Studies on DNA replication initiation in Saccharomyces cerevisiae
Date
2013-04-03
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Abstract
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.
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.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Keywords
DNA, transkriptsioon (biol.), molekulaaraspektid, DNA, transcription (biol.), molecular aspects