DNA-herstelprocessen in levende cellen
Wim Vermeulen
- afd Genetica
DNA is constantly at risk of being damaged by a variety of environmental influences, such as UV-irradiation by sunlight and toxic chemicals, such as cigarette smoke and car combustion gas. Non-removed DNA lesions cause cellular malfunction eventually leading to cell death, a process that contributes to the aging of organisms. In addition, DNA damage may also cause mutations in coding genes. When essential genes are mutated vital functions of the cell are affected, including the regulation of cell growth. Uncontrolled cell growth is an important step in the process of cancer development. To circumvent these deleterious consequences of DNA damage each living cell contains different DNA repair mechanisms to remove these DNA damages.
A large number of enzymes constantly search for DNA damage with specific repair enzymes for different types of damage. If damage is detected these enzymes bind to it and subsequently recruit other enzymes that repair the damage.
The importance of an intact DNA repair system becomes specifically clear in patients suffering from the rare genetic disease xeroderma pigmentosum. These patients are extremely sensitive to solar exposure and develop numerous skin tumors at young age, due to a defect in the repair mechanism that removes DNA damage caused UV-light.
Most of our knowledge on the different DNA repair processes is based on biochemical analysis after extraction of enzymes from cells and analysis in the test-tube. Little is known, however, about the way DNA is maintained and repaired in its most relevant context: the living cell. Inside live cells different repair processes compete with each other and other vital cellular processes are deregulated by the presence of DNA lesion. The current challenge in DNA repair research is to gain knowledge on how these processes are regulated and communicate with each other. It is of great importance to get insight in how DNA repair is organized in living cells, how it prevents mutation induction and avoids cancer in an intact organism.
In 1995 a green fluorescent protein was isolated from the jellyfish Aequoria victoria. Methods were developed to (genetically) attach this protein to cellular enzymes. Using modern high-resolution microscopes, the location and the movement of these fluorescently labelled proteins can be visualised in living cells and even in entire organisms. Since then, the research of enzyme activity in living cells has gone through a revolution, in which our laboratory played an important role.
The research focus in the laboratory is currently applying these techniques to dissect the precise reaction mechanism of the enzyme machinery that repairs UV-damage. We have developed sophisticated procedures to determine the movement of proteins and enzyme reaction kinetics in living cells. We have started to study the behaviour of a large number of repair proteins. Currently, new techniques are being developed which allows direct determination on how the repair factors interact with each other and how they communicate with gene transcription and DNA replication. In addition, we adapt and modify procedures that allow us to investigate how the different enzymes find damage in regions where DNA is very tightly packed. Very recently, we have generated a mouse model that expresses such a fluorescently tagged DNA repair protein in all cells of the organism; this allows us to study the relationship between DNA repair efficiency in different cell types and carcinogenesis.