i) Development of new assay systems to detect and quantify DNA abasic (apurinic/apyrimidinic, AP) sites;
ii) Correlation between AP site frequencies and biological effects; characterization of the endocellular DNA repair processes active on AP sites and study of their mutagenic effects in mammalian cells in culture;
iii) Validation of these new assay systems for their ability to detect low frequencies of AP sites in different cell types and tissues.
The major and most common damage to cellular DNA is the loss of bases leading to abasic sites. This loss may be spontaneous and is enhanced by a variety of DNA damages, such as alkylation of the base and attack by hydroxyl radicals generated by ionizing radiation. AP sites are also the result of the activity of DNA glycosylases which remove modified bases. AP sites are "non-informational" DNA lesions with potential cytotoxic and mutagenic effects.
Many attempts have been made in the past to assay AP site formation, but their detection is still limited to indirect assays, like alkaline sucrose gradients and alkaline elution, which do not allow either the measurement of low frequency occurring AP sites or the localization of these lesions at gene level. There is thus a need to develop novel methodologies to detect AP sites in DNA. The assay systems examined in this project are based (a) on the substrate recognition properties of AP-endonucleases of different source, and (b) on the development of immunological assays, to identify and quantify AP sites at cell and/or gene level.
Abasic sites are common intermediate products in base excision repair, but several aspects of this repair mechanism in mammalian cells are still undefined. Mammalian cell extracts active for the excision repair will be used to investigate the in vitro repair of DNA abasic sites. Moreover, the mutagenic potential of abasic sites in mammalian cells in culture will be established by using shuttle vector molecules containing multiple or single AP sites.
The AP site detection systems which exhibit adequate sensitivity will be employed to determine low frequencies of AP sites in different cell type and tissues. This work will produce scientific information helpful for risk assessment and it may also generate new approaches for biomonitoring studies.
Call for proposalData not available
Funding SchemeCSC - Cost-sharing contracts
2300 RA Leiden