Abstract: Our research projects focus on understanding the biological consequences that result from replication of damaged genomes. The presence of damage in template DNA during replication is the major source of point mutations, the initiating cause of Cancer. Human cells respond to genotoxic stress by inducing a complex response known as the DNA Damage Response (DDR). Our team investigates the effect of lesions on normal DNA metabolism from bacterial to human cells.
Lesion Tolerance pathways / How cells tolerate lesions during replication?
Despite efficient repair mechanisms, the presence of unrepaired lesions at the replication fork is a frequent event in all dividing cells. Cells possess two major strategies to tolerate lesion in their DNA: i) Translesion Synthesis (TLS) where specialized DNA polymerases insert a few nucleotides opposite the lesion with the possibility of introducing a mutation; ii) Damage Avoidance (DA) strategies that are error-free as they rely on mechanisms related to homologous recombination with the sister chromatid. The balance between these two strategies is very important since it defines the level of mutagenesis during lesion bypass. We have developed a novel technology that allows us to insert a single lesion at a specific site of the bacterial chromosome. We can thus monitor in vivo and quantitatively the partition between Translesion Synthesis (TLS) and Damage Avoidance (DA) in different genetic backgrounds, in order to define the genes that regulate the choice among these pathways. For several replication-blocking lesions, we demonstrate that DA events massively outweigh TLS events and that DA events are highly dependent upon the presence of the RecA protein. More recently, in order to mimic a natural situation of genotoxic stress, we followed the fate of a site-specific lesion present among randomly distributed lesions in the rest of the chromosome. The data show that lesion tolerance events are executed in chronological order, with TLS coming first, followed by DA leading to the important conclusion that cells favor genetic diversity before insuring survival. We are also analyzing the early replication intermediates that form near the lesion site, at the molecular level (qPCR, 2D-gels, Electron Microscopy), to define the molecular mechanisms involved in lesion tolerance. We plan to apply the same approach to eukaryotic cells.
The human DNA Damage Response (DDR)
The human DNA Damage Response (DDR) encompasses an intricate network of proteins that sense lesions in DNA and respond by inducing metabolic pathways essentially aimed at repairing the lesions (DNA Repair pathways) and at holding cell cycle progression (DNA Damage checkpoint pathways). In case of high or non-repairable damage, a programmed cell death pathway may be activated. Classical genetic and biochemical approaches have been implemented in order to identify the proteins that sense and signal the presence of lesions in DNA in the context of chromatin. In recent years, the methodologies involved in chromatin exploration are essentially limited to ChIP (chromatin immuno-precipitation) and derived approaches. These approaches efficiently allow identify all DNA sequences to which a protein-of-interest binds. Methods to achieve the reverse, i.e. the isolation and identification of all proteins bound to a DNA locus-of-interest are not yet available. We are presently developing a novel method to isolate native protein complexes as they assemble in vivo in a locus-specific manner. The approach is based on the capture by means of an oligonucleotide probe that forms a triple helix with a sequence tag (TFT) that is located nearby the locus-of-interest (see figure below). Triple helix formation is compatible with the nucleosome structure of chromatin. This methodology, IDDAP (for Identification of DNA Damage Associated Proteins) allows proteins complexes that assemble at lesion sites during repair or replication to be isolated and analyzed. Typical DNA lesions (UV-induced lesions, cis-Pt, chemical carcinogens, cross-links…) are introduced into plasmids in vitro, transfected into human cells and allowed to be repaired and/or replicated for various periods of time before being re-isolated for analysis of the proteins assembled at lesion sites by proteomics. This method will be further developed for the isolation of any fragment of chromatin-of-interest.
Schematic view of the Triple Helix Capture methodology. Protein complexes associated to a specific fragment of DNA can be captured through the formation of a stabilized triple helix between a nearby Triplex Forming Tag sequence (TFT in blue) and a triplex forming oligonucleotide (TFO in red). The TFO contains a long spacer and a biotin residue that can be trapped by streptavidin-coated magnetic beads.
From left to right: Yoshihiko Yagi, Robert Fuchs, Asako Isogawa, Shingo Fujii, Karel Naiman, Gaëlle Philippin, Luisa Laureti, Vincent Pagès