Mechanisms of error-free lesion tolerance – GENOBLOCK

The methodology to introduce a single lesion in E. coli chromosome works as follow: a non-replicating plasmid construct containing the single lesion of interest and a genetic marker on its opposite strand is integrated at a specific locus of the bacterial chromosome by the mean of the phage lambda site specific recombinase. Following insertion of the specific lesion, we can monitor DNA damage tolerance events (i.e. TLS and DA) as well as a failure to recover a blocked replication fork that leads to cell death. The technique has proven its robustness and we are now ready to exploit it to reach our two main objectives.

We have pursued the genetic study of DNA damage tolerance pathway.
i) Using separation of function mutants of RecA, we investigated the genetic control of pathway choice between TLS and DA in E. coli. In a strain with wild type RecA activity, the extent of TLS across replication blocking lesions is generally low while DA is used extensively. Interestingly, recA alleles that are partially impaired in D-loop formation confer a decrease in DA and a concomitant increase in TLS. This allowed us to conclude the pathway choice between error-prone TLS and error-free DA is controlled by the efficiency of homologous recombination. These results were published in Nucleic Acids Res.
ii) Using a new strategy to specifically monitor the exchange of genetic information from the non-damaged chromatid to the damaged sister chromatid, we were able to evidence a new DA mechanism that we named «damaged chromatid loss«. This strategy allows cell survival and proliferation. These results were published in Plos Genetics. We also showed the involvement of RecFOR and for the first time of RecBCD in process of gap repair. These results appear in the Plos Genetics article and are also discussed in a review article in Current Genetics.
iii) We further explored the role played by RecBCD in the process of gap repair. We showed that the complex played a role not only in homologous recombination, but also in TLS. By using several point mutations that inactivate specific enzymatic activities of the complex, we showed that RecBCD plays a non-catalytic role in gap repair, seemingly by preserving the integrity of the fork and allowing an efficient bypass of the lesion. These results were published in Nucleic Acids Research (2017).
iv) We are exploring the effect of the proximity of two DNA lesions in the genome on the balance between DA and TLS..
v) We succeeded in integrating a single DNA lesion into the yeast genome, and have initiated the exploration of DNA damage tolerance in this organism.

We are going to pursue the study of lesion tolerance and focus on the molecular mechanisms that are involved. We also wish to develop in vitro biochemical approaches in order to confirm the models that arise from our genetic approach.
We are also going to study these mechanisms in eukaryotic cells, focusing on the bypass of UV lesion in the yeast S. cerevisiae.

Laureti, L., Demol, J., Fuchs, R. P., and Pagès, V. (2015) Bacterial Proliferation: Keep Dividing and Don't Mind the Gap. PLoS Genet 11: e1005757

Pagès, V. (2016) Single-strand gap repair involves both RecF and RecBCD pathways. Curr. Genet. doi: 10.1007/s00294-016-0575-5

Naiman, K., Pagès, V., & Fuchs, R. P. (2016) A defect in homologous recombination leads to increased translesion synthesis in E. coli. Nucleic Acids Res. doi: 10.1093/nar/gkw488

Laureti, L., Lee, L., Philippin, G., & Pagès, V. (2017) A non-catalytic role of RecBCD in homology directed gap repair and translesion synthesis. Nucleic Acids Research. doi.org/10.1093/nar/gkx217

Pagès, V. and Fuchs, R.P. (2017) Inserting site specific DNA lesions into whole genomes. Methods in Molecular Biology. in press

The encounter of a replication fork with a blocking DNA lesion is a common event that cells need to address properly to preserve genome integrity. Cells possess two main strategies to restore transiently blocked replication forks: Translesion synthesis (TLS) and Damage Avoidance (DA) pathways. While TLS pathways are error-prone and are the major source of point mutations, DA pathways are error-free as they rely on mechanisms related to homologous recombination with the sister chromatid. Whereas TLS has been extensively studied over the past 15 years after the discovery of the specialized translesion DNA polymerases, the genetics and the molecular mechanisms that trigger Damage Avoidance remain poorly defined. While it is important to understand how mutations arise, it is essential to understand the mechanisms that are employed by the cell to avoid mutations. Especially in human cells, where mutagenesis results in genomic instability that is responsible for ageing, and that leads to severe pathologies such as cancer, numerous neurodegenerative diseases and diseases linked to trinucleotide expansions. Moreover, the balance between TLS and DA is very important since it defines the level of mutagenesis during lesion bypass. Until today, studies on the consequences of lesion in DNA in vivo are usually limited to the analysis of induced mutations. However, the respective proportion of TLS versus DA events within DNA damage tolerance events is presently not known.
The aim of our project is to investigate the process of DA and how is regulated the partition between DA and TLS, using a methodology that allows to introduce a single lesion at a specific locus in the genome of a living cell. We recently published the proof of concept and the first set of results (Pagès et al., Nucleic Acid Res 2012) and showed that following a robust and precise integration of a damaged DNA, our system allows for the first time to monitor both TLS and DA events that occur in vivo. More recently, we published a new set of data (Naiman et al., PNAS 2014) where we followed the fate of a specific lesion that we insert in the genome among lesion randomly distributed over the rest of the chromosome to mimic a natural genotoxic stress situation. This lead us to the conclusion that lesion tolerance events are executed in a chronological order, with TLS coming first, followed by DA.
The data we have obtained so far prove the robustness of the technique and its great potential that we want to exploit following 2 main objectives:
1) Pursuing the genetic study of DNA damage tolerance and understanding the molecular mechanisms of DA in bacteria. We expect to better understand the structure of a replication fork encountering a lesion and therefore the molecular mechanisms of lesion tolerance. Combining molecular approaches in vivo with a genetic approach, we will define the factors responsible for the different possible structures of the replication fork, and therefore the outcome in term of lesion bypass mechanisms. Especially, we will identify proteins involved in the error-free Damage Avoidance process and determine their impact in the structure of the replication fork.
2) Adapting the system to eukaryotic cells to better understand any deregulation in the balance between TLS and DA that can lead to genome instability responsible for several pathologies. With this approach, we will for the first time monitor lesion tolerance mechanisms (both TLS and DA) of defined lesions in the chromosomal context in human cells. We will therefore be able to understand the molecular mechanisms of numerous pathologies related to genome instability. This is very important since it will not only expand the basic knowledge in cell biology, but has the potential to uncover many new pharmacological target for the treatment of a variety of diseases.