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Obravnavanje heterokromatina med replikacijo DNK

Obravnavanje heterokromatina med replikacijo DNK



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Heterokromatin je prisoten vzdolž kromosoma (nezvito stanje). Z visoko kondenzirano strukturo glede na evhromatin RNA polimeraza ne more priti v bazne pare DNK v heterokromatinu in začeti transkripcijo.

vprašanje: Med replikacijo DNK je potrebno replicirati tudi heterokromatin, saj je del kromosoma. Zato bi rad vprašal, ali lahko topoizomeraze same odvijejo to ekstremno super-zvijanje ali pa so drugi encimi/mehanizmi vključeni v odvijanje heterokromatina med replikacijo DNK.

(To vprašanje imam v mislih, ker nisem prepričan, ali je mogoče isti mehanizem odvijanja iz topoizomeraze uporabiti za super navitje, ki nastane v že kondenziranem heterokromatinu v primerjavi z evhromatinom) Hvala.


Meje v celiciin razvojna biologija

Pripadnost urednikov in recenzentov je zadnja, navedena v njihovih raziskovalnih profilih Loop in morda ne odraža njihovega položaja v času pregleda.


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    Replikacija DNK skozi okolje kromatina

    Zbijanje genoma v jedrski prostor se doseže z ovijanjem DNK okoli oktamernih sklopov histonskih proteinov, da se tvorijo nukleosomi, temeljna ponavljajoča se enota kromatina. Poleg zagotavljanja sredstva za namestitev večjih genomov v celico je kromatinizacija DNK ključno sredstvo, s katerim celica uravnava dostop do genoma. Čeprav je bila kompleksna vloga, ki jo ima kromatin pri transkripciji genov, cenjena že dolgo, je zdaj tudi očitno, da so ključni vidiki replikacije DNK povezani z biologijo kromatina. Ta pregled se bo osredotočil na nedavni napredek v našem razumevanju, kako okolje kromatina vpliva na ključne vidike replikacije DNK.

    Ta članek je del tematske izdaje „Modifikatorji in preoblikovalci kromatina pri popravljanju in signalizaciji DNK“.

    1. Uvod v izvor replikacije

    Visoko zvesto podvajanje genoma je osnovna zahteva za sposobnost preživetja organizma. Tako je razumevanje, kako genom kodira informacije za lastno podvajanje, glavno vprašanje na področju replikacije DNK. Prvotni cilj zgodnjih študij je bil ugotoviti, kako se replikacija začne. Izhodiščno delo v Escherichia coli je razkrilo, da se sinteza DNK začne z ene same točke na kromosomu (izvor) in poteka dvosmerno, dokler se dve replikacijski vilici ne združita in končata [1]. The E. coli kromosomski izvor replikacije (oriC) je določen z enim samim ohranjenim zaporednim elementom, ki ga nato vežejo komponente bakterijskega replisoma pred začetkom sinteze [2].

    V nasprotju s situacijo pri bakterijah evkarionti uporabljajo več izvorov, ki jih je treba sprožiti v rednih intervalih, da učinkovito replicirajo večje kromosome med vsako S-fazo [3]. Izvor brstečih kvasovk je omejen z ohranjenimi elementi zaporedja, imenovanimi avtonomno replicirano zaporedje (ARS) [4]. Na teh mestih se zgodita dva bistvena, ločena koraka aktivacije izvora: licenciranje izvora in sprožitev izvora. Licenciranje je posredovano s sestavljanjem kompleksa pred replikacijo (pre-RC), ki vključuje vezavo heksamernega kompleksa za prepoznavanje izvora (ORC) na izvor, čemur sledi nalaganje helikaznega kompleksa za vzdrževanje mini kromosomov (MCM). na DNK z ORC, skupaj s Cdc6 in Cdt1. Naložen, a neaktiven kompleks helikaze MCM je sestavljen iz dvojnega heksamera beljakovin MCM2-7, ki obkrožajo dvoverižno DNK [5,6]. V G1 ima vsak licencirani izvor možnost, da se sproži v naslednji S-fazi, vendar je sprožitev izvora odvisna od združevanja več dodatnih dejavnikov, vključno s CDC45 in GINS, ki ju uravnavata dve proteinski kinazi, regulirani v celičnem ciklu: Dbf4 -odvisna kinaza (DDK) in ciklin odvisna kinaza (CDK) [7]. Po aktivaciji lahko kompleks CDC45-MCM2-7-GINS (CMG) začne s translokacijo in odvijanjem starševskega genoma, kar omogoča sintezo DNK z replikativnimi DNK polimerazami.

    2. Kromatin in začetek replikacije DNK

    Časovna ločitev pred-RC sestavljanja in aktivacije predstavlja zaščito pred prekomernim podvajanjem, vendar specifične determinante, ki narekujejo, kateri izvor se sproži v kateri koli dani S-fazi, niso popolnoma razumljene. Pri kvasovkah število zaporedij, ki lahko določajo izvor, veliko presega dejansko število izvorov, uporabljenih v S-fazi [8], v celicah sesalcev pa je v dani celici dejansko uporabljenih manj kot 10 % licenciranih izvorov [9].

    Številni dokazi kažejo na pomembno vlogo kromatina pri uravnavanju replikacije DNK. V primeru specifikacije porekla, nedavno razvita in vitro sistemi replikacije iz brstečih kvasovk so poudarili, kako kromatin pomaga določiti mesta iniciacije [10,11]. Za razliko od situacije in vivo, pred-RC sestavljanje in iniciacija na goli predlogni DNK nista odvisna od specifičnih iniciatorskih sekvenc [12,13]. Vendar pa kromatinizacija predloge dramatično vpliva na sposobnost ORC, da se stabilno veže na DNK, tako da je učinkovita vezava in in vitro replikacija zahteva konsenzno zaporedje (ACS), ki je visoko afinitetno mesto za vezavo ORC [10,11]. Ta ugotovitev se dobro ujema s podatki iz brstečih kvasovk, kjer se zdi, da je struktura kromatina blizu izvora replikacije strogo nadzorovana: izvori replikacije imajo stereotipno razporeditev nukleosomov, osredotočeno na nukleosomsko osiromašeno regijo, ustanovljeno s proteini, ki vežejo zaporedje specifičnih DNK, in ORC [ 14] funkcija izvora je dejansko zavirana s posegom sosednjih nukleosomov v nukleosomsko osiromašeno regijo [15,16].

