Projects

Projects: Mechanisms for DNA repair and genomic (in)stability

Background

Repair of DNA damage is essential for protection against cancer and other age related diseases. DNA damage generated by ionizing radiation, simple alcylating agents or endogenously hydrolytic and oxidative processes is corrected by the base excision repair (BER) pathway(s).

Previous work include:

i) Overexpression of DNA glycosylases in mammalian cells. DNA glycosylases initiate the base excision repair pathway by removing the damaged base, and was shown to be a limiting factor for cell survival following induced DNA damage.

ii) Reconstituting the pathway(s) for base excision repair, in vitro, with purified human proteins. This work also lead to the identification of a human enzyme, XPG, stimulating the BER pathway. XPG is one of the genes causing the severe syndrome Xeroderma Pigmentosum, and is required for the repair of UV-induced DNA damage.

iii) Recent work includes the construction and analysis of a mice deficient for the OGG1 gene. This gene is the major enzyme for removal of the mutagenic oxidized lesion, 8oxoGua. Consequently, the OGG1 KO mice have an increased G to T transvertion mutation frequency and accumulate 8-oxoG in nuclear and mitochondria DNA. However, no obvious phenotype was observed. Interestingly, mouse embryo fibroblasts from the OGG1 KO mouse can still, efficiently, remove 8-oxoG from transcribed sequences.

Future projects

Currently two different approaches are used for identification of new genefunctions for repair and regulation of the mammalian genome.

i) The publication of the human DNA sequence revealed several open reading frames with possible DNA repair domains. Cloning and overexpression of candidate repair enzymes have recently resulted in the characterization of a human DNA damage specific endonuclease.

ii) Sensitive E.coli mutants have been very valuable for the cloning of homologues gene functions from eucaryotic cells by functional complementation. We have produced human cDNA libraries for expression in S.cerevisiae. Several defined S.cerevisiae mutants for genomic instability exist, and complementation of this defect should allow us to identify homologues genefunction with human origin.

iii) Finally, genes involved in maintenance of genome stability and structure are deleted from the mouse genome by homologous recombination (e.g. generation of knockout mice).

Goals

Identify and characterize genfunctions required for genomic stability and for protection against cancer. Targeted mutations of DNA repair genes in mice.

Illustration of the 5'-endonuclease activity of the FEN-1 enzyme (Five ENdonuclease-1)