the site where the DNA helix enters and leaves the histone octamer.

• When H1 is present on the nucleosomes, 166 nucleotide pairs of DNA are protected from micrococcal nuclease digestion, compared with 146 nucleotide pairs for nucleosomes lacking H1.

The 30-nm chromatin fiber

• A model to explain how the "beads-on-a-string" form of nucleosomes is packed to form the 30-nm fiber seen in electron micrographs. This type of packing requires one molecule of histone H1 per nucleosome (not shown).

Nucleosome-free regions in 30-nm fibers.

• A schematic section of chromatin illustrating the interruption of its regular nucleosomal structure by short regions where the chromosomal DNA is unusually vulnerable to digestion by DNase I.

• At each of these nuclease-hypersensitive sites, a nucleosome appears to have been excluded from the DNA by one or more sequence-specific DNA-binding proteins.

Model of chromatin packing

• This schematic drawing shows some of the many orders of chromatin packing postulated to give rise to the highly condensed mitotic chromosome.

Human karyotype

• This map was determined at the prometaphase stage of mitosis. Chromosomes 1 through 22 are labeled in the approximate order of their size.

• A diploid cell contains two of each of these autosomes plus two sex chromosomes - two X chromosomes (female) or an X and a Y chromosome (male). The 850 bands shown here are G bands, which stain with reagents that appear to be specific for A-T-rich DNA sequences.

Funkce buněčného jádra

• replikace DNA

• transkripce

• processing, splicing RNA

• tvorba a transport ribosomálních podjednotek

• transport mRNA do cytosolu

Replikace

• Replikace – v S-fázi buněčného cyklu (synthesis)

• Zdvojení nejenom DNA, ale i histonů a jaderných proteinů.

• Na konci S-fáze (8hodin) – dvě kopie chromozomu spojené centromerou

• Semikonzervativní

Replication origins

• Replication origins tend to be activated in clusters (called replication units) of perhaps 20 to 80 origins.

• New replication units seem to be activated throughout the S phase until all of the DNA is replicated.

• Within a replication unit, individual origins are spaced at intervals of 30,000 to 300,000 nucleotide pairs from one another

• Different Regions on the Same Chromosome Replicate at Distinct Times

• Highly Condensed Chromatin Replicates Late, While Genes in Active Chromatin Replicate Early

Helicase function

• The assay used to test for DNA helicase enzymes. A short DNA fragment is annealed to a long DNA single strand to form a region of DNA double helix. The double helix is melted as the helicase runs along the DNA single strand, releasing the short DNA fragment in a reaction that requires the presence of both the helicase protein and ATP. The movement of the helicase is powered by its ATP hydrolysis.

DNA replication initiating proteins

• The major types of proteins involved in the formation of replication forks at the E. coli and bacteriophage lambda replication origins are indicated. Subsequent steps result in the initiation of three more DNA chains by a pathway that is not yet clear. For E. coli DNA replication, the major initiator protein is the dnaA protein; for both lambda and E. coli, the primosome is composed of the dnaB (DNA helicase) and dnaG (DNA primase) proteins.

The structure of a DNA replication fork

Because both daughter DNA strands (colored) are synthesized in the

5'- to-3' direction, the DNA synthesized on the lagging strand must

be made initially as a series of short DNA molecules,

called Okazaki fragments.

RNA primer synthesis

A schematic view of the reaction catalyzed by DNA primase, the enzyme that synthesizes the short RNA primers made on the lagging strand. Unlike DNA polymerase, this enzyme can start a new polynucleotide chain by joining two nucleoside triphosphates together. The primase stops after a short polynucleotide has been synthesized and makes the 3' end of this primer available for the DNA polymerase.

The synthesis of the DNA fragments on the lagging

strand

• In eucaryotes the RNA primers are made at intervals spaced by about 200 nucleotides on the lagging strand, and each RNA primer is 10 nucleotides long. This primer is erased by a special DNA repair enzyme that recognizes an RNA strand in an RNA/DNA helix and excises it; this leaves a gap that is filled in by DNA polymerase and DNA ligase, as we saw for the DNA repair process

Replication• The fork is drawn to emphasize its similarity to the bacterial replication fork, although both forks utilize the same basic components, the mammalian fork differs in two important respects.

• First, it makes use of two DNA polymerases, one for the leading strand and one for the lagging strand. It seems likely that the leading-strand polymerase is designed to keep a tight hold on the DNA, whereas that on the lagging strand must be able to release the template and then rebind each time that a new Okazaki fragment is synthesized.

• Second, the mammalian DNA primase is a subunit of the lagging-strand DNA polymerase, while that of bacteria is associated with the DNA helicase.

A replication fork in three dimensions

The two-dimensional structure has been altered by folding the DNA on the lagging strand to bring the lagging-strand DNA polymerase molecule into a complex with the leading-strand DNA polymerase molecule. This folding process also brings the 3' end of each completed Okazaki fragment close to the start site for the next Okazaki fragment. Because the lagging-strand DNA polymerase molecule is held to the rest of the replication proteins, it can be reused to synthesize successive Okazaki fragments; thus it is about to let go of its completed DNA fragment and move to the RNA primer that will be synthesized nearby, as required to start the next DNA fragment.

