Emilia Botello

Quinolones are one of the types of antibiotics with higher resistance rates in the last years. At molecular level, quinolones block type II topoisomerases producing double strand breaks (DSBs). These DSBs could play a double role, as inductors of the quinolone bactericidal effects but also as mediators of the resistance and tolerance mechanisms. In this work we have studied the molecular pathways responsible for DSBs repair in the quinolone susceptibility: the stalled replication fork reversal (recombination-dependent) (RFR), the SOS response induction, the translesional DNA synthesis (TLS) and the nucleotide excision repair mechanisms (NER). For this reason, at the European University in Madrid, we analysed the minimal inhibitory concentration (MIC) to three different quinolones in Escherichia coli mutant strains coming from different type culture collections. recA, recBC, priA and lexA mutants showed a significant reduction on the MIC values for all quinolones tested. No significant changes were observed on mutant strains for TLS and NER. These data indicate that in the presence of quinolones, RFR mechanisms and the SOS response could be involved in the quinolone susceptibility.

Cyclooxygenases (prostaglandin-endoperoxide synthases, (EC 1.14.99.1) 1 and 2 (COX-1 and COX-2)) are key enzymes with a highly functional and pharmacological relevance. Genetic variations in the corresponding genes PTGS1 and PTGS2 are related to diverse human disorders and adverse drug reactions. Although COX-2 is highly inducible, most genetic association studies have focused on coding region gene variants. The aim of this study is to analyze the genetic variants modifying transcription factor binding sites in human PTGS genes based on the combined use of bioinformatics with 1,000 genomes data and replication by next generation sequencing. Updated information on gene sequences and variants was obtained from the 1,000 genomes website and from a replication sequencing study. Of the 570 upstream PTGS1 gene variants, 43 altered binding sites, either by disrupting existing sequences or by creating new binding sites. The most relevant are the SNP rs72769722, which creates a new binding site for NFKB, and the SNPs rs73559017 and rs76403914, both disrupting binding sites for CDX1. Of the 682 upstream PTGS2 gene variants, 31 altered binding sites, the most relevant being rs689466 and rs20417, which disrupt binding sequences for MYB and E2F, respectively; rs689462 which creates a new binding site for POU3F2; and a haplotype combining the SNPs rs34984585+rs10911904, which creates a new binding site for SRY. This study provides a detailed catalog of variant and invariant transcription factor binding sites for PTGS genes and related haplotypes. This information can be useful to identify potential genetic targets for studies related to COX enzymes.

An upshift of 10 degrees C or more in the growth temperature of an Escherichia coli culture causes induction of extra rounds of chromosome replication. This stress replication initiates at oriC but has functional requirements different from those of cyclic replication. We named this phenomenon heat-induced replication (HIR). Analysis of HIR in bacterial strains that had complete or partial oriC deletions and were suppressed by F integration showed that no sequence outside oriC is used for HIR. Analysis of a number of oriC mutants showed that deletion of the L-13-mer, which makes oriC inactive for cyclic replication, was the only mutation studied that inactivated HIR. The requirement for this sequence was strictly correlated with Benham's theoretical stress-induced DNA duplex destabilization. oriC mutations at DnaA, FIS, or IHF binding sites showed normal HIR activation, but DnaA was required for HIR. We suggest that strand opening for HIR initiation occurs due to heat-induced destabilization of the L-13-mer, and the stable oligomeric DnaA-single-stranded oriC complex might be required only to load the replicative helicase DnaB.

A temperature upshift of 10 or more degrees in the growth temperature of a bacterial culture causes induction of extra rounds of chromosome replication. This heat‐induced replication (HIR) initiates at oriC, is transitory, requires RNase H1 and RecA proteins and requires neither RNA polymerase activity nor de novo protein synthesis. The number of origins activated by heat is growth rate and temperature differential dependent. An origin activation higher than 20% increases the DNA:mass ratio around twofold, and this value is kept constant for the subsequent generations of growth at 41°C. We have also shown that HIR is neither related to SDR nor induced by the heat shock response. We suggest that a thermodynamic alteration of oriC structure or of membrane fluidity could explain the observed HIR.