Transcription of AAT•ATT Triplet Repeats in Escherichia coli Is Silenced by H-NS and IS1E Transposition

Abstract

BackgroundThe trinucleotide repeats AAT•ATT are simple DNA sequences that potentially form different types of non-B DNA secondary structures and cause genomic instabilities in vivo.Methodology and Principal FindingsThe molecular mechanism underlying the maintenance of a 24-triplet AAT•ATT repeat was examined in E.coli by cloning the repeats into the EcoRI site in plasmid pUC18 and into the attB site on the E.coli genome. Either the AAT or the ATT strand acted as lagging strand template in a replication fork. Propagations of the repeats in either orientation on plasmids did not affect colony morphology when triplet repeat transcription using the lacZ promoter was repressed either by supplementing LacIQ in trans or by adding glucose into the medium. In contrast, transparent colonies were formed by inducing transcription of the repeats, suggesting that transcription of AAT•ATT repeats was toxic to cell growth. Meanwhile, significant IS1E transposition events were observed both into the triplet repeats region proximal to the promoter side, the promoter region of the lacZ gene, and into the AAT•ATT region itself. Transposition reversed the transparent colony phenotype back into healthy, convex colonies. In contrast, transcription of an 8-triplet AAT•ATT repeat in either orientation on plasmids did not produce significant changes in cell morphology and did not promote IS1E transposition events. We further found that a role of IS1E transposition into plasmids was to inhibit transcription through the repeats, which was influenced by the presence of the H-NS protein, but not of its paralogue StpA.Conclusions and SignificanceOur findings thus suggest that the longer AAT•ATT triplet repeats in E.coli become vulnerable after transcription. H-NS and its facilitated IS1E transposition can silence long triplet repeats transcription and preserve cell growth and survival.

Ribonuclease H1-targeted R-loops in surface antigen gene expression sites can direct trypanosome immune evasion

“…Within the ES the 70 bp repeats appear to be a pronounced site of VSG RNA-DNA hybrid accumulation, given the strongest R-loop ES enrichment is seen in this location in Tbrh1 -/- mutants. R-loops may form more readily on the 70 bp repeats due to their sequence composition: individual 70 bp repeats show considerable size and sequence variation but are, in part, comprised of (TRR) repeats [15, 75] that can become non-H bonded [76] and promote recombination [77] when transcribed. Increased DRIP-seq signal across the active VSG ES of Tbrh1 -/- mutants might therefore be most readily explained by R-loops forming initially in the 70 bp repeats and extending back toward the RNA Pol I promoter due to retrograde spreading of the VSG ES transcription blockade.…”

RibonucleaseH1-targeted R-loops in surface antigen gene expression sites can direct trypanosome immune evasion

“…Within the ES the 70 bp repeats appear to be a pronounced site of VSG RNA-DNA hybrid accumulation, given the strongest R-loop ES enrichment is seen in this location in Tbrh1 -/- mutants. R-loops may form more readily on the 70 bp repeats due to their sequence composition: individual 70 bp repeats show considerable size and sequence variation but are, in part, comprised of (TRR) repeats (15, 66) that can become non-H bonded (67) and promote recombination (68) when transcribed. Increased DRIP-seq signal in the active VSG ES of Tbrh1 -/- mutants is therefore most readily explained by R-loops forming initially in the 70 bp repeats and extending back toward the RNA Pol I promoter due to retrograde spreading of the VSG ES transcription blockade.…”

The Gratuitous Repair on Undamaged DNA Misfold

“…Interestingly we have recently found that trinucleotide repeats AAT can also adopt curved DNA in E.coli, which can be repressed by H-NS and its stimulated IS1E transposition (Pan et al, 2010) …”