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Chlorouracil

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Published on 4 August 2016

Systematic substitution of thymine by 5-chlorouracil in the DNA of Escherichia coli

Genome-scale modification of base usage in nucleic acids has apparently not evolved in any known living cells, possibly because of a chemical barrier that natural biodiversity cannot overcome. To achieve a genome wide change in DNA composition we executed an experimental plan consisting of the construction of E. coli selection strains blocked in the biosynthesis of the canonical DNA base thymine and long-term cultivation of these strains in the presence of growing concentrations of the non-natural thymine homologue 5-cholorouracil. Strain evolution during the cultivation process, conducted in the GM3 automated culture devices operational at Genoscope, led to cells showing thymine to 5-chlorouracil substitution levels of up to 99 % in their genomes. This change in DNA base composition was perfectly compatible with cell proliferation.

Genome sequencing of different 5-chlorouracil dependent lineages and comparison of their mutation patterns reveals some common characteristics: A high number of point mutations per genome (between 120 and more than 1500, were detected, 35% of them being silent. This suggests a direct mutagenic effect of the thymine homologue 5-chlorouracil, most probably through mispairing with guanine. In addition, various mutations have been found in genes involved in DNA repair mechanisms. At least some of them are likely to entail inactivation of the respective protein and probably have contributed to the high mutation rate. Also, they might account for the drastic differences in the mutation patterns observed for the different lineages analyzed.

Very few genes are mutated in all the different lineages obtained. However mutations were highly frequent in some gene classes coding for functionally related proteins. These involved enzymes active in the correction of base mispairings, enzymes of the nucleic acid metabolism (polymerases, helicases) and various transcriptional regulators.

Experiments are currently conducted in order to explore the mechanism of cellular adaptation to an artificial base in DNA. Further long term cultivation processes have allowed the selection of 5-chlorouracil dependent mutants showing impaired growth on the natural base thymine, pointing to the possibility that descendants could be evolved for which thymine would be a xenobiotic compound.

Références:

Marlière, P., Patrouix, J., Döring, V., Herdewijn, P., Tricot, S., Cruveiller, S., Bouzon, M. & Mutzel, R. 2011 Chemical evolution of a Bacterium’s GenomeAngewandte Chemie International Edition, 50: 7109-7114