    Medtem ko se brsteči kvas opira na elemente zaporedja DNK za specifikacijo izvora, je situacija v metazoa veliko bolj zapletena in manj razumljena [17]. Prve zemljevide časovne replikacije za celoten genom Drosophila ugotovili, da je presenetljiva korelacija med časom replikacije in gensko aktivnostjo zgodnjih replicirajočih regij genoma sovpadala z večjo verjetnostjo genske aktivnosti na ravni celotnega genoma [18]. V skladu s tem novejše delo v Drosophila, Caenorhabditis elegans in celice sesalcev je pokazalo, da so mesta iniciacije replikacije označena z istimi histonskimi modifikacijami, ki jih običajno najdemo na mestih aktivne transkripcije genov [19–22]. Pri ljudeh Miotto et al. pregledali več kot 50 000 ORC-vezajočih mest v celotnem genomu, da bi razlikovali značilnosti kromatina, povezane s selektivno vezavo ORC, in ugotovili, da so največji napovedovalec vzorcev vezave ORC dostopne kromatinske regije, razvrščene kot preobčutljiva mesta za DNazo I (DHS) [19]. Na podlagi te povezave z DHS je bila vezava ORC povezana tudi s transkripcijsko aktivnostjo in je pokazala obogatitev histonskih modifikacij, ki so značilne za aktivni kromatin, in sicer acetilacijo histona H3 pri lizinu 27 (H3K27ac) in dimetilacijo histona H3 pri lizinu 4 (H3K4me2). ). Nobenih drugih dejavnikov ni bilo ugotovljeno, da bi tako napovedovali vezavo ORC, kar kaže na selektivnost lokalizacije ORC in vivo je večinoma posledica oportunistične vezave na dostopne regije DNK, ki jo vzpostavijo kromatinski faktorji, povezani z gensko ekspresijo, in ne prek neposredne interakcije s faktorjem, ki veže DNK, ali specifično modifikacijo histona [19].

    Kljub temu obstajajo številni dokazi, da ORC izvaja specifične interakcije s histonskimi modifikacijami in zlasti z metilacijskimi stanji histona H4 lizina 20 (H4K20me1/me2/me3) [23]. Pri sesalcih je metilacija H4K20 odvisna od treh znanih encimov: PR-Set7 (Set8) je odgovoren za H4K20me1, Suv4-20H1 katalizira H4K20me2 in Suv4-20H2 katalizira H4K20me3. Od teh je najbolj dinamično stanje metilacije H4K20me1, katerega ravni dosežejo vrhunec med fazo M in vztrajno upadajo, dokler ne dosežejo najnižje ravni v fazi S, te spremembe se odražajo z enakimi nihanji ravni Set8 [23,24]. Začetna poročila o celicah sesalcev so odkrila hude replikacijske napake, povezane s stabilizacijo ali izčrpanjem H4K20me1, kar kaže na vlogo Set8 ali H4K20me1 pri uravnavanju replikacije DNK [25]. Dinamične ravni Set8 (in s tem monometilacija na H4K20) se pripisujejo njegovi razgradnji z ligazo E3 Cul4-Ddb1 z interakcijo s PCNA, homotrimernim obročem, ki deluje kot faktor procesivnosti za številne replizomske proteine ​​[26]. Celice, ki izražajo mutantno obliko Set8, ki ne deluje v interakciji s PCNA in je Cul4-Ddb1 ne more razgraditi, kažejo nestabilnost genoma, povezano s ponovno replikacijo, kar kaže, da je dinamično stanje metilacije H4K20 pomembno za uravnavanje podvajanja genoma [26]. Učinek, ki ga ima motnja stanja metilacije H4K20 na replikacijo DNK, je pojasnil Reinbergov laboratorij, ki je pokazal, da Suv4-20H1, ki katalizira H4K20me2, olajša nalaganje ORC [27].

    V podporo vlogi metilacije H4K20 pri replikaciji DNK je ugotovitev, da ima podenota ORC1 bromo sosednjo homološko domeno (BAH), ki je evolucijsko ohranjen motiv za prepoznavanje kromatina, ki ga najdemo tudi na faktorju utišanja kromatina Sir3 [28,29]. Ta domena BAH omogoča interakcijo metazoan ORC1 z metiliranim H4K20, pri čemer ima pomembno prednost H4K20me2 pred H4K20me1 in H4K20me3 [29]. Na podlagi dokazov, da dodatne podenote ORC kažejo manjšo vezavo na kromatin v mutantnih celicah ORC1-BAH, interakcija s H4K20me2 verjetno pomaga stabilizirati povezavo kompleksa ORC s kromatinom [29]. Dejansko je replikacija DNK v mutantih ORC1-BAH okvarjena, pri čemer velik odstotek celic kaže zapoznel vstop v S fazo, fenotip, ki ga opazimo tudi v celicah brez H4K20me2 metiltransferaze, Suv4-20H1 [29,30].

    Kljub temu naše razumevanje zaposlovanja ORC z metilacijo H4K20 ostaja nepopolno. Prvič, profiliranje veznih mest ORC in izvora replikacije ne kaže močne obogatitve za metilacijo H4K20 [19,20], drugič, metilacija H4K20 je povezana z različnimi biološkimi rezultati, vključno s popravilom DNK, utišanjem transkripcije in vzpostavitvijo višjega reda. struktura kromatina [23]. Na primer, stanja metilacije H4K20 pomagajo ločiti repliciran kromatin od neponovljenega kromatina med fazo S [31], s čimer pripravijo stroje za popravilo HR, da vežejo na novo podvojeni kromatin za popravilo navzdol, ta pomožna funkcija metilacije H4K20 bo podrobno opisana v naslednjem razdelku. Glede na številne vloge pri ohranjanju stabilnosti genoma je lahko zapozneli vstop v fazo S v mutantnih celicah Set8 in Suv4-20 povezan z njihovo vlogo pri odzivu na poškodbe DNK ali popravljanju homologne rekombinacije (HR), ne pa s pomanjkanjem licenciranja izvora in iniciacije. Dejansko z uporabo kombinacije citoloških, genetskih in testov neposredne replikacije v Drosophila, je skupina MacAlpine ugotovila, da čeprav sta Set8 in H4K20me1 pomembna za napredovanje celičnega cikla, aktivacija izvora ni bila prizadeta brez Set8 ali H4K20me1 [32]. Pravzaprav več kot 50 %. Drosophila mutanti z alaninskimi substitucijami pri H4K20 so bili sposobni preživeti, v popolnem nasprotju s smrtonosnim fenotipom izpada Set8, kar kaže, da bi lahko bili replikacijski stres ali fenotipi poškodbe DNK, o katerih so poročali v mutantnih celicah Set8, povezani z drugimi tarčami metilacije Set8, vključno s p53 in PCNA [32]. –34].

