DNA repair and tolerance pathways involved in the processing of a single lesion on the chromosome of Escherichia coli

MARGARA LUCÍA M; PAGÉS VINCENT; MONTI MARIELA R

Congreso; LV Reunión Anual de SAIB y Conferencia de la PABMB; 2019

The genome is constantly exposed to DNA damaging agents that alter the integrity of the DNA molecule. Most of these lesions are removed by the repair mechanism called Nucleotide Excision Repair (NER). However, some lesions might escape the repair process and perturb DNA synthesis by the replicative DNA Polymerase (Pol), resulting in cell lethality. To survive DNA damage, cells have evolved Damage Tolerance (DT) pathways such as the Translesion Synthesis (TLS), which involves specialized Pols capable of inserting a nucleotide opposite the lesion. In this work, we studied both repair and tolerance pathways implied in the processing of a single alkylation lesion (N2-furfurylguanine, FF) inserted site-specifically in the chromosome of Escherichia coli. The main results were: i-TLS was highly favored over other DT pathways; ii- Pol IV, but not Pol II and Pol V, catalyzed the TLS reaction; iii- Pol IV incorporated the correct nucleotide opposite the FF lesion and thus, the TLS reaction is error-free; iv-the proofreading subunit of the replicative Pol III prevented the Pol IV-catalyzed TLS, indicating a competition between exonucleolytic excision and TLS; v-the FF lesion was not removed by the NER, which is the main repair pathway implicated in the reversion of the alkylating damage; vi- the postreplicative Mismatch Repair (MMR) pathway was able to repair this damage. Finally, no differences in the repair or TLS of the FF lesion were detected when the damaged base was located in the leading or the lagging strand of replication. Our finding showed that Pol IV-catalyzed TLS and MMR become the main tolerance and repair pathways used by E. coli cells to deal with FF lesions on the chromosome.