Final Activity Report Summary - ARCHREP (Influence of genome architecture on the induction of damage in DNA by UVA, and the repair of that damage)
UVA radiation (315-400 nm) is a major genotoxic thread of our environment. Epidemiological data and recent research have demonstrated that UVA exposure is directly linked to the development of non-melanoma skin cancer. Although UVA is known to cause DNA damage the detailed molecular mechanism is not well understood. It is accepted that UVA irradiation has no direct effect on DNA, but acts via unknown endogenous photo-sensitizers. These sensitizers must absorb electromagnetic radiation in the range between 315 and 400 nm and transform the absorbed energy to reactive oxygen species (ROS). These ROS in turn damage the DNA. Since the ROS have a very limited life time and range of action the endogenous sensitizers are expected to be in proximity to DNA - a component of chromatin.
The aim of this project was to map UVA induced DNA damage with high resolution and thus identify possible structures containing UVA photosensitizers. A second aspect of the project was focused on the interplay between chromatin structure and DNA repair. Since DNA is highly organised in the cell nucleus, this organisation must have an influence on the induction and repair of (UVA induced) DNA damage.
The initial approach to use DNA damage specific antibodies to detect DNA alterations with high resolution techniques proved not to be feasible. Therefore we established poly-ADP-ribose as an early marker for UVA induced DNA damage. Using this marker we could identify the majority of UVA induced DNA damage in heterochromatin. Additionally we could show a slower speed of repair of damage in heterochromatin.
In a second set of experiments we could demonstrate that heterochromatin protein 1 (HP1) is recruited to sites of localised DNA damage. Further analysis revealed that this accumulation is much slower than the known accumulation of repair factors. Additionally we could show that the accumulation of HP1 at sites of DNA damage is not dependent on the known interaction partner histone H3, tri-methylated at the K9 residue.
Additionally we could demonstrate that UVA exposure alters chromatin structure at an intermediate level. Using the chromosome conformation capture technique, we were able to show that UVA stabilises some minor existing loops, while destabilising some major ones. In contrast UVC seems to completely destroy many loops. This effect is reversible and the original state is restored within 24 hours.
The aim of this project was to map UVA induced DNA damage with high resolution and thus identify possible structures containing UVA photosensitizers. A second aspect of the project was focused on the interplay between chromatin structure and DNA repair. Since DNA is highly organised in the cell nucleus, this organisation must have an influence on the induction and repair of (UVA induced) DNA damage.
The initial approach to use DNA damage specific antibodies to detect DNA alterations with high resolution techniques proved not to be feasible. Therefore we established poly-ADP-ribose as an early marker for UVA induced DNA damage. Using this marker we could identify the majority of UVA induced DNA damage in heterochromatin. Additionally we could show a slower speed of repair of damage in heterochromatin.
In a second set of experiments we could demonstrate that heterochromatin protein 1 (HP1) is recruited to sites of localised DNA damage. Further analysis revealed that this accumulation is much slower than the known accumulation of repair factors. Additionally we could show that the accumulation of HP1 at sites of DNA damage is not dependent on the known interaction partner histone H3, tri-methylated at the K9 residue.
Additionally we could demonstrate that UVA exposure alters chromatin structure at an intermediate level. Using the chromosome conformation capture technique, we were able to show that UVA stabilises some minor existing loops, while destabilising some major ones. In contrast UVC seems to completely destroy many loops. This effect is reversible and the original state is restored within 24 hours.