    3. Replikacija v času in prostoru

    Medtem ko organizacija nukleosomov in histonske modifikacije, povezane s transkripcijo, jasno vplivajo na to, kje lahko nastane izvor, je kromosomski kontekst pomemben pri določanju, kdaj se izvor sproži med fazo S. Pri brstečih kvasovkah je preprosto s premikanjem danega izvora replikacije iz regije s pozno replikacijo v regijo zgodnje replikacije kromosoma mogoče podaljšati čas žganja [35]. Poleg tega bi lahko z usmerjanjem modifikatorjev kromatina, kot so histonacetilaze ali deacetilaze na določena mesta na kromosomu, povečali ali zmanjšali izvorno učinkovitost ali verjetnost iniciacije [36,37]. Tako določeni izvori niso vnaprej programirani, da se sprožijo zgodaj ali pozno med S-fazo, lokalno okolje kromatina pa bi lahko dovolilo ali omejilo sprožitev izvora.

    Okolje kromatina je neločljivo povezano z organizacijo kromosomov znotraj jedra [38]. Delo iz Gilbertovega laboratorija na celicah vretenčarjev je zaznamovala velike kromosomske domene z enotnim in ponovljivim časom replikacije. Te domene je mogoče razdeliti v dva razreda: konstantne časovne regije (CTR), za katere je značilno sorazmerno sinhrono in dosledno sprožitev tesno razporejenih izvornih grozdov iz celice v celico, in časovna prehodna območja (TTR), ki predstavljajo regije, kjer se čas replikacije premika od zgodnjega do poznega. [9,39,40]. Presenetljivo je, da se preslikava CTR-jev prekriva s predhodno preslikanimi topološko asociacijskimi domenami (TAD), ki so kromosomske regije, ki dovoljujejo specifične in pogoste interakcije znotraj definirane ločene regije kromosoma in preprečujejo interakcijo s sosednjimi domenami (slika 1) [38,39 ,41]. Tako se zdi, da je program, s katerim se bo genom repliciral, določen s strukturno organizacijo kromatina v jedru in znano je, da je ta struktura vzpostavljena veliko preden celice vstopijo v fazo S [42]. Prizadevanja za razumevanje, kako je določen program replikacije, bodo verjetno odvisna od zahtevne naloge razumevanja mehanizmov, s katerimi so kromosomi organizirani v jedru, na primer pri brstečih kvasovkah beljakovine vilic Fkh1/2 pomagajo pri zgodnjem sprožitvi izvora s spodbujanjem izvora. združevanje v tridimenzionalni prostor [43]. V metazoa protein Rif1 potencialno vpliva na čas replikacije z posredovanjem interakcij med pozno replicirajočimi regijami in jedrno lamino [44,45].

    Slika 1. Struktura topološko povezane domene (TAD) in odnos do izvora replikacije. Predstavitev regije kromosoma, ki vsebuje TAD. TAD vsebujejo elemente kromatina, kot so genski promotorji in ojačevalci, ki pogosto medsebojno delujejo (upodobljeni kot zelene črte). TAD so ločeni z mejami TAD. kromatin znotraj enega TAD redko sodeluje s kromatinom iz drugega. Izvori podvajanja so obogateni na mejah TAD (označenih z modrimi ovali), vendar se natančna mesta iniciacije razlikujejo od celice do celice znotraj populacije, kar povzroči območja iniciacije replikacije, ki so na dnu prikazana v sivi barvi.

    Ugotovitev, da jedrska organizacija razmejuje široka področja v programu za določanje časa replikacije, je pomemben razvoj, vendar pa dodatne informacije razkrijejo kartiranje izvorne lokacije visoke ločljivosti in učinkovitosti v človeških celicah. Zaporedje Okazakijevih fragmentov (OF) omogoča merjenje smeri replikacijskih vilic in predstavlja uporabno sredstvo za preslikavo dinamike replikacije pri visoki ločljivosti [20,46,47]. Skupina Hyrien je s sekvenciranjem OF razkrila, da je iniciacija v človeških celicah pogosto omejena na specifične regije kromosomov, ki obsegajo desetine kilobaz [20]. Natančna mesta iniciacije znotraj teh con se razlikujejo od celice do celice znotraj populacije. V skladu s predlagano vlogo organizacije kromatina višjega reda pri funkciji izvora replikacije [40] je preslikava OF pokazala, da izvori replikacije zgodnjega sprožitve običajno prekrivajo meje TAD [20]. Kot bi bilo pričakovati iz te korelacije, je biologija meja TAD in izvora replikacije podobna: oba sta na splošno povezana z aktivnimi geni, vendar genska aktivnost ni strogo zahtevana niti ni napovedna [38,40].

    Kako bi lahko določili izvor in meje TAD? Odgovor še zdaleč ni jasen, vendar potencialni namig izhaja iz ugotovitve, da so številni TAD dosledno razmejeni skozi različne vrste celic in razvojne stopnje. Ti "konstitutivni" TAD so pogosto omejeni s tako imenovanimi "gospodarskimi" geni [41], katerih dosledna ekspresija v cikličnih celicah jih loči od inducibilnih genov s spremenljivo ekspresijo. Takšna povezava je logična: celice morajo verjetno vedno izražati gospodinjske gene, ko se replicirajo, torej ko je izvor aktiven. Povezava med ekspresijo genov in replikacijo DNK je nadalje prikazana v nedavnem poročilu, ki raziskuje uporabo izvora replikacije pri razvoju C. elegans zarodki [21]. Tu, podobno kot pri človeških celicah, se replikacija začne s širokih območij [20], vendar je središče izvora omejeno s histonskimi modifikacijama H3K27ac in H3K4me2, ki jih najdemo na genskih promotorjih in ojačevalcih [48], pred kratkim pa so bili obogateni pri vezavi ORC. mesta v človeških celicah [19]. V bistvu imajo vsi izvori te spremembe in velika večina mest sprememb je izvor. Tako sta programa za prepisovanje in replikacijo verjetno medsebojno povezana. Kako se lahko s to ureditvijo izvede povezava programa za replikacijo in programa za transkripcijo? Odgovor se skriva v ugotovitvi, da so geni nenaključno porazdeljeni po genomu: tisti poleg izvora replikacije so močno pristranski glede genov, ki se izražajo med rastjo, kar nujno vključuje gospodinjske gene. S to ureditvijo C. elegans embrionalne celice se lahko razmnožujejo v 20 minutah in hkrati izražajo gene, potrebne za rast [21].

