Structure of eukaryotic genome, its replication and gene expression Molecular Biology RNDr. Jan Hošek, Ph.D hosekj@vfu.cz Structure of eukaryotic genome > Nucleus consists of chromatin > dsDNA > histones > nonhistones = proteins of non-histonic character > Chromosomes contain linear dsDNA > Nucleus is divided by mitosis > Cell wall consists of cellulose (plants) or chitin (fungi), animal cells have no cell wall > They have organelles - mitochondria and plastids > Endoplasmic reticulum, Golgi system, and lysosomes Animal cells: nucleus and mitochondria Plant cells: nucleus, mitochondria and chloroplasts > chromosomal (AKA nuclear) DNA (nDNA) > mitochondrial DNA (mtDNA) > chloroplast DNA (ctDNA) > plasmids > Stainable material, which forms the nucleus of eukaryotic cells > dsDNA, histories, nonhistones According to the ability to be stained by basic dyes and degree of condensation we distinguish: > euchromatin - weakly stainable, decondensed, "transcriptionally active" > heterochromatin - strongly stainable, condensed, "transcriptionally inactive" heterochromatin https://www.studyblue.com/notes/note/ri/3-chromosomes/deck/4743713 - Constantly in heterochromatin stage - centromeres and telomeres - One of X chromosome in women Facultative - Switches between heterochromatin and euchromatin on the base of oncogenetic development of organism Chromatin condensation 1) Basic structure = interphase = decondensed 10-nm chromatin fiber (beads-on-a-string) 2) 30-nm chromatin fibre 3) Chromatin in mitotic phase = mitotic chromosomes Meté phase <;h romosomc 1400 nm Condensed scaffold-associated form Emended scaffold-associated form 300 nm \\ 30-nm chromatin fiber of packed nucleosomes / / f i i "Beads-on-a-string" c form of chromatin I Shon region of DNA double-helix /V\AA/V 2 nm Chromatin condensation mmmmtö&t „naked" dsDNA "beads-on-a-string" form nucleosomes 30 nm solenoid relaxed form of chromosome condensed region of chromosome Si^SBNSSIB? mitotic chromosome Chromatin component 1) Histones > Centre is globular, ends are flexible and filamentous > High content of arginine and histidine > 5 species = H1, H2A, H2B, H3 and H4 2) Nonhistones > RNA polymerase and other enzymes usable in transcription > HMG1 and HMG2 - bind to unusual DNA structures > HMG3 a HMG4 - bind to histone core especially in transcriptionally active regions > The basic unit of chromatin > octamer of histones (H2A, H2B, H3, H4)2 > One molecule of histone H1 > DNA segment 200 bp long, which is wound about 2 times around the octamer of histones nucleosome histone core core DNA (146 bp) / linker DNA (20-60 bp) H2A- yellow, H2B red, H3 blue, H4 green Nature 389: 251-260 (1997) Nuclmsome fibn > 10nm chromatin fibre > its individual items form nucleosome cores connected by long linear dsDNA > visible by microscope -H1 + H1 H1 30nm chromatin fibre 30nm chromatin fibn > It is created by the condensation of nucleosome fibre caused by histone H1 > It binds to protein scaffold (nonhistones, e.g. topoisomerase II) Stacking for packing.Possible modes of how a chromatin fiber folds into helical or columnar forms are shown. Waaas Published by AAAS Chromatin domain > loops of 30nm chromatin fibre attached to protein scaffold > there is one molecule of topoisomerase II in base of each loop = change of topology during replication and transcription > each domain has one ori locus DNA protein scaffold connecting region Mitotic chromosomes > They originate by condensation of 30nm chromatin fibres > They are formed during mitosis or meiosis > Condensation of 30nm to 600-700nm chromatin fibres, which constitute the structure of chromosomes > In chromosomes, the chromatin is in the stage of the highest condensation and is transcriptionally inactive sister chromatids telomeres Chromosomal (nuclear) DMA > a single linear molecule of dsDNA > the number of bp of haploid cell is about 1.34 x107- 1.5 