DNA replikatsiooni initsiatsiooni uurimine Saccharomyces cerevisiaes

Väitekirja elektrooniline versioon ei sisalda publikatsioone.DNA kui eluslooduse keskne molekul kannab endas informatsiooni mis on vajalik organismi ülesehitamiseks ja funktsioneerimiseks. Selleks, et DNA-s olevast informatsioonist oleks võimalik luua elusat olendit, olgu see siis bakter või inimene, on esmalt vajalik seda informatsiooni lugeda ja edasi toimetada. Molekulaarses mõttes tähendab see seda, et DNA molekuli peal toimub pidev sagimine. Informatsiooni lugevad molekulaarsed masinad liiguvad üksteise järel mööda kromosoome ja kirjutavad selles sisalduva informatsiooni uude molekuli - RNA-sse. Saadud RNA molekulide alusel aga sünteesitakse elusate organismide peamised ehituskivid ehk valgud. Valkude ehitamiseks mõeldud informatsiooni lugemise taustal on aga vajalik tagada ka rakkude jagunemine. Selleks tuleb DNA-s sisalduv informatsioon edasi kanda kõikidesse tütarrakkudesse ja enne rakkude jagunemist olemasolevatest DNA molekulidest sünteesima uue koopia. Taaskord tuleb esmalt lugeda vanas DNA molekulis olev informatsioon ja selle alusel sünteesida uus. Siinkohal tuleb aga meeles pidada, et sünteesi aluseks oleval DNA-l on juba suur hulk teisi molekule, mis aitavad organismil funktsioneerida. DNA paljundamise masinavärk peab aga sellises olukorras, kus DNA juba on pidevas kasutuses, suutma ülima täpsusega sünteesida uue identse molekuli. Ühe eesmärgina antud uurimustöö raames uuritigi protsesse mis võimaldavad DNA täpse paljundamise sellises situatsioonis. Leidsime, et DNA kopeerimise algatamine on RNA-de pideva sünteesimise tõttu tõsiselt häiritud ja selle tõttu peab seda protsessi igas rakus korduvalt uuesti alustama. Õnneks ei kaota DNA kopeerimise masinad, mis on ajutiselt DNA molekulilt eemaldatud oma võimet uut DNA-d sünteesida. Lisaks DNA kasutamise suurele intensiivsusele raskendab DNA kasutamist ka selle molekuli suurus. Näiteks inimese DNA kogupikkus on 3 meetrit, see tuleb aga mahutada 100 mikromeetri pikkustesse rakkudesse. Selle võimaldamiseks on DNA tihedalt kokku pakitud, analoogselt niidile mis on keeratud ümber niidirulli. DNA kopeerimise algatamiseks tuleb seega esmalt leida piirkonnad mille kokkupakkimise aste on väiksem ja millele on võimalik hõlpsalt juurde pääseda. Seda protsessi uurides leidsime, et rakkudes hoitakse DNA kopeerimise algatamiseks mõeldud alad aktiivselt ligipääsetavatena. Kui neid piirkondi liigutada teistesse kromosoomidesse, jäävad nad endiselt avatuks. Sellise olukorra tagamiseks seonduvad DNA-le spetsiifilised valgud mis takistavad selle kokku pakkimist. Kui need valgud eemaldada või muteerida DNA piirkondi kuhu nad seonduvad, on DNA kopeerimise algatamine häiritud.DNA carries the information needed for building and functioning of an organism. In order to achieve this, information embeded in DNA has to be first read and edited. Molecularly, it means that multiple molecular machines are moving along chromosomes and transcribing the information into an other molecule – RNA. Based on RNA in turn, the basic building blocks of life, proteins, are being synthesized. In addition to building proteins, it is also vital for an organism to be able to divide its cells. In order to to that, the DNA content of a cell has to be duplicated. Once again, the molecular machines have to move along the chromosomes, first read and then write the information into a new DNA molecule. But remember, there is already a lot of traffic on DNA – the molecules that help to transcribe the information for building proteins. Therefore, the DNA replication machinery has to be able to cope with these obstructions and accurately copy the DNA sequence. One of the goals in this study was to determine how DNA replication manages to cope in this situation. We found that, indeed, the DNA replication initiation is disrupted from constant synthesis of the RNA molecules. On the other hand, the DNA replication machinery does not lose its functionality after being temporarily removed from DNA, and can reform shortly after. The reformed molecules can then successfully finish the job. In addition to the intensity of DNA usage, also the length of the DNA molecule makes it more difficult to copy it. For example, the length of human DNA is about 3 meters and it has to be packed into 100 micrometer long cells. In order to achieve this, DNA has to be heavily packed. Therefore, in cells the string of DNA is wound around little barrels of proteins to minimize its size. This in turn makes parts of DNA inaccessible and consequently the DNA replication machinery can not efficiently start the synthesis. When studying this phenomenon, we found that certain regions of chromosomes are being kept in a constantly unpacked state in order to efficiently start DNA synthesis. These regions are being kept in an unpacked state even when artificially moved to different chromosomes. This process is dependent on specific proteins, that bind DNA and manage to inhibit its packing. When these proteins are removed, or the DNA regions where they bind are mutated, the start of DNA replication is heavily disturbed

Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase

Activation of the Mcm2–7 replicative DNA helicase is the committed step in eukaryotic DNA replication initiation. Although Mcm2–7 activation requires binding of the helicase-activating proteins Cdc45 and GINS (forming the CMG complex), an additional protein, Mcm10, drives initial origin DNA unwinding by an unknown mechanism. We show that Mcm10 binds a conserved motif located between the oligonucleotide/oligosaccharide fold (OB-fold) and A subdomain of Mcm2. Although buried in the interface between these domains in Mcm2–7 structures, mutations predicted to separate the domains and expose this motif restore growth to conditional-lethal MCM10 mutant cells. We found that, in addition to stimulating initial DNA unwinding, Mcm10 stabilizes Cdc45 and GINS association with Mcm2–7 and stimulates replication elongation in vivo and in vitro. Furthermore, we identified a lethal allele of MCM10 that stimulates initial DNA unwinding but is defective in replication elongation and CMG binding. Our findings expand the roles of Mcm10 during DNA replication and suggest a new model for Mcm10 function as an activator of the CMG complex throughout DNA replication.National Cancer Institute (U.S.) (Grant P30-CA1405

Acetylation of H3 K56 Is Required for RNA Polymerase II Transcript Elongation through Heterochromatin in Yeast▿

In Saccharomyces cerevisiae SIR proteins mediate transcriptional silencing, forming heterochromatin structures at repressed loci. Although recruitment of transcription initiation factors can occur even to promoters packed in heterochromatin, it is unclear whether heterochromatin inhibits RNA polymerase II (RNAPII) transcript elongation. To clarify this issue, we recruited SIR proteins to the coding region of an inducible gene and characterized the effects of the heterochromatic structure on transcription. Surprisingly, RNAPII is fully competent for transcription initiation and elongation at the locus, leading to significant loss of heterochromatin proteins from the region. A search for auxiliary factors required for transcript elongation through the heterochromatic locus revealed that two proteins involved in histone H3 lysine 56 acetylation, Rtt109 and Asf1, are needed for efficient transcript elongation by RNAPII. The efficiency of transcription through heterochromatin is also impaired in a strain carrying the K56R mutation in histone H3. Our results show that H3 K56 modification is required for efficient transcription of heterochromatic locus by RNAPII, and we propose that transcription-coupled incorporation of H3 acetylated K56 (acK56) into chromatin is needed for efficient opening of heterochromatic loci for transcription

DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7

© 2021, eLife Sciences Publications Ltd. All rights reserved. The committed step of eukaryotic DNA replication occurs when the pairs of Mcm2-7 replicative helicases that license each replication origin are activated. Helicase activation requires the recruitment of Cdc45 and GINS to Mcm2-7, forming Cdc45-Mcm2-7-GINS complexes (CMGs). Using single-molecule biochemical assays to monitor CMG formation, we found that Cdc45 and GINS are recruited to loaded Mcm2-7 in two stages. Initially, Cdc45, GINS, and likely additional proteins are recruited to unstructured Mcm2-7 N-terminal tails in a Dbf4-dependent kinase (DDK)-dependent manner, forming Cdc45-tail-GINS intermediates (CtGs). DDK phosphorylation of multiple phosphorylation sites on the Mcm2-7 tails modulates the number of CtGs formed per Mcm2-7. In a second, inefficient event, a subset of CtGs transfer their Cdc45 and GINS components to form CMGs. Importantly, higher CtG multiplicity increases the frequency of CMG formation. Our findings reveal molecular mechanisms sensitizing helicase activation to DDK levels with implications for control of replication origin efficiency and timing

