Supervisor: TÜ MRI vanemteadur Arnold Kristjuhan.
Opponent: Doktor John Diffley, Ühendkuningriigi Vähiuurimise Keskus (Cancer Research UK), Londoni Uurimisinstituut, Suurbritannia.
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.