Roles for the transcription elongation factor NusA in both DNA repair and damage tolerance pathways in Escherichia coli

Abstract

We report observations suggesting that the transcription elongation factor NusA promotes a previously unrecognized class of transcription-coupled repair (TCR) in addition to its previously proposed role in recruiting translesion synthesis (TLS) DNA polymerases to gaps encountered during transcription. Earlier, we reported that NusA physically and genetically interacts with the TLS DNA polymerase DinB (DNA pol IV). We find that Escherichia coli nusA11(ts) mutant strains, at the permissive temperature, are highly sensitive to nitrofurazone (NFZ) and 4-nitroquinolone-1-oxide but not to UV radiation. Gene expression profiling suggests that this sensitivity is unlikely to be due to an indirect effect on gene expression affecting a known DNA repair or damage tolerance pathway. We demonstrate that an N(2)-furfuryl-dG (N(2)-f-dG) lesion, a structural analog of the principal lesion generated by NFZ, blocks transcription by E. coli RNA polymerase (RNAP) when present in the transcribed strand, but not when present in the nontranscribed strand. Our genetic analysis suggests that NusA participates in a nucleotide excision repair (NER)-dependent process to promote NFZ resistance. We provide evidence that transcription plays a role in the repair of NFZ-induced lesions through the isolation of RNAP mutants that display altered ability to survive NFZ exposure. We propose that NusA participates in an alternative class of TCR involved in the identification and removal of a class of lesion, such as the N(2)-f-dG lesion, which are accurately and efficiently bypassed by DinB in addition to recruiting DinB for TLS at gaps encountered by RNAP.

Fig. 1.

nusA11 mutants are specifically sensitive to NFZ and 4-NQO. (A) Percent survival of strains treated with 0–15 μM NFZ. All graphs in this figure's experiments were performed at the permissive temperature (30 °C), and error bars represent the SD determined from at least three independent cultures. (B) Percent survival of strains treated with 0–17.5 μM 4-NQO. At 30 °C the sensitivity of the ΔdinB strain to NFZ and 4-NQO is less than the degree of sensitivity observed at 37 °C (10). (C) Percent survival of strains irradiated with 0–45 J/m² UV. (D) Percent survival of strains treated with 0–0.08% MMS.

Fig. 2.

Comparison of nusA11 and ΔnusA mutations in MDS42. (A–C) Percent survival of strains treated with the DNA-damaging agents NFZ (μM), UV, and MMS, respectively, at 30 °C. For all graphs in this figure, error bars represent the SD determined from at least three independent cultures. (D–F) Percent survival of strains treated with the DNA-damaging agents NFZ (μM), UV, and MMS, respectively, at 37 °C.

Fig. 3.

E. coli RNA polymerase does not bypass template strand gaps or a N²-f-dG lesion. (A) Schematic of experimental design. Three oligonucleotides, one containing the N²-f-dG lesion or an undamaged proxy, are ligated together to generate the transcribed strand (T) using the nontranscribed strand (NT) as a scaffold. A 9-ntd noncomplementary region between the (T)DNA and (NT)DNA allows for the annealing of an RNA primer (green) to initiate transcription. For each nucleic acid scaffold, purified RNAP, UTP, and [³²P]αGTP are added to radiolabel the RNA transcript and extend to G12. Because of the limiting ATP left over from the ligation reaction, we observe the addition of several nucleotides to the transcript (first lane of B). The addition of excess cold ATP, UTP, and GTP extends the RNA to the G27 position (second lane of B). Addition of CTP allows for transcription through the lesion or proxy to the end of the scaffold in the full-length/undamaged template (third lane of B; band labeled RO). “X” indicates the site of N²-furfuryl-dG lesion or proxy dG, nucleotide colored in red represents the position of the 1-ntd gap, and nucleotides colored in blue represent the position of the 14-ntd gap. Positions labeled in B represent the extension of RNA primer as marked, underneath the templating base, in schematic. (B) For each template (labeled at bottom), the first lane is the transcription reaction after addition of UTP, [³²P]GTP, and limiting ATP to allow labeling and extension to G12; the second lane is the reaction after the addition of excess ATP, UTP, and GTP; and the third lane is the reaction after the addition of CTP. All lanes represent 1-min time points. The asterisk represents the product formed on the 1-ntd gap.