Recruitment of Fkh1 to replication origins requires precisely positioned Fkh1/2 binding sites and concurrent assembly of the pre-replicative complex

<div><p>In budding yeast, activation of many DNA replication origins is regulated by their chromatin environment, whereas others fire in early S phase regardless of their chromosomal location. Several location-independent origins contain at least two divergently oriented binding sites for Forkhead (Fkh) transcription factors in close proximity to their ARS consensus sequence. To explore whether recruitment of Forkhead proteins to replication origins is dependent on the spatial arrangement of Fkh1/2 binding sites, we changed the spacing and orientation of the sites in early replication origins <i>ARS305</i> and <i>ARS607</i>. We followed recruitment of the Fkh1 protein to origins by chromatin immunoprecipitation and tested the ability of these origins to fire in early S phase. Our results demonstrate that precise spatial and directional arrangement of Fkh1/2 sites is crucial for efficient binding of the Fkh1 protein and for early firing of the origins. We also show that recruitment of Fkh1 to the origins depends on formation of the pre-replicative complex (pre-RC) and loading of the Mcm2-7 helicase, indicating that the origins are regulated by cooperative action of Fkh1 and the pre-RC. These results reveal that DNA binding of Forkhead factors does not depend merely on the presence of its binding sites but on their precise arrangement and is strongly influenced by other protein complexes in the vicinity.</p></div

Alterations of distance between the Fkh1/2 binding sites in <i>ARS607</i>.

<p><b>(A)</b> Schematic representation of <i>ARS607</i> origins inserted into the <i>GAL</i>-<i>VPS13</i> locus. Approximate locations of Fkh1/2 consensus binding sites (blue boxes) and the ACS (pink box) are indicated. In all constructs except wt <i>ARS607</i> the ACS-distal (3’) Fkh1/2 binding site was mutated (red X) and a new Fkh1/2 site was introduced at various distances from the ACS-proximal (5’) site. <b>(B)</b> Depiction of modified <i>ARS607</i> origins with small insertions and deletions between Fkh1/2 binding sites. Approximate locations of Fkh1/2 consensus binding sites (blue boxes), the ACS (pink box), poly-A track and sites of nucleotide insertions or deletions are indicated. Detailed sequences of all modified origin loci are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006588#pgen.1006588.s004" target="_blank">S4 Fig</a>. <b>(C)</b> Fkh1 binding to <i>ARS607</i> with altered distances between Fkh1/2 sites as determined by ChIP assay. Fkh1 occupancy at mutant <i>ARS607</i> is shown relative to its binding to the native <i>ARS607</i> locus in the same strain. A strain with no ARS in the <i>VPS13</i> locus and the native late-replicating origin <i>ARS522</i> that contains no Fkh1/2 binding sites are shown as controls (<i>VPS13</i> and <i>ARS522</i>, respectively). <b>(D)</b> Relative copy number of <i>VPS13</i>-<i>ARS607</i> DNA in HU-arrested cells. Cells were arrested in G1 with α-factor and then released into HU-containing media for 45 and 75 minutes. Graphs show the ratio of <i>VPS13</i>-<i>ARS607</i> and late-replicating <i>ARS522</i> loci, the ratio in G1-arrested cells was set as 1. Relative copy number of the native <i>ARS607</i> locus is shown as control. Full data for each strain is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006588#pgen.1006588.s001" target="_blank">S1 Fig</a>. <b>(E)</b> Mcm4 binding to <i>ARS607</i> loci with altered distances between Fkh1/2 sites as determined by ChIP assay. Mcm4 occupancy at mutant <i>ARS607</i> is shown relative to its binding to the native <i>ARS607</i> origin in the same strain. A strain with no ARS in the <i>VPS13</i> locus and the native origin <i>ARS522</i> are shown as controls (<i>VPS13</i> and <i>ARS522</i>, respectively).</p