x1010 > only 10% of mammal genome bears the genetic information > most structural genes are from 1 x 104 to 2 x 106 bp long - considerable part is created by regularoty sequences Repeats in nuclear DNA > short tandem repeats - not in prokaryotes > dispersed repeats - not in prokaryotes > 25% - 50% structural genes as unique sequence > the rest as gene repeats = repetitions has function of a gene > Gene family = is a set of several similar genes, formed by duplication of a single original gene, and generally with similar biochemical functions. E.g. the genes for haemoglobin subunits. The term pseudogene. > Tandem gene repeats = directly adjacent, the term spacer (intergenic sequence, separates genes or a group of genes), genes transcribed to 5S-rRNA, genes for tRNA and genes for histones. The repeats provide sufficient amounts for the cell requirements. > Dispersed gene repeats = their copies are dispersed at different positions of haploid genome, genes transcribed to tRNA, snRNA and others. > is a dysfunctional relative of known genes that lost their protein-coding ability or are otherwise no longer expressed in the cell > usually has no introns Organisation of chromatin® in nucleu > Localisation of chromosomes is not accidental > Crowding of region with similar function or activity DOI: 10.1038/nsmb.2474 The local packing of chromatin h consistent with the behavior oi lobuh Chromosome scale Closed Megabase scale Fractal globule E. Lieberman-Aiden et al., Science 326, 289-293 (2009) https://unlockinglifescode.org/the-genome-ball DMA replication in eukaryotes plication of eukmryotic geno > replication of mitochondrial and chloroplat DNA > replication of nuclear chromosomes > semiconservative and semidiscontinuous > initiation, elongation, and termination > only in S phase of the cellular cycle transcription, translation, metabolism Replication of nuclear chromosom Origin Origin Origin —1_I_1_ I In contrast to prokaryotic cells the eukaryotic replication proceeds on several places in time Chromosome is a couple of replicons, it has more ori sequences (mammals 30.000-50.000) Euchromatin replicates earlier to heterochromatin 0 old nukleosomes •oV*o oo0o°- strand newly synthesises nucleosomes beginning of bidirectional replication Eukaryotic DNA-polymerase: a, p, 5 and £ • DNA polymerase a - in the complex of primase synthetises Okazaki fragments, it does not possess 3-5'exonuclease activity (proof reading) • DNA polymerase ß - synthesis of short fragments during DNA reparation • DNA polymerase v - synthesis of mitochondrial DNA • DNA polymerase 5 - synthesis of leading strand and completing of lagging strand • DNA polymerase £ - probably synthesis of leading strand DNA Ligase http ://yxsj .baiduyy.com/ http://www.ppu.mrc.ac.uk/researc h/?pid= 1012&sub 1 =research Historie Acceptor/Donor Parental histories Asf1 New histories ■Asf1 A. GrothetaL, Science 318,1928-1931 (2007) H3-H4 partitioning during nucteosome assembly D. Ray-Gallet et al., Science 328,56- 57 (2010) New nucleosomes result from de novo assembly using newly synthesized H3 and H4 histones in the form of two H3-H4 dimers; after association with two H2A-H2B dimers, the result is a nucleosome containing only new H3-H4 dimers. Mixed particles will form using a newly synthesized H3-H4 dimer and an H3-H4 dimer recycled from a disrupted parental nucleosome. Old nucleosomes will form either by self-reassociation of two H3-H4 dimers recycled from a transiently disrupted parental nucleosome, or according to the generally accepted view, by inheritance of a stable H3-H4 tetramer from a parental nucleosome. Nam Nucleosome assembling is associated with hydrolysis of E. Luk et al., Cell 143, 725-736, November 24, 2010 imipi B template DNA 3 5 RNA primer removing Okazaki fragments leading strand + 3 replication of spacers incompletely replicated DNA, missing nucleotides , the end