Single-strand binding proteins

• Because each protein molecule prefers to bind next to a previously bound molecule (cooperative binding) long rows of this protein will form on a DNA single strand. This cooperative binding straightens out the DNA template and facilitates the DNA polymerization process. The "hairpin helices" shown in the bare single-stranded DNA result from a chance matching of short regions of complementary nucleotide sequence.

Sliding clamp

• (A) The structure of the sliding clamp from E. coli, with a DNA helix added to indicate how the protein fits around DNA. A similar protein is present in eucaryotic cells.

• (B) Schematic illustration of how the clamp is thought to hold a moving DNA polymerase molecule on the DNA.

DNA topoisomerase I

As indicated, these enzymes form a transient covalent bond with DNA so as to allow free rotation about the covalent bonds linked to the blue phosphate.

DNA topoisomerase II

• An example of a DNA-helix-passing reaction catalyzed by a type II DNA topoisomerase.

• Unlike type I topoisomerases, these enzymes require ATP hydrolysis for their function, and some of the bacterial versions can introduce superhelical tension into DNA.

• Type II topoisomerases are largely confined to proliferating cells in eucaryotes; partly for that reason, they have been popular targets for anticancer drugs.

Model of nucleosome separation

• Speculative model showing how a nucleosome might open up to permit DNA replication.

• After the replication fork passes, the nucleosome reassembles. In this way the histones of the nucleosome core remain permanently bound to the DNA. Although in this diagram the old nucleosome has been inherited intact by the DNA helix made on the leading strand, there is evidence that an intact nucleosome can be inherited by either daughter DNA molecule. Moreover, this is only one of many possible models.

Telomere replication

• The figure outlines the reactions involved in the formation of the repeating G-rich sequences that form the ends of chromosomes (telomeres) of diverse eucaryotic organisms.

• The incomplete, newly synthesized strand is the strand made on the lagging side of a replication fork. As indicated, the telomerase is a protein-RNA complex that carries an RNA template for synthesizing a repeating, G-rich telomere DNA sequence. These repeats are GGGTTA in humans.

• The lagging strand is presumed to be completed by DNA polymerase a, which carries primase as one of its subunits.

Summary

DNA replication begins with the loading of a DNA helicase onto the DNA by an initiator protein bound to a replication origin. A replication bubble forms at such an origin as two replication forks move away from each other. During S phase in higher eucaryotes, neighboring replication origins appear to be activated in clusters known as replication units, with the origins spaced an average of about 100,000 nucleotide pairs apart. Since the replication fork moves at about 50 nucleotides per second, only about an hour should be required to complete the DNA synthesis in a replication unit. Throughout a typical 8-hour S phase different replication units are activated in a sequence determined in part by the structure of the chromatin, the most condensed regions of chromatin being replicated last. After the replication fork passes, chromatin structure is re-formed by the addition of new histones and other chromosomal proteins to the old histones inherited on the daughter DNA molecules. A DNA re-replication block of unknown nature acts locally to prevent a second round of replication from occurring until a chromosome has passed through mitosis; this block is needed to ensure that each region of the DNA is replicated only once in each S phase. The problem of replicating the ends of chromosomes is solved by a specialized end structure (the telomere) and an enzyme (telomerase) that extends this structure using an RNA template that is part of the telomerase.

Léčiva směřující do buněčného jádra

• Inhibice biosyntézy NK - analoga• Poškození struktury a funkce NK – alkylace,

interkalace, inhibice topoizomeráz • Alterace mikrotubulárních proteinů

Léčiva ovlivňující dělení buněk:

Cytostatika, imunosupresiva, antivirotika

Inhibice biosyntézy NK - analoga

Analoga: aktivovány a inkorporovány do NK – zástava replikace, transkripce, nesprávný kód.

Indikace: nejčastěji hematolgické malignity

Dělíme na:

• Analoga kyseliny listové

• Purinová analoga

• Pyrimidinová analoga

Analoga kyseliny listové

Způsobí inhibici dihydrofolátreduktázy, která jinak redukuje

kys. listovou na tetrahydrolistovou, která je nepostradatelným

kofaktorem pro biosyntézu purinů (dTMP), tedy DNA.

Methotrexát (MTX)

• Onkologie – hematolgické malignity (ALL, lymfomy), osteosarkomy, karcinomy ORL, intrathekální aplikace

• NÚ – mukozitidy, leukopenie, plicní komplikace.

