Transcription infidelity and genome integrity: the parallax view

Abstract

It was recently shown that removal of GreA, a transcription fidelity factor, enhances DNA break repair. This counterintuitive result, arising from unresolved backtracked RNA polymerase impeding DNA resection and thereby facilitating RecA-loading, leads to an interesting corollary: error-free full-length transcripts and broken chromosomes. Therefore, transcription fidelity may compromise genomic integrity.

Keywords: Backtracked RNA polymerase; DNA break repair; DNA resection; DksA; Epimutation; Genetic noise; Genomic integrity; GreAB; RecBCD; Transcription fidelity.

Figure 1.

The absence of GreA increases survival to PHL treatment by the RecBCD-RecA pathway. (a) Average survival data of indicated mutant on 2 μg ml⁻¹ PHL (compared to non-PHL) for two days at 37°; for detailed results see Sivaramakrishnan et al. [12]. Red bars indicate strains with deleted or non-functional greA alleles; striped red bar indicates over-expression of DksA, which we suggest leads to increased survival by preventing GreA function through DksA successfully competing for RNAP binding to the exclusion of GreA. (b) DSB repair is initiated by RecBCD which resects DNA ends and then loads RecA to promote strand exchange. During transcription RNAP can become backtracked, creating a barrier to RecBCD resection. In the presence of GreA, transcription restarts and the barrier to RecBCD is resolved allowing continued resection leading to inefficient DNA repair. In the absence of GreA, the backtracked RNAP barrier remains causing RecBCD to pause and resection to cease allowing RecA loading and increased DNA repair[12].

Figure 2.

Heritable stochastic switching in the E. coli lac operon: a forward epimutation system. (a) The lac operon comprises an autocatalytic positive feedback loop (the presence of LacY permease will produce more permease) allowing a heritable all-or-none epigenetic switch at a maintenance concentration of inducer. LacY is presented in green because the lacA gene is replaced by gfp, and the lac operon is now lacZYA::gfp, so when permease is made GFP will also be made and the cell will fluoresce green. Stochastic events that lead to a transient depletion of repressor (in red) within a cell will result in a burst of LacY permease expression and the presence of permease will activate the positive feedback loop, so that the new induced state will be heritably maintained, mimicking mutation[22]. While it is the presence of LacY permease that allows the positive feedback loop to initiate and continue, it is the faithful transcription of the lacI gene that dictates cell fate. (b) Wild-type cells that were originally ON (open circles) or OFF (closed circles) were sub-cultured and grown in media containing various concentrations of inducer thio-methylgalactoside (TMG). The shaded area highlights the maintenance concentration of inducer for these strains; hysteresis and bistability in this system is observed [21,24]. (c) When OFF cells are grown in the maintenance concentration of inducer, the absence of GreA and GreB (blue flow cytometry histograms) increases the proportion of ON cells with respect to wild-type cell levels (red flow cytometry histograms) [20,21]. Each line represents an independent histogram of 10,000 cells. The increase in stochastic switch frequency is 38-fold over wild-type level[21].