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. 2010 May 14;141(4):595-605.
doi: 10.1016/j.cell.2010.03.036.

The transcription factor DksA prevents conflicts between DNA replication and transcription machinery

Ashley K Tehranchi  1 , Matthew D Blankschien, Yan Zhang, Jennifer A Halliday, Anjana Srivatsan, Jia Peng, Christophe Herman, Jue D Wang
Affiliations

The transcription factor DksA prevents conflicts between DNA replication and transcription machinery

Ashley K Tehranchi et al. Cell. .

Abstract

Actively dividing cells perform robust and accurate DNA replication during fluctuating nutrient availability, yet factors that prevent disruption of replication remain largely unknown. Here we report that DksA, a nutrient-responsive transcription factor, ensures replication completion in Escherichia coli by removing transcription roadblocks. In the absence of DksA, replication is rapidly arrested upon amino acid starvation. This arrest requires active transcription and is alleviated by RNA polymerase mutants that compensate for DksA activity. This replication arrest occurs independently of exogenous DNA damage, yet it induces the DNA-damage response and recruits the main recombination protein RecA. This function of DksA is independent of its transcription initiation activity but requires its less-studied transcription elongation activity. Finally, GreA/B elongation factors also prevent replication arrest during nutrient stress. We conclude that transcription elongation factors alleviate fundamental conflicts between replication and transcription, thereby protecting replication fork progression and DNA integrity.

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Figures

Figure 1
Figure 1
(A). Schematic of the experimental strategy for monitoring replication using whole-genome microarrays. Cells were grown at 30°C and shifted to 42°C to inhibit replication initiation and then shifted back to 30°C. 5 min. after replication initiation, cultures were treated with 0.5 mg/ml SHX and collected 20 min. later. Relative DNA levels (log2) were determined by co-hybridization of Cy5-labeled replicating DNA and Cy3-labeled pre-initiation reference DNA (t=0) to microarrays, and plotted as a function of gene positions. Blue ovals: chromosomes at different stages of replication, with the expected gene dosage profiles depicted graphically alongside; black solid ovals: replisomes. (B). Schematic of the theoretical gene dosage distribution of a replicating cell. Blue lines: DNA strands; black ovals: replisomes; x axis: gene position with oriC in the middle, and terC at each end; y axis: gene dosage. (C). The actual microarray profile of untreated dnaC2 cells 25 min. after initiation of replication. x axis: gene position; y axis: ratio of gene dosage in the sample relative to a synchronized pre-initiation reference; yellow window: the transition of gene dosage ratio from 1 to 2, representing the range of positions of the replication forks; arrows: the average positions of the forks.
Figure 2
Figure 2
Replication elongation is inhibited by amino acid starvation in the absence of DksA. (A, C) SHX, but not RHX, inhibits cell growth, as measured by absorbance at 600 nm. Cells were treated with SHX or RHX (0.5 mg/ml) at t=0. (B, D) Overlay of microarray profiles of SHX-treated (red) and untreated (black) dksA+ (B) and ΔdksA (D) cells. Microarray profiles were obtained as described in Fig. 1. (E, F) Wild-type and ΔdksA cells were grown at 37°C and treated with SHX at t=0. The ratio of DNA content relative to t=0 is plotted against time after SHX addition. ΔdksA cells grow slower than the wild-type cells and the increase of DNA content in ΔdksA cells is also slower, because replication initiates slowly to coordinate with slow growth. Black: untreated. Red: SHX-treated. Arrows: addition of SHX. (G). DNA replication was monitored by measuring the rate of incorporation of 3H-thymidine. Cells were grown and treated as in Fig. 2A, C. Results are normalized against t=0. See also Figure S1.
Figure 3
Figure 3
Amino acid starvation in the absence of DksA increases localization of RecA. Control and ΔdksA cells expressing the RecA-GFP fusion protein were treated with SHX as in Fig. 2A, C, and samples were taken for microscopy after 40 min. (A) The percentage of cells with at least one RecA-GFP focus. (B) The number of RecA-GFP foci per cell length (μm). (C to F) Micrographs of dksA+ (C, E) and ΔdksA (D, F) cells during normal growth (C, D) and after SHX-treatment (E, F). In (C), the nucleoid-associated RecA-GFP are indicated with white arrows. Scale bar: 5μm.
Figure 4
Figure 4
ΔdksA cells exhibit induction of SOS response. (A). Cleavage of the SOS repressor LexA. Cells with the indicated genotypes were treated with SHX. At the indicated times after treatment, cells were collected and full length LexA (25 kDa) were detected by Western blotting. (B) Quantification of LexA band intensities of wild-type and ΔdksA cells after SHX treatment. (C). Microarray gene expression analysis reveals strong induction of SOS response in ΔdksA cells 40 min. after SHX-treatment. Relative expression (SHX-treated vs. untreated) of selected members of the SOS regulon are plotted. (D). Untreated ΔdksA cells exhibit mild SOS response, monitored using a GFP-reporter under a SOS-inducible promoter. See also Figure S2.
Figure 5
Figure 5
DksA prevents replication arrest upon amino acid starvation by affecting transcription. (A). Rifampicin (Rif) releases the replication block imposed by starvation. Rif (0.3mg/ml) was added 2 min. before the addition of SHX. Results are normalized against untreated cells. (B). rpoB* (rpoB111) relieves the starvation-induced inhibition of replication elongation in ΔdksA cells. The rate of H3-thy incorporation (normalized against that of untreated cells) is plotted against the time after SHX-addition. (C, D). The total DNA contents in rpoB* (C) and rpoB* ΔdksA cells (D) were plotted as in Fig. 2E, F. Arrow: addition of SHX. Results are normalized against t=0. (E). rpoB* partially relieves UV-sensitivity of a ΔdksA strain in a ΔruvB background. Cells were exposed to indicated doses of UV (J/m2) and counted 48 hours later. (F). Overlay of synchronized replication profiles of W3110 dnaC2 (black) and W3110 dnaC2 ΔrelA (green) cells upon SHX-treatment. Cells were grown in M9 medium supplemented with threonine and synchronized as described in Fig. 1A. 10 min. after replication initiation, cells were treated with SHX and samples were collected 25 min. See also Figure S3.
Figure 6
Figure 6
DksA prevents starvation-induced replication arrest by affecting transcription elongation. (A) Conserved aspartic acid residues of DksA are required for the inhibition of the rrnB P1 promoter activity. The β-galactosidase activity of the rrnB P1-lacZ promoter fusion in the ΔdksA background is plotted against cell density. pDksA, plasmid-borne wild-type DksA; pDksANN, plasmid-borne DksANN; pBA169, control plasmid. Expression of plasmids was induced by 0.1mM IPTG. (B) The rate of H3-thy incorporation in a ΔdksA strain with control (pBA169), wild-type DksA (pDksA) or DksANN (pDksANN) is plotted as a function of time after SHX addition. (C) DksANN significantly rescues the UV sensitivity of ΔdksA ΔruvB cells. Cells were plated on LB/ampicillin/IPTG and tested as described in Fig. 5E. (D). Nucleoid morphology of ΔdksA cells with pBA169, pDksA or pDksANN as revealed by DAPI-staining. (E, F) The average fluorescence intensities (E) and cell lengths (F) of ΔdksA cells with pBA169, pDksA or pDksANN. These strains either have a wild-type lexA (dark bars) or lexA3(Ind−) (light bars). (G) rpoB3595 and rpoB2 relieve the starvation-induced replication