replication problem Telomerase = ribonukleoprotein - RNA acts as a template, protein has catalytic function FSm ©í mmmmi B ^mág 5'■ 5* S'- Telomerase elongates the 3-end Formation of hairpin and RNA primer Replication of complementary strand and removing of hairpin ttgggg a- Telomere Template TTGGGGTTGGGG 1. Hairpin created; rna Primer added RNA Primer 5' 3> GGGG 2.Hairpin extended; RNA primer removed TTGGGGTTGGGG ■5' 31- AAGGGG y 3. Gaptilled TTGGGGTTGGGG AACCCCAAGGGG TTGGGG 4. Hairpin removed _ Replicated Telomere aacccc _ litt[^://\\ \\ \\ .ncKii.Oílii/[niln\'otV~inccloaii/[)hc431 /onkar ychrom/eukaryo3 .htm Missing DNA on ,. lagging strand c c -■- Telomerase with its own RNA template A A A T C C C 1. End is unreplicated. 2. Telomerase extends unreplicated end. 3. Again, telomerase extends unreplicated end. DNA polymerase RNA primer ( A A U C C C Sliding clamp 4. Lagging strand is completed. O 2011 Pwwon C auction. Inc. http://masteringyourwaytomedscho TABLE 11.5 Telomeric Repeat Sequences Within Selected Organisms Group Examples TGlomGric Repeat Sequence Mammals Humans TTAGGG Slime molds Physarum, Didymium TTAGGG Dtctyosteiium Filamentous fungi Neurospora TTAGGG Budding yeast Saccharomyces cerevisiae TG(1-3) Ciliates Tetrahymena TTGGGG Paramecium TTGGG(T/G) Euplotes IIIIGGGG Higher plants Arabtdopsis TTTAGGG http://reasonandscience.heavenforum.org/t2263-the-telomerase-enzyme ctlon problem How Telomeres Solve the End-Protection Problem "in .1 ■ L m - Tfit tfrii (rl t^ffOM (tVUnUfifMf hJfrt fie pMefibU to be flrifLlfctfi 1w i DH1 Mill *nu£l »*«tfirt be pMfecled tori MIMIC DMA dlmtqe IKpanfe pittUiys. CtflMV***, «ii Wright pwnwiHlyAwlIri »* Mil qrtft and ^tempts to 'repair ti« ends hduU hiw dHKUllfis Gramme -irfagri^ Tt* erd-prcfcttiari pnttan is sailed by praieto-DUA umpkief oiled letaffieres. studies -.. . showed 1hal 1 hesharlt3 rich repeat* iTamHieends ol'yeasl chromosomes um su Hicinri 1a slahi t«e a bnearplasrnid >;j, jJi Since"Oi01 t1 has hecame dar lhi1 {] sdi KpdiH cap -(he ends d' nutfl eulajy-:rb: thnrnutttinu.-l, in: K>J in ^ muTumbmn dnimnsames thai aidinTTAfXHl repeals. Second, the mechanism hy which lelameric DNA I; maintained was mlwd when l:l .:■ hum and (IreideT showed ihallelamerJG DMA b synthesized hy leVimrrase. TeVmLeHsei e a. re t,™ e transcTijrl as e lhi1 adds idomeric jepeata to the j" ends deadi chromosome {Tj. In doing so, tekimeHse malen up kir the sfr[ricominj= d' ÄendoniKerra-tive DNA jeplicajion, which cannol compkfc the synthesis ol" chromosome aids, CMier samtians la this aid leplicalian prahlem ekL-rt, ntibnlv in D.tj.yyVi Jj and ofha dipl-ann-i, "tuI il e now dou lhal idomerase e -(he main method hy which eTikaryates avoid sequencelass al the ends ol'lheücluorTiosaTnes. ti hs hsen suswelled thai earrv euiarvoles l-. I a n ii'. I ,-i 'I .j - il . ■ 1 1.1 ■ 1 .J ■ ■11 . ■. La ■ 11-. (he aid replication pre in km •.J'/ The tier acqui-i tern a I'le'tnneE.'k- ncil only salved the end .'-h. ] pra'ilem 'jul einuTed ihe jnesence d'lhe simr sequence Hal drramosame aid.■; Oiks alllaameies in the cell had the same sequence, tdamenc1 DNA hmdrnj lacvus cauld ewi he, thererw em'nlbi j ceus 1ci di-ün /insh nitunl dnamosame aids Iir-un sites d' DNA damage. Ibe [nd-PiottiLkjii Pi.jLltiii Research on tin ihhd majciT issue in leVnneie hi oVigy, how idomoes sah« ihe end unleclvm pra'ilenL, stagnated until ihe ]99Ci_ Iba end jiKiteclKin jiKi'nleni lirsl surläced early las1 coi fury, when MuDer and McCItitIckI unserved a Uircriiy, Nu\ Hf ■ ■ ". IE*. =-■■■ :-■ _-_ .. 