    V somatskih celicah se v bližini aktivno prepisanih genov zgodi le podskupina dogodkov iniciacije replikacije. Dejansko je morda najbolj zanimiv vidik visokoločljivosti preslikavanja izvora človeške replikacije ta, da obsežne regije genoma, ki se replicirajo pozno v fazi S, niso odvisne od iniciacije iz specifičnih con [20]. Takšne pozne regije so na splošno transkripcijsko neaktivne in so obogatene s heterokromatinom. Zdi se, da začetek pozne replikacijskih domen narekuje kaskada sprožitvenih dogodkov izvora replikacije, ki se začnejo iz zgodnje sprožene regije [20]. Licenčni izvori, ki so razpršeni po regiji pozne replikacije, se verjetno sprožijo zaradi replikacijskih vilic, ki posegajo iz zgodnje sproženih regij – morda s ponovno uporabo omejujočih faktorjev replikacije. Dejansko je znano, da so dejavniki, potrebni za začetek podvajanja, omejujoči in bi omogočili, da se podmnožica izvorov sproži istočasno [49,50]. Tako je lokalni nadzor replikacije v poznih regijah deloma odvisen od pravočasne replikacije zgodnjih regij. Ti podatki skupaj podpirajo model, v katerem imajo izvori replikacije, ki so razdeljeni znotraj svojih kromosomskih domen, različne verjetnosti sprožitve na začetku faze S in da se verjetnost sprožitve izvora povečuje z napredovanjem faze S [51]. Izvori, ki se sprožijo zgodaj in bolj enotno, imajo verjetno večjo verjetnost vžiga na podlagi permisivnih kromosomskih značilnosti: bližina gospodinjskih genov in meje TAD, DHS in dostopni kromatin.

    Zakaj so genomi ločeni v zgodnje in pozno replicirane regije? Ena od razlag bi bila, da bi takšna časovna segregacija pri izvornem sprožitvi omogočila celični presnovi, da zagotovi dosledne količine metabolitov za učinkovito replikacijo. Dejansko je rast brstečih sevov kvasovk, ki hkrati sprožijo zgodnje in pozne izvore, delno omejena s ravnmi dNTP [50]. Drugič, ločitev replikacije na domene omogoča celicam, da se učinkoviteje spopadajo s stresom podvajanja. Težave, ki se pojavijo v zgodnji fazi S, lahko sprožijo kontrolno točko faze S in zavirajo začetek novih izvorov replikacije na oddaljenih mestih [52]. Tako lahko celice, ko se pojavijo težave v eni regiji kromosoma, zagotovijo, da so težave rešene pred dokončanjem replikacije preostalega genoma [9]. Končno lahko široka delitev genoma na regije z zgodnjo in pozno replikacijo zagotovi preprosto sredstvo za povečanje robustnosti, s katero se domene histonskih modifikacij in kromatinskih stanj ponovno vzpostavijo po fazi S. Na primer, pri brstečih kvasovkah je acetil-CoA intrinzično povezan z rastjo in ravnmi acetil-CoA in vrhom acetilacije histona na začetku faze S in nato pade skozi S fazo [53,54]. Tako lahko zgodnje replikacijske regije, ki so tipično transkripcijsko aktivne, spodbujajo povečano acetilacijo na novo sestavljenega kromatina in s tem označujejo transkripcijsko aktivne regije za naslednjo generacijo. V skladu s to hipotezo so eksperimenti z mikroinjekcijami v človeških celicah pokazali, da je sestavljanje transkripcijsko kompetentnega kromatina odvisno od časa injiciranja, pri čemer se DNK, injiciran zgodaj v S-fazi, sestavi v acetilirani kromatin in se izrazi na višjih ravneh [55,56 ]. Časovna ločitev replikacije aktivnega in potlačenega kromatina lahko zato okrepi različne vrste kromatina.

    4. Replikacija s kromatinom: novi pogledi na helikazo

    Ko se izvor sproži, je osrednje vprašanje pri razumevanju, kako replikacijska vilica poteka skozi genom, razkriti, kako kompleks helikaze CMG odvija kromatinizirano DNK. Nova spoznanja na tem področju so prišla iz strukturnih podatkov, ki kažejo, da helikaza CMG napreduje skozi kromatin v nasprotni smeri od tistega, kar se je prej mislilo [57]. Replikativna helikaza MCM2-7 je sestavljena iz heksamernega obroča, ki v kombinaciji s petimi dodatnimi faktorji sestavlja 11-podenotni kompleks CMG [58]. Vsak heksamerni obroč je sestavljen iz dveh nivojev, ki obsega C-terminalno domeno (CTD), ki vsebuje ATP-vezovna mesta in motor, ki poganja translokacijo in odvijanje DNK, ter N-terminalno domeno (NTD). Ko se naložijo na DNK v G1, so dvojni heksameri MCM2-7 orientirani na način NTD proti NTD, tako da so motorične CTD domene obrnjene druga od druge [5,6]. Na podlagi orientacije dvojnih heksamerov v G1 je veljalo, da so po aktivaciji kompleksi CMG preprosto migrirali drug od drugega s CTD na vodilnem koncu 3′–5′ translokacije [59]. By using cryo-EM to visualize CMG on forked DNA substrates, Georgescu et al. were able to capture the helicase in ‘translocation mode' surprisingly, their structures revealed that the NTD is proximal to the fork and the CTD motor trails behind (figure 2). This finding is important for a number of reasons: first, because the leading strand polymerase (ɛ) associates with the CTD and polymerase α/primase, associates with the NTD [60], this orientation of CMG logically positions each polymerase for synthesis on their associated strands: polymerase ɛ can synthesize the leading strand as its template emerges from the CMG. Second, this orientation of CMG minimizes the amount of exposed single-stranded DNA on the lagging strand as the lagging-strand template, unwound at the front of the replication fork, can be primed by polymerase α/primase. Third, the model reveals an elegant quality control mechanism ensuring that each hexamer associates with the opposite strand of DNA before separating [57]. As CMGs are loaded onto double-stranded DNA prior to initiation and translocate on single-stranded DNA, the ‘NTD first' orientation implies that the hexameric rings must pass one another during initiation, which is only possible once both hexamers encircle single-stranded DNA. Finally, the new translocation orientation of CMG potentially reveals new modes for chromatin disassembly and parental histone recycling. The threading of the 3′ end of the DNA through the leading NTD positions the recently characterized MCM2 histone-binding domain at the very front of the CMG [61,62]. MCM2 chaperones H3:H4 tetramers both in vitro in in vivo by wrapping around the tetramer, much like nucleosomal DNA [61,62]. Therefore, the new model indicates that MCM2 could play a major role in disassembling parental nucleosomes in front of the replication fork (figure 3). The authors also speculate that the new position of the lagging-strand machinery at the leading edge of the CMG increases the likelihood that parental nucleosome deposition would preferentially occur on the lagging strand, opening up intriguing mechanisms for chromatin state inheritance and nucleosome assembly. However, with the exception of the remarkable, yet poorly understood inheritance of histone proteins in Drosophila male germline stem cells [63], there is little evidence of biased segregation of parental histones to the daughter genomes [64,65]. This may reflect the inherent asymmetry of the replication fork imparts little bias on histone segregation or, that mechanisms have evolved—perhaps employing specific histone chaperones—to ensure the equal passage of parental histone to the daughter genomes.