Fig. 4.

Interactions with NER and a role for transcription. (A) Far-Western blot demonstrates that NusA interacts with UvrA. Cell lysates harboring the empty vector (lane 1) or overexpressing UvrA (lane 2) were separated by SDS/PAGE, transferred to a PVDF membrane, and incubated with purified NusA. α-NusA antibodies detected the binding of NusA to a 100-kDa migrating protein specifically in the UvrA (103 kDa) overexpressing lane. (B) Percent survival of strains treated with 0–15 μM NFZ at 30 °C. Squares, wild type/AB1157; circles, nusA11 (SEC164); inverted triangles, ΔuvrA (SEC 316); diamonds, nusA11ΔuvrA (SEC318). In this and all graphs in this figure, error bars represent the SD determined from at least three independent cultures. (C) nusA11 and Δmfd strains display an additive phenotype with respect to NFZ (filled bars) or 4-NQO (striped bars) sensitivity at 10 μM at 30 °C. (D) Percent survival of strains, MDS42, ΔnusA, Δmfd (SEC1629), and ΔnusA Δmfd double mutants (SEC1276), to UV irradiation demonstrates that the ΔnusA Δmfd double mutant is much more sensitive than either of the single mutants. (E) Sensitivity of rpoB mutants expressed in AB1157 to 10 μM NFZ at 37 °C. (F) Sensitivity of rpoB mutants expressed in a Δmfd background to 10 μM NFZ at 37 °C. (G) Sensitivity of rpoB mutants expressed in a nusA11 mutant background to 12.5 μM NFZ at 30 °C. (H) Sensitivity of rpoB mutants expressed in a ΔuvrA background to 10 μM NFZ at 37 °C. Despite differences in the survival of ΔuvrA strains expressing the rpoB variants, the ability of each rpoB mutant to confer NFZ^S or NFZ^R as observed in a uvrA⁺ strain background is lost.

Fig. 5.

nusA11 cells display phenotypes of altered DNA processing in stationary phase. (A–C) Representative micrographs of stationary-phase wild-type (AB1157) (SEC677) (A), lexA(Def) (SEC678) (B), and nusA11 cells (SEC679) (C). Number of cells counted (n) was 731 for wild type, 330 for lexA(Def), and 402 for nusA11. Cell outlines (red) were visualized with the vital membrane stain FM4-64, and SOS induction was monitored from P_sulA-GFP fusion integrated at an ectopic locus on the chromosome (34) (green). (D–F) Representative micrographs of stationary-phase wild-type (AB1157) cells (n = 2,000) (D), wild-type (AB1157) cells irradiated with 25 J/m² UV and left to recover in the dark for 10 min (n = 509) (E), and stationary-phase nusA11 cells (n = 362) (F). Cell outlines (red) were visualized with the vital membrane stain FM4-64, and RecA-GFP foci are shown in green. RecA-GFP translational fusion is chromosomally expressed from endogenous locus (35).

Fig. 6.

Models of NusA involvement in two distinct and previously unrecognized pathways: transcription-coupled translesion synthesis (TC-TLS) and an alternative class of transcription-coupled repair. (A) Model of TC-TLS. NusA, associated with elongating RNA polymerases, can recruit TLS polymerases to fill in gaps opposite to lesions in the transcribed strand to allow for the continuation of transcription. (B) An alternative class of TCR, NusA-dependent TCR, where NusA participates in a previously unrecognized branch of the TCR pathway. NusA is capable of recruiting NER to sites of stalled RNAPs to repair DNA lesions on the transcribed strand.