Studies of molecular mechanisms of telomere maintenance in Naumovozyma castellii

Abstract: POPULAR SCIENTIFIC SUMMARYI was on my way home, sitting on the bus. My tired eyes swept through the pages of the local newspaper and that was the moment I met Lisa. She had previously been diagnosed with cancer. She had recovered and was back to her daily life. Unfortunately the cancer has come back and it has spread to several organs. Lisa as a mother of two kids, as a wife, as a daughter, as a sister, as a friend and as a colleague has less than one year left for this world. What are the reasons that our body loses control over this horrible disease? It is not my intention to go through every single reason here. Instead, I would like to discuss when and why the smallest building blocks of our body become immortal. The human body is composed of building blocks that are called cells. A cell represents a basic unit of life in our body. Most of these cells have similar life cycle like us. They grow, age and die. This cycle continuously occurs until a cell changes its behavior and become immortal. How do they become immortal or why are we mortal? The answer relies on our DNA, the decision maker. DNA is individual-specific and store all information about us. DNA is packaged into chromosomes. Human chromosomes are linear and thus have ends. Before a cell divides, the chromosomes are replicated to make an extra copy for the new cell. This replication is done by DNA polymerase. The DNA polymerase copies the chromosomes and thus we can think of it as a photocopy machine. In each cell division a part of the DNA will be lost from the ends of the chromosome due to incomplete DNA replication. When this gradual loss of the DNA reaches a critical point, the cells will stop to divide and eventually die. This process is one of the reasons for aging. The complete DNA replication is ensured by the help of another kind of synthesis machinery in human germ cells (egg and sperm). This replication machinery is called telomerase. Telomerase is specialized to replicate the ends of chromosomes and thus prevents DNA loss from the ends. The ends of the chromosome are composed of tandem repeats, which are called telomeres. These repeats do not contain essential information but are the guardians for the stable chromosomes. Telomerase is not active in most cells in the human body. A cell will divide a certain number of times and will eventually die when the telomeres are not replicated by telomerase. Cancer cells escape this death and counteract the telomere loss by either re-activating telomerase or by alternative ways. Most cancer cells (85-90%) re-activate telomerase to become immortal. The remaining 10-15% of cancer cells use alternative ways to maintain the telomeres. These alternative ways are not dependent on the telomerase. In my PhD project I have studied how telomeres are maintained with and without telomerase. In my studies, I have used the yeast Naumovozyma castellii which is a relative of the yeast that we use in baking and in alcohol production. The yeast N. castellii shares similarities with human telomere biology. Its telomeric DNA is similar with humans and the activity of its telomerase is comparable with the human telomerase. In contrast to humans, yeasts have active telomerase from birth to death. Therefore, it was advantageous to conduct my research on the yeast N. castellii. The first part of my thesis is focused on a detailed characterization of telomerase. I have designed an experimental strategy to understand how telomerase recognizes and elongates the ends of chromosomes. Chemically synthesized DNA stretches were used to mimic the telomere structure of a yeast cell. When telomerase was incubated with the chemically synthesized DNA stretch together with additional ingredients, telomerase would act in a similar way as in the cell. I have changed the composition and the length of these chemically synthesized DNA stretches to monitor the activity of telomerase under extreme conditions. These detailed studies have shown that multiple DNA interactions occurred with telomerase and they influence the activity of telomerase. These interactions had regulatory roles in the decision of the replication of a telomeric DNA by telomerase. The results from the characterization studies are very crucial in understanding the nature of telomerase and how it would act in a cancer cell. They are also beneficial in development of anti-ageing therapy that can help to reduce or postpone the aging process. The second part of my thesis is focused on how telomeres are elongated in the absence of the telomerase. 10-15% of cancer cells replicate their telomeres without the involvement of telomerase. Instead they activate alternative ways. I aimed to study factors that are required to support immortality in the absence of telomerase. When the yeast telomerase fails to elongate the ends of chromosomes, cells will have a similar aging profile like most human cells. Their telomeres slowly shorten; they grow slowly and eventually die. Intriguingly, a few of them escape form this death and survive. These telomerase-negative cells are called ´´survivors´´. More interestingly when we inactivated telomerase in the yeast N. castellii, telomerase-negative cells did not die and had similar growth rate like normal cells. They were able to activate the alternative ways very easily and quickly. I have looked more closely at factors that are required for the very easily and quickly activated survival mechanism. I showed how they re-arrange the ends of their chromosomes after the inactivation of telomerase. Comparison studies with chromosomal structure of a normal cell showed that the yeast N. castellii has specific DNA elements that might contribute to the cell survival strategies in the absence of telomerase. In order to develop an efficient anti-cancer therapy, we have to consider the alternative ways that are used in the maintenance of the telomeres. Cancer cells gain immortality either by reactivating telomerase or activating the alternative ways. In my PhD studies, I investigated both perspectives in order to put a step forward in developing better strategies to fight with cancer. Unfortunately, my research would not affect Lisa’s life but eventually it will affect other people's lives.