    Figure 2. A model for helicase activation and separation. See main text for details.

    Figure 3. Dynamics at the replication fork. Representation of the replication fork progressing through chromatin. For simplicity, several proteins are omitted and only proteins discussed in the text are included. Za podrobnosti si oglejte besedilo.

    5. In vitro replication of chromatin template

    Progression of the replication fork through chromatin is also stimulated by an array of other factors that probably alter the structure of chromatin. Here newly developed in vitro systems are beginning to reveal important clues. Uporaba an in vitro replication system with purified components Kurat et al., were able to identify several proteins specifically associated with replicating chromatin [11]. Most prominent were histone chaperones (FACT, Nhp6, Asf1), chromatin remodelling complexes (INO80, Isw1), and histone acetyltransferase complexes (NuA4, SAGA). Importantly, efficient replication of chromatinized DNA required FACT, which is consistent with the noted role for FACT in promoting transcription from chromatinized templates and the general understanding of how FACT can disrupt the structural integrity of the nucleosome [66–68]. FACT associates with the replisome progression complex, [69] and interacts with multiple components at the replication fork, including DNA polymerase α [70] and the MCM2 N-terminal tail, where it forms a salt resistant complex with histones [71]. In the light of the recent findings regarding the orientation of translocating CMG [57] these results would place FACT at the leading edge of the helicase where it would presumably collaborate with the MCM2 tail to mediate the unwinding of parental histones (figure 3). Such a scenario is supported by the discovery that histones captured from FACT-MCM2 complexes lacked acetylation of histone H3 at lysine 56 [71] which is a modification found on newly synthesized histones [72].

    Kurat et al. also showed that the ATP-dependent chromatin remodelling complexes INO80 and ISW1a, and histone acetyltransferases, SAGA and NuA4, were all required in order to achieve rates of replication comparable with those measured in vivo [11]. Of these factors, Isw1 and INO80 have been implicated in various aspects of DNA replication: in budding yeast, Isw1 repositions nucleosomes on nascent DNA [73] and in human cells an ISW1-related complex (ACF1-ISWI) promotes efficient replication of heterochromatin [74]. INO80 has also been shown to interact with replication origins and the replication fork [75] and depletion of INO80 results in slowed replication fork progression [76]. Moreover, there is increasing evidence that INO80 is required for replication restart after fork stalling and can function in a pathway to evict RNA polymerase II from chromatin [77] upon replication stress [78]. However, with the exception of FACT, it remains to be determined whether the factors examined by Kurat et al. are specifically targeted to replication forks and, if so, how they function in replication fork progression. Indeed, Devbhandari et al., who established a similar in vitro system achieved replication on chromatinized templates in the absence of FACT and many of the stimulatory factors described by Kurat et al. their reactions contained the histone chaperone NAP1 and Isw1, which were included to assemble nucleosomes on the template DNA. These factors presumably also stimulated replication through chromatin and nucleosome assembly on the nascent DNA. Thus, while many proteins appear capable of stimulating replication, it appears that no single factor is specifically required for replication through templates.

    6. Chromatin regulates lagging-strand synthesis

    The in vitro systems described by Devbhandari et al., and Kurat et al. also noted a profound alteration in lagging-strand synthesis when replication occurred on chromatin templates. The frequency at which the polymerase α/primase complex initiates each OF will influence the ultimate length of OFs produced by the replisome. On naked DNA templates, polymerase α/primase acts distributively: OFs become shorter—hence were more frequently initiated—with increasing amounts of polymerase α/primase [79]. When replication occurred through chromatin, Kurat et al., noted that the initiation frequency became much less sensitive to the concentration of polymerase α/primase—indicating that polymerase α/primase may act processively in the context of chromatin [11]. It is unclear how chromatin should influence the frequency of OF initiation: one mechanism would be that chromatin somehow helps sequester polymerase α/primase to the replisome but another interesting possibility is that MCM helicase progression may slow each time a nucleosome is encountered. If the rate-limiting step in replication fork progression is assumed to be the unwinding of nucleosomal DNA ahead of the replication fork, then initiation of OFs by polymerase α/primase may occur as the fork slows from one nucleosome to the next. Thus, the initiation site and the frequency of initiation events (hence the ultimate length of the OF) could, at least in part, be dictated by nucleosome structure and how many nucleosomes the fork moves through each cycle.

    Aside from the frequency of initiation, the lengths of OFs are also dictated by a processing reaction in which polymerase δ simultaneously extends the 3′ end of a nascent OF and triggers the degradation of the 5′ end of the preceding OF [80]. Repeated cycles of extension and DNA cleavage produce a nick that migrates away from the replication fork and can be sealed by DNA ligase I [80]. This reaction—known as strand displacement synthesis—relies upon structure specific nucleases such as Fen1 to degrade the RNA or DNA displaced by polymerase δ (figure 3). Devbhandari et al. incorporated the basic components for OF processing, including the flap endonuclease Fen1 and DNA ligase I in their in vitro system [10]. Interestingly, they noted that while the replication of naked plasmid DNA occurred robustly, much of the synthesized DNA was greater than unit length—meaning that unconstrained synthesis was occurring (most probably by polymerase δ) and only a fraction of the lagging-strand products were small and competent for ligation [10]. Replication of chromatinized templates dramatically suppressed the extent of DNA polymerization, resulting in short lagging-strand products that were readily ligated. Since the DNA replication reactions were conducted with an excess of core histones, Nap1 and Isw1 (which are potent nucleosome assembly factors [81]) the suppression of polymerase δ is most readily explained by the reassembly of nucleosomes on the lagging strand, which presumably prevent extensive strand displacement synthesis by polymerase δ [10]. These data support earlier results from budding yeast which showed that nucleosome assembly on daughter strands is required for optimal processing of OFs in vivo [47].

