Tomas Lindahl

Molecular genetics and biochemistry of DNA repair in mammalian cells

Environmental agents can cause damage to the structure of DNA, leading to genetic instability and cancer. These DNA damaging agents include ionizing radiation and many anti-cancer drugs. However, one of the major threats to the genetic integrity of living cells comes from endogenous damage, due to the unavoidable exposure of DNA to reactive molecules such as active oxygen and water during the course of normal cellular metabolism.

The laboratory is interested in the repair pathways that form the major line of defence against such damage. Novel DNA repair enzymes have been identified, and their cloning and expression have allowed detailed mechanistic studies. Different sub-pathways of DNA base excision-repair have been reconstituted with purified proteins and DNA substrates containing defined lesions, specific protein-protein interactions have been analysed that co-ordinate sequential catalytic steps, and the effects of chromatin structure have been evaluated. Such biochemical approaches are correlated with genetic data through the analysis of mutant cell lines derived from ionizing radiation-sensitive or cancer-prone individuals, as well as the generation and characterisation of knock-out mice deficient in specific repair functions. We have also established null mouse embryo fibroblast cell lines that can be used to analyse the repair of induced DNA damage and modified responses to genotoxic agents, including certain cancer therapeutic drugs.

One of the most abundant miscoding DNA base lesions is uracil, resulting from hydrolytic deamination of cytosine; we have identified a second major uracil-DNA glycosylase in mammalian cells as the candidate anti-mutator enzyme excising this damage. Analysis of spontaneous mutation frequency and tumour incidence in knock-out mouse models, and the identification of causative gene mutations in human tumour cell lines, will now evaluate the contribution of these enzymes to maintaining the low mutation rate necessary for normal cellular proliferation and the avoidance of cancer. Such approaches are identifying interesting overlaps between DNA repair, recombination and other biological processes, such as the functioning of the immune system.

Enzymatic demethylation of DNA is important to counteract the effects of cytotoxic alkylating agents. Epigenetic control of gene expression may also require active demethylation of promoter sequences silenced by methylation. We are investigating the enzymatic mechanisms of these processes. In recent work, we have shown that iron-dependent DNA demethylases are active in both bacteria and mammalian cells to directly revert toxic alkylation damage by oxidation and release of the methyl moieties. Two human nuclear enzymes with this action spectrum are being investigated. These DNA dioxygenases not only provide protection against alkylating agents, but unfortunately may also counteract effective chemotherapy in vivo.

References

1. H. Nilsen, G. Stamp, S. Andersen, G. Hrivnak, H.E. Krokan, T. Lindahl and D.E. Barnes. Gene-targeted mice lacking the Ung uracil-DNA glycosylase develop B-cell lymphomas. Oncogene 22: 5381-5386 (2003).
   
   
2. M. Morita, G. Stamp, P. Robins, A. Dulic, I. Rosewell, G. Hrivnak, G. Daly, T. Lindahl and D.E. Barnes. Gene-targeted mice lacking the Trex1 (DNaseIII) 3' → 5' DNA exonuclease develop inflammatory myocarditis. Mol. Cell. Biol. 24: 6719-6727 (2004).
   
   
3. P. Koivisto, T. Duncan, T. Lindahl and B. Sedgwick. Minimal methylated substrate and extended substrate range of Escherichia coli AlkB protein, a 1-methyladenine-DNA dioxygenase. J. Biol. Chem. 278: 44348-44354 (2003).
   
   
4. P. Koivisto, P. Robins, T. Lindahl and B. Sedgwick. Demethylation of 3-methylthymine in DNA by bacterial and human DNA dioxygenases. J. Biol. Chem. 279: 40470-40474 (2004).
   
   
5. D.E. Barnes and T. Lindahl. Repair and Genetic Consequences of Endogenous DNA Base Damage in Mammalian Cells. Annu. Rev. Genet. 38: 445-76 (2004).