Mfd translocase is necessary and sufficient for transcription-coupled repair in Escherichia coli

Abstract

Nucleotide excision repair in Escherichia coli is stimulated by transcription, specifically in the transcribed strand. Previously, it was shown that this transcription-coupled repair (TCR) is mediated by the Mfd translocase. Recently, it was proposed that in fact the majority of TCR in E. coli is catalyzed by a second pathway ("backtracking-mediated TCR") that is dependent on the UvrD helicase and the guanosine pentaphosphate (ppGpp) alarmone/stringent response regulator. Recently, we reported that as measured by the excision repair-sequencing (XR-seq), UvrD plays no role in TCR genome-wide. Here, we tested the role of ppGpp and UvrD in TCR genome-wide and in the lacZ operon using the XR-seq method, which directly measures repair. We found that the mfd mutation abolishes TCR genome-wide and in the lacZ operon. In contrast, the relA^-spoT^- mutant deficient in ppGpp synthesis carries out normal TCR. We conclude that UvrD and ppGpp play no role in TCR in E. coli.

Keywords: DNA repair; DNA sequencing; Escherichia coli (E. coli); Mfd; UvrD; genomics; lac operon; nucleotide excision repair; ppGpp; transcription-coupled repair.

Figure 1.

The effects of mutations on genome-wide TCR profiles. The TCR values, measured as the TS/NTS repair signal of all the E. coli genes, grouped and colored by expression quartiles, are plotted as stacked (A) and separated (B) histograms. The x axis shows log2-scaled TS/NTS values normalized by the TT content of each strand per gene. The y axis shows the total gene number. In the stacked histogram (A), the x axis scale was focused (−5 to 5) to show the majority of the genes and misses the outliers that can be viewed in panel B.

Figure 2.

The contribution of ppGpp, UvrD, and Mfd to the XR-seq profiles in the highly expressed genes. The repair screenshots show normalized read numbers for both DNA strands of the RNA polymerase β subunit (rpoB), polyribonucleotide nucleotidyltransferase (pnp), and DNA polymerase I (polA) genes from top to bottom. The rows with red bars correspond to the TS of pnp and NTS of rpoB and polA. The rows with blue bars correspond to the TS of rpoB and polA and NTS of pnp. The y axis was scaled to show 50 RPM (reads per million mapped reads), and some hotspots with high repair values remain off-scale. The screen shots are derived from the data set of the first experiment. The TS/NTS ratios of the two experiments (Exp. 1 and Exp. 2) are horizontally plotted in the right-hand panes. +, protein or alarmone present; −, absent.

Figure 3.

TCR profiles of the lacZ gene for six E. coli strains with and without IPTG induction. The presence of glucose and IPTG is indicated by the filled black circles in the left column. The XR-seq bars (red for TS and blue for NTS) exhibit the repair levels on the y axis, which is scaled to 50 RPM (reads per million mapped reads). The TS/NTS ratios, which quantitatively represent TCR, from two experiments (Exp. 1 and Exp. 2) are horizontally plotted. β-Galactosidase (β-gal) activities are shown in Miller units with the standard deviations. The XR-seq signal shown in green represents TCR independent of Mfd. It is within the lac operator O₁ and indicates inhibition of repair by bound lac repressor, which cannot be overcome by Mfd.

Figure 4.

Time-course subtractive analysis of the TS/NTS in lacZ gene. Results were obtained with E. coli uvrD⁻ phr⁻ cells irradiated with 60 J/m² UV and allowed to repair for 1, 3, 10, 30, and 90 min. On the y axis, each data point represents a TS/NTS value derived from the change in reads seen at successive time points. The x axis shows the time intervals; for example, the initial time-point (1 min) reads were subtracted from 3-min reads to obtain the reads plotted for the 1-min to 3-min time interval. Each data point from two experiments (Exp. 1 and Exp. 2) is plotted separately. Blue and red colors indicate plus and minus IPTG induction, respectively.