1 1. I -.hi: inctKin nelween Ihe nehavkiiol len chrammoiTie en± ajid lelameirs. Muler kiund thai cmnnvisanies Liclcinu their natural ends ■were urcnhJe; MrClnnadt documenled the pro nensity ol'hiolOTeikl%hu1na1telanieies,Vi ruse. However, ihe KjTJ enemol" the end jDolertionpoh Jem. lerrnined ahscuie until ihe piincipjes al" the DNA dmugeiesparex- were revealed in the l'ffijs The IhTriinsHJhl camewhoi Aui.ftik, Roihsldn, and Orr Weaver lound 1ha1 bnear DNA brtai duced tttki eulajyalic cells is unstihle "because the DNA lecomirine th 1 he gertzme 0). t e now clear lhal ijitroduced linear DNA I alls victim la two irnjiarnrrl DNA repair palhways thai moid 'runken diramosames: 1 - i >l ■ n direcled lepair ^1 ID and nanhomaVisaus end 3 (HI IFJ). The observation thai DNA ends I - ■ known as l-il.--.il ■ .1 ■ ■ axe pni cessed hv these DNA lejnir leadions raised the qiM.Tl km 'Hi""whether 1he iuIuxlI ends d' diximci somea are also al-bcLed hy ILDR and Ml IT J, and il -■ ■ . ■ ■- 1 ■ A seonnd question arose i'nm the worl ol' llarlweTl and Wernerl, who lound thai huddmj ■. :■ L .:■ ii." 1. T'.-fj'JP miis ladled fci airesl the cell drvEion cycle in resporce tG I ■ 1 I. strand "meals ljna.« path Hg. 1 ibe eni-pnriettVn peUem. Wtwi j fnjfMmJlün thomtiscfne treibt top t tie exposed DMA (nils cm HtihdAt im signattig pitoviiyf *be AW jnd ATI LVjc» fMmttfi} toJAAnreAtoeMlihrEton Qfllt md *m todüH Hl dHrtlt it* broken ohwnasuie t t&iH^ icpirWilbyane tA Imh imtereMDHA Kpeli pjttiwJr/i tNHE] Jnd Wlvrihuring tefc to antiriiie tiev dnristons urito *n totott aernme. Be pHstt\#tA9ttstDta.iMuagt itspbtafiumiifi poses i pM> km 1w tw ends «1 hnaw rtnmuwnt! ttlhtt**^ bWinm) bethE« «6rMW *H DNA dwii^t fioriJiVit] « CeiA itfitit M (eJafi***? utfuildfc« disaslroiiL .vummjlün tobrneres sotre ttris end-pratoclian praUem tmau* tie use oi J tehnwe-sptcfficiiiäleto i*npMT eflei%i! it 4 it mered AakU* «* Hoop)» Mlooetw eraure ti M H1 out pa/ttviatfi Hsfniiri btodud. 11 NCA/EMBER20Ü? SCltlN« T. de Lange Science 326, 948-952 (2009) Fig. 1: The end-protection problem Double-strand break V i Telomere ft x J iL Activates DNA damage response pathways Cell cycle arrest ATM kinase ATR kinase DNA repair Homology-directed repair (HDR) Nonhomologous end joining (NHEJ) Protected from DNA damage response pathways T. de Lange Science 326, 948-952 (2009) Fig. 2 Mammalian telomeres T. de Lange Science 326, 948-952 (2009) Fig. 3 Different components of shelterin are dedicated to different aspects of the end-protection problem B KU70/80 k. » Loss of TRF2 ♦ t-loop opens Loss of P0T1 NHEJ i Exposed single-stranded DNA KU70/80 M (_) Cell cycle arrest (apoptosis/senescence) \ J s T. de Lange Science 326, 948-952 (2009) Waaa Fig. 4 Different solutions to the end protection problem Mammals POT1 Ku NHEJ OTHERWISE Dicentric chromosomes and genome instability OTHERWISE 1vsi 111— I i v v I O i— HDR Terminal deletions and telomere length changes OTHERWISE G/S or G2/M arrest and apoptosis/ senescence Budding yeast Rlf1 Rif2 Rap1 15' Cdc13 Stn1/Ten1 OTHERWISE NHEJ Dicentric chromosomes and genome instability HDR OTHERWISE Telomere length changes OTHERWISE G2/M arrest OTHERWISE de Lange Science 326, 948-952 (2009) TERMINATION ZONE {TERs) A Precatenane resolution B Fork fusion Fork pausing \ Asymmetric <2> fork Catenation Resolution karyoii ion co CO CD CM O O o C\J "05 _o o E CD CD C^> d O OPolu #Polt76 (^Pausing element o ■q >< u O a (A) Precatenane resolution: Top2 mediates fork progression at the TER zone by resolving precatenanes behind the forks. (B) Fork fusion: the right fork stalls at a pausing site (pausing element, red symbol) and emerges with an asymmetric conformation. The leading polymerase (black oval) and the lagging apparatus (yellow oval) are shown. (C) Catenation: Top2 then resolves the last catenation at TERs before DNA segregation, allowing chromosome resolution. Other factors involve in termination of replication were described in 2014 - topoisomerase II participates on it and the proces is regulated by ubiquitination. Disassembly required. Once two converging replisomes complete DNA replication, they are targeted sequentially by SCFDia2 and Cdc48, resulting in CMG disassembly. Science S P Bell Science 2014;346:418-419 Waaas