Single strand gap repair: The presynaptic phase plays a pivotal role in modulating lesion tolerance pathways.
Laureti L, Lee L, Philippin G, Kahi M, Pagès V
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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:
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.
Very recently, we have developed a similar approach in eukaryotic cells using the yeast Saccharomyces cerevisiae. Inserting a single lesion in the yeast’s genome allows us to explore the mechanisms of DNA damage tolerance in this organism.
Bacterial colonies: following the insertion of a single DNA lesion, bacteria that bypassed the lesion by TLS appear as blue colonies.
Team publications
Laureti L, Lee L, Philippin G, Kahi M, Pagès V
Guervilly JH, Blin M, Laureti L, Baudelet E, Audebert S, Gaillard PH
Masłowska KH, Villafañez F, Laureti L, Iwai S, Pagès V