• Leukovorin – antidotum, ochrana

• Imunosupresivum

Purinová analoga

Analoga guaninu - 6-merkaptopurin – leukemie, MDS

- 6-thioguanin - AML

- azathioprim – imunosupresivum

- alopurinol – antiuratikum

Analoga adeninu - fludarabin – i do RNA – CML

- kladribin – TCL, NHL

Pyrimidinová analoga

Fluorované pyrimidiny – 5-FU – KrK, GIT

- Kapecitabin – p.o. 5-FU

Analoga cytidinu - cytosinarabinosid –AML

- gemcitabin – NSCLC

Nucleoside analogues

Nucleoside analogues interfere directly with DNA replication, impede it indirectly by limiting the synthesis of deoxy-nucleotide triphosphate precursors, or cause strand breaks after incorporation into DNA. 5-fluorouracil (5-FU) is a widely employed member of this class, which acts mainly by inhibition of thymidylate synthase. Methotrexate is not a nucleoside analogue, but also interferes with deoxy-nucleotide biosynthesis by inhibiting dihydrofolate reductase. Thus, both compounds diminish the level of dTTP, the nucleotide precursor specifically needed for DNA replication.

Poškození struktury a funkce NK

Účinek: poškození struktury a funkce NK má za následek inhibici replikace a transkripce.

Indikace: nejčastější cytostatika, solidní tumory

Dělíme dle mechanismu poškození NK na:

• alkylace – kovalentní vazba

• interkalace – nekovalentní vazba

• inhibice topoizomeráz

Alkylační látky

Deriváty platiny – cisplatina, karboplatina, oxaliplatina- koordinační sloučeniny Pt- gynekol. malignity, NSCLC, ORL, KrK...- NÚ – nefro-, neuro- a ototoxicita

Oxazafosforiny – cyklofosfamid - ALL, solidní tumory- imunosupresivum- NÚ – hemor. cystitida (akrolein)

Interkalátory

Antracykliny – daunorubicin, doxorubicin, epirubicin- cytostatikum se vmezeří do DNA dvojšroubovice a váže se H můstky- tvorba volných radikálů- hematol. malignity, ca prsu,.......

- NÚ – kardiotoxicita- liposomální doxorubicin (PEG) – stejný

účinek, nižší nežádoucí účinky

Aktinomyciny – aktinomycin D- protinádorové ATB (Streptomycety)- sarkomy, tu varlat, Wilmsův tu

Inhibitory topoizomeráz

Topoizomerázy jsou nukleární enzymy důležité pro replikaci – riziko překroucení dvojšroubovice a vznik zlomů.

• Topoizomeráza I – váže se na jedno vlákno DNA, rozpojí ho, uvolní se pnutí, a opět vlákno spojí.

• Topoizomeráza II – váže se na obě vlákna DNA, způsobí jejich přerušení a opětovné spojení a umožní separaci chromozomů při mitoze.

• Účinek – působení zlomů, alterace DNA

Inhibitory topoizomeráz

• Inhibitory topoizomerázy I – deriváty camptotecinu (africký keř Camptoteca

accuminata)

- irinotekan, topotekan

- KrK, ca ovaria....

• Inhibitory topoizomerázy II

- tenipozid, etopozid

- hematologické malig., NHL, NSCLC, testikulární nádory, sarkomy...)

. Topoisomerases are necessary for DNA replication (as well as for transcription), since they relax the torsional stress that is caused by the unwinding of the DNA helix. Topoisomerase I enzymes reversibly insert a single-strand break, allow the DNA strands to swivel around each other, and re-ligate the strand-break. Topoisomerase II enzymes catalyze a more dramatic reaction, in which a double strand break is reversibly introduced and another DNA helix (or a distant part of the same helix) is passed through, before the ends are resealed by the enzyme. This is a more fundamental reaction, which in addition to relaxing torsional stress allows the untangling of DNA knots and loops. In this fashion, DNA replication is inhibited and DNA is fragmented, more efficiently than by topoisomerase I inhibitors.

Inhibitory topoizomeráz

Alterace mikrotubulárního proteinu

Mitotické jedy – alterace mikrotubulů poškodí funkci dělícího vřeténka – omezená migrace chromosomů při mitóze. Většina omezuje syntézu tubulinu.

Polymerizace – v rovnováze s - depolymerizaci

• Inhibitory polymerizace – inhibice syntézy

• Inhibitory depolymerizace – inhibice rozpadu

Inhibitory polymerizace

• Vinkristin, vinblastin, vinorelbin – (Vinca rosea)

• Blok mitózy v metafázi – nepravidelně rozptýlené metafazické chromozomy

• Poškození funkce buněčných membrán.

• Hematol. malignity, solidní tumory...

Inhibitory depolymerizace

• Taxany - paklitaxel, docetaxel

• Tvorba mikrotubulů – svazků, hvězdic

• Blok buněčného cyklu, indukce apoptózy (bcl2)

• Ca ovaria, prsu, NSCLC, GIT, ORL

Molecular Biology of the CellMolecular Biology of the Cell, , Fourth EditionFourth Edition Bruce Alberts, Alexander Johnson, Julian Lewis, Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter Martin Raff, Keith Roberts, Peter Walter 28/02/2002 28/02/2002 1616 pages 1616 pages

Úvod do mÚvod do moleolekulární biologiekulární biologieStanislav Rosypal, 4. vydání, 2006Stanislav Rosypal, 4. vydání, 2006__________________________________________________________

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MUDr. Josef Srovnal

Laboratoř experimentální medicíny DK FN a LF UP Olomouc

Tel: +420 585 853 225

Email: josef.srovnal@seznam.cz

www.lem.ocol.cz