    The requirement for nucleosome assembly to constrain DNA synthesis on the lagging strand potentially provides a means to ensure the removal of error prone DNA synthesized by DNA polymerase α while preventing excessive strand displacement synthesis by polymerase δ [47]. In addition, inhibition of polymerase δ by newly assembled nucleosomes may allow components of the lagging-strand machinery to be recycled from one OF to the next and, in doing so, allow the fidelity of nucleosome assembly to be communicated to the replication fork. In this scenario, defects in nucleosome assembly on the lagging strand result in extensive strand displacement synthesis by polymerase δ. Thus, in the absence of timely nucleosome assembly, the synthesis of each OF would be slowed, which may ultimately slow the replication fork—allowing the rate of nucleosome assembly to be coupled with the rate of DNA synthesis. Studies in mammalian cells indicate that replication fork progression through chromatin requires efficient delivery of newly synthesized histones [82] and replication forks are slowed when the nucleosome assembly is impaired [83]. With this reasoning, it is worth considering whether some of the stimulatory effects on DNA synthesis of histone chaperones and chromatin remodelling enzymes seen in vitro replication systems may be attributed to the promotion of nucleosome assembly and efficient lagging-strand synthesis.

    7. Priming for recombination during DNA replication

    Even in the absence of exogenous DNA damage, faithful completion of DNA replication relies upon the HR pathway to protect stalled replication forks or to restart collapsed forks [84]. DNA replication produces sister chromosomes, but because chromosomes are replicated at different times during S phase, an interesting question is how do cells know when they have a sister with which to repair? Recent data suggests that events occurring at the replication fork help the HR machinery discriminate between replicating and non-replicating chromatin [31]. TONSL-MMS22 L is a heterodimeric complex capable of interacting with histones as well as the histone chaperones Asf1 and MCM2 via the TONSL ankyrin repeat domain (ARD) [85,86]. Structural characterization shows that the binding of soluble histones H3-H4 by TONSL bridges the connection to ASF1 and MCM2, creating a co-chaperone complex prior to deposition of histones during replication coupled chromatin assembly [31]. The co-chaperone complex is dependent upon a number of interactions on the histone H4 tail, including lysine 20 (H4K20), the methylation of which is associated with replication and repair processes that maintain genome integrity [23]. Importantly, H4K20 methylation (H4K20me) abolishes TONSL binding to nucleosomes, suggesting that TONSL-MMS22 L specifically recognizes unmodified histones at H4K20. Since newly synthesized histones deposited in S phase are unmethylated at K20 [72], TONSL-MMS22 L is thus likely to bind to replication forks and nascent chromatin. However, TONSL-MMS22 L recruitment to chromatin occurs within a narrow temporal window as H4K20 is methylated by the histone methyltransferase SET8 [23] in late S phase. Based on this timely recruitment of TONSL-MMS22L and its known role as a mediator of HR, the recognition of nascent chromatin by the complex probably promotes expedient HR repair at compromised replication forks [31,85,86]. Indeed, a subsequent report provided direct evidence for the recruitment of TONSL-MMS22 L to collapsed replication forks and its promotion of Rad51-dependent recombination [87]. Presumably, recruitment of TONSL-MMS22 L to replication forks, allows efficient interaction with RPA, which coats stretches of single-stranded DNA formed during collapse of replication forks [87]. While it is known that the TONSL-MMS22 L heterodimer is required for RAD51 foci formation upon DNA damage [85,86], it is also required to recruit RAD51 to stalled replication forks [87]. An intact TONSL-MMS22 L heterodimer forms a tight interaction with two molecules of RAD51 and was shown to facilitate strand exchange by reducing the affinity of RAD51 for double-stranded DNA, a function similar to the tumour suppressor BRCA2 [87–89]. These findings illustrate an interesting new paradigm, in which replication coupled chromatin assembly provides an opportunity to prime newly replicated daughter genomes with repair complexes in the event of replication stress or DNA damage. In the light of these findings, it would be of interest to identify whether complexes similar to TONSL-MMS22 L are able to recognize chromatin specific transitions as a signal to load the DNA repair components best suited to fix the damage within the specific cell-cycle phase.

    8. Conclusion

    The advances in biochemical characterization of the replisome and its components have reinforced our understanding of how integrated the passage of the replication fork is with chromatin dynamics. Indeed, while in vitro systems have shown that DNA can be efficiently replicated in the absence of chromatin, it is clear that the presence of nucleosomes on the template DNA can constrain sites of initiation and the processivity of the DNA polymerases. But rather than a simple impediment, chromatin should more realistically be viewed as a modulator that can fine tune many aspects of DNA replication. Novi in vitro systems offer tantalizing insights into how replication occurs on chromatin, yet they remain incomplete. Most notably, neither report examined components of the replication-associated chromatin assembly system that couples nucleosome assembly, mediated in part through the histone chaperone CAF-1, to the replication fork through PCNA. Once this pathway is included, it may be possible to faithfully recapitulate nucleosome assembly on nascent DNA in vitro. But a considerable challenge with the biochemical systems will be to achieve a stoichiometry of the components, which faithfully recapitulates the situation in vivo. Thus, more quantitative assays that interrogate in vivo replication will be needed to supplement the in vitro sistemov.

    From the demonstration that ORC binds to accessible DNaseI hypersensitive sites, to the association of replication timing with higher-order chromatin folding, it is apparent that DNA replication is profoundly influenced by chromatin organization. Given that the same chromatin features are implicated in gene transcription and DNA replication, some immediate challenges will be to disentangle causal relationships between the processes at play. This may prove challenging as perturbation of one process will probably affect the other nevertheless the realization that chromatin structures typically associated with gene transcription are also used in DNA replication may provide a new perspective from which we may better understand how and why such chromatin structures are established and maintained. While ORC-binding represents the critical first step in origin licensing in G1, it is yet unknown how ORC is targeted to chromatin or whether chromatin structural changes precede ORC-binding and MCM loading. Most certainly, a deeper understanding of how chromatin is organized within the nucleus and the factors responsible for such organization will prove valuable to many aspects of genome research.


    Vsebina

    Constitutive heterochromatin is found more commonly in the periphery of the nucleus attached to the nuclear membrane. This concentrates the euchromatic DNA in the center of the nucleus where it can be actively transcribed. During mitosis it is believed that constitutive heterochromatin is necessary for proper segregation of sister chromatids and centromere function. [6] The repeat sequences found at the pericentromeres are not conserved throughout many species and depend more on epigenetic modifications for regulation, while telomeres show more conserved sequences. [2]

    Constitutive heterochromatin was thought to be relatively devoid of genes, but researchers have found more than 450 genes in the heterochromatic DNA of Drosophila melanogaster. [5] These regions are highly condensed and epigenetically modified to prevent transcription. For the genes to be transcribed, they must have a mechanism to overcome the silencing that occurs in the rest of the heterochromatin. There are many proposed models for how the genes in these regions are expressed, including the insulation, denial, integration, exploitation, and TE restraining models. [ potrebno pojasnilo ]

    When genes are placed near a region of constitutive heterochromatin, their transcription is usually silenced. This is known as position-effect variegation and can lead to a mosaic phenotype.

    Constitutive heterochromatin is replicated late in S phase of the cell cycle and does not participate in meiotic recombination.

    Histone modifications are one of the main ways that the cell condenses constitutive heterochromatin. [7] The three most common modifications in constitutive heterochromatin are histone hypoacetylation, histone H3-Lys9 methylation (H3K9), and cytosine methylation. These modifications are also found in other types of DNA, but much less frequently. Cytosine methylation is the most common type, although it is not found in all eukaryotes. In humans there is increased methylation at the centromeres and telomeres, which are composed of constitutive heterochromatin. These modifications can persist through both mitosis and meiosis and are heritable.

    SUV39H1 is a histone methyltransferase that methylates H3K9, providing a binding site for heterochromatin protein 1 (HP1). HP1 is involved in the chromatin condensing process that makes DNA inaccessible for transcription. [8] [9]

    Genetic disorders that result from mutations involving the constitutive heterochromatin tend to affect cell differentiation and are inherited in an autosomal recessive pattern. [6] Disorders include Roberts syndrome and ICF syndrome.

    Some cancers are associated with anomalies in constitutive heterochromatin and the proteins involved in its formation and maintenance. Breast cancer is linked to a decrease in the HP1 alpha protein, while non-Hodgkin's lymphoma is linked to hypomethylation of the genome and especially of satellite regions. [ potreben navedba ]


    Vsebina

    Chromatin is found in two varieties: euchromatin and heterochromatin. [7] Originally, the two forms were distinguished cytologically by how intensely they get stained – the euchromatin is less intense, while heterochromatin stains intensely, indicating tighter packing. Heterochromatin is usually localized to the periphery of the nucleus. Despite this early dichotomy, recent evidence in both animals [8] and plants [9] has suggested that there are more than two distinct heterochromatin states, and it may in fact exist in four or five 'states', each marked by different combinations of epigenetic marks.

    Heterochromatin mainly consists of genetically inactive satellite sequences, [10] and many genes are repressed to various extents, although some cannot be expressed in euchromatin at all. [11] Both centromeres and telomeres are heterochromatic, as is the Barr body of the second, inactivated X-chromosome in a female.

    Heterochromatin has been associated with several functions, from gene regulation to the protection of chromosome integrity [12] some of these roles can be attributed to the dense packing of DNA, which makes it less accessible to protein factors that usually bind DNA or its associated factors. For example, naked double-stranded DNA ends would usually be interpreted by the cell as damaged or viral DNA, triggering cell cycle arrest, DNA repair or destruction of the fragment, such as by endonucleases in bacteria.

    Some regions of chromatin are very densely packed with fibers that display a condition comparable to that of the chromosome at mitosis. Heterochromatin is generally clonally inherited when a cell divides, the two daughter cells typically contain heterochromatin within the same regions of DNA, resulting in epigenetic inheritance. Variations cause heterochromatin to encroach on adjacent genes or recede from genes at the extremes of domains. Transcribable material may be repressed by being positioned (in cis) at these boundary domains. This gives rise to expression levels that vary from cell to cell, [13] which may be demonstrated by position-effect variegation. [14] Insulator sequences may act as a barrier in rare cases where constitutive heterochromatin and highly active genes are juxtaposed (e.g. the 5'HS4 insulator upstream of the chicken β-globin locus, [15] and loci in two Saccharomyces spp. [16] [17] ).

    All cells of a given species package the same regions of DNA in constitutive heterochromatin, and thus in all cells, any genes contained within the constitutive heterochromatin will be poorly expressed. For example, all human chromosomes 1, 9, 16, and the Y-chromosome contain large regions of constitutive heterochromatin. In most organisms, constitutive heterochromatin occurs around the chromosome centromere and near telomeres.

    The regions of DNA packaged in facultative heterochromatin will not be consistent between the cell types within a species, and thus a sequence in one cell that is packaged in facultative heterochromatin (and the genes within are poorly expressed) may be packaged in euchromatin in another cell (and the genes within are no longer silenced). However, the formation of facultative heterochromatin is regulated, and is often associated with morphogenesis or differentiation. An example of facultative heterochromatin is X chromosome inactivation in female mammals: one X chromosome is packaged as facultative heterochromatin and silenced, while the other X chromosome is packaged as euchromatin and expressed.

    Among the molecular components that appear to regulate the spreading of heterochromatin are the Polycomb-group proteins and non-coding genes such as Xist. The mechanism for such spreading is still a matter of controversy. [18] The polycomb repressive complexes PRC1 and PRC2 regulate chromatin compaction and gene expression and have a fundamental role in developmental processes. PRC-mediated epigenetic aberrations are linked to genome instability and malignancy and play a role in the DNA damage response, DNA repair and in the fidelity of replication. [19]

    Saccharomyces cerevisiae, or budding yeast, is a model eukaryote and its heterochromatin has been defined thoroughly. Although most of its genome can be characterized as euchromatin, S. cerevisiae has regions of DNA that are transcribed very poorly. These loci are the so-called silent mating type loci (HML and HMR), the rDNA (encoding ribosomal RNA), and the sub-telomeric regions. Fission yeast (Schizosaccharomyces pombe) uses another mechanism for heterochromatin formation at its centromeres. Gene silencing at this location depends on components of the RNAi pathway. Double-stranded RNA is believed to result in silencing of the region through a series of steps.

    In the fission yeast Schizosaccharomyces pombe, two RNAi complexes, the RITS complex and the RNA-directed RNA polymerase complex (RDRC), are part of an RNAi machinery involved in the initiation, propagation and maintenance of heterochromatin assembly. These two complexes localize in a siRNA-dependent manner on chromosomes, at the site of heterochromatin assembly. RNA polymerase II synthesizes a transcript that serves as a platform to recruit RITS, RDRC and possibly other complexes required for heterochromatin assembly. [20] [21] Both RNAi and an exosome-dependent RNA degradation process contribute to heterochromatic gene silencing. These mechanisms of Schizosaccharomyces pombe may occur in other eukaryotes. [22] A large RNA structure called RevCen has also been implicated in the production of siRNAs to mediate heterochromatin formation in some fission yeast. [23]


    Heterochromatin loss as a determinant of progerin-induced DNA damage in Hutchinson–Gilford Progeria

    Oliver Dreesen, PhD, Cell Ageing, Skin Research Institute Singapore, 8A Biomedical Grove, #06-06 Immunos, 138648 Singapore, Singapore.

    Developmental and Regenerative Biology, Institute of Medical Biology, Singapore, Singapore

    Cell Ageing, Skin Research Institute Singapore, Singapore, Singapore

    Cell Ageing, Skin Research Institute Singapore, Singapore, Singapore

    A*STAR Microscopy Platform, Singapore, Singapore

    Cell Ageing, Skin Research Institute Singapore, Singapore, Singapore

    Developmental and Regenerative Biology, Institute of Medical Biology, Singapore, Singapore

    Cell Ageing, Skin Research Institute Singapore, Singapore, Singapore

    Oliver Dreesen, PhD, Cell Ageing, Skin Research Institute Singapore, 8A Biomedical Grove, #06-06 Immunos, 138648 Singapore, Singapore.

    Povzetek

    Hutchinson–Gilford progeria is a premature aging syndrome caused by a truncated form of lamin A called progerin. Progerin expression results in a variety of cellular defects including heterochromatin loss, DNA damage, impaired proliferation and premature senescence. It remains unclear how these different progerin-induced phenotypes are temporally and mechanistically linked. To address these questions, we use a doxycycline-inducible system to restrict progerin expression to different stages of the cell cycle. We find that progerin expression leads to rapid and widespread loss of heterochromatin in G1-arrested cells, without causing DNA damage. In contrast, progerin triggers DNA damage exclusively during late stages of DNA replication, when heterochromatin is normally replicated, and preferentially in cells that have lost heterochromatin. Importantly, removal of progerin from G1-arrested cells restores heterochromatin levels and results in no permanent proliferative impediment. Taken together, these results delineate the chain of events that starts with progerin expression and ultimately results in premature senescence. Moreover, they provide a proof of principle that removal of progerin from quiescent cells restores heterochromatin levels and their proliferative capacity to normal levels.


    WHAT NEEDS TO BE DONE?

    Although the broad outline and many important details of DNA replication have been identified, many important aspects of this central process remain to be discovered. In large part, we still do not know how origins function. How do origin-binding proteins organize the DNA? Exactly how do helicase loaders function? How and when does the MCM helicase transition from encircling dsDNA to encircling ssDNA? The functions of several proteins required for origin activation and priming in eukaryotes are still shrouded in mystery. Priming and replisome assembly require numerous proteins that lack homologs in bacteria. What are the functions of Sld2, Sld3, Dbp11, Mcm10, GINS, and Cdc45 and how is their function influenced by phosphorylation? We lack an understanding of how the multiple origins in eukaryotes are coordinated and how the domain structure is established and maintained through multiple cell divisions. For example, just what are nuclear foci and how are replication foci organized within them? Are origins within one replicon clustered into one focus? Once replication forks are established, we know little about how they are regulated. If one replication fork in a focus were to stop, would it halt the other forks within that focus? How do replisomes move through nucleosomes, especially in highly condensed DNA and how are the parental nucleosomes inherited to the sister chromatids? How do replisomes deal with cohesin rings and how are these loaded? We have barely scratched the surface on questions surrounding the interface of replication with repair and recombination. For example, how can replication forks form during break-induced replication in S and G2 phase when the MCMs are thought to be loaded only in G1? How do checkpoint mechanisms act on moving replication forks? The newly revealed coordination of DNA metabolism with chromatin establishment, gene silencing, and epigenetic control is only beginning to be explored. Most of what we know about DNA replication has been learned in organisms with stable karyotypes and ploidy. However some organisms, particularly microbial eukaryotes, have extreme variations in ploidy and variable numbers of chromosomes. What mechanisms exist to facilitate this yet maintain order in this apparent chaos? Finally, and perhaps most important, some types of human disease, including certain cancers, have their basis in replication. Clearly many important questions remain, despite the enormous progress of recent years. We hold hope that understanding the mechanistic details of these processes may lead to cures, or at least treatment of human disease in the future.


    Leemor Joshua-Tor

    Ph.D., The Weizmann Institute of Science, 1991

    [email protected] | (516) 367-8821

    Our cells depend on thousands of proteins and nucleic acids that function as tiny machines: molecules that build, fold, cut, destroy, and transport all of the molecules essential for life. My group is discovering how these molecular machines work, looking at interactions between individual atoms to understand how they activate gene expression, DNA replication, and small RNA biology.

    V Leemor Joshua-Tor’s lab, researchers study the molecular basis of nucleic acid regulatory processes using the tools of structural biology and biochemistry. One such regulatory process is RNA interference (RNAi), in which a small double-stranded RNA triggers gene silencing. Joshua-Tor and her team offered critical insight when they solved the crystal structure of the Argonaute protein and identified it as the long-sought Slicer. They then went on to explore the mechanism of the slicing event. The structure of human Argonaute 2 (hAgo2) bound to a microRNA (miRNA) guide allowed Joshua-Tor and her colleagues to understand how mRNA is cleaved during RNAi. This year, members of the Joshua-Tor lab explored the function of a very similar protein, called Argonaute 1, that has no slicing ability, even though it is almost identical in structure to the slicing hAgo2. Using biochemical methods and mutational analysis, they were able to identify key parts of the protein that are required for slicing activity. The lab also studies the generation of PIWI-interacting RNAs (piRNAs), which serve to protect the genome of germ cells. With colleagues in the Hannon lab, Joshua-Tor’s team also determined the structure and function of Zucchini, a key nuclease in the initial generation of piRNAs in fruit flies. In other work, the lab is exploring the mechanisms of heterochromatin formation and gene silencing through the study of a protein complex called RNA-induced initiation of transcriptional gene silencing (RITS). Joshua-Tor is also well known for her work on the E1 helicase enzyme, which acts to unwind DNA strands during the DNA replication process.

    Dr. Leemor Joshua-Tor honored with Mildred Cohn Award from ASBMB
    Member of the National Academy of Sciences
    Member of the American Academy of Arts and Sciences
    2018 Mildred Cohn Award in Biological Chemistry, ASBMB
    2014 ACE Women’s Network, New York, Women in Science and Education Leadership Award
    Fellow of the American Association for the Advancement of Science (AAAS)
    2007 Dorothy Crowfoot Hodgkin Award (Inaugural award), The Protein Society
    1996 Beckman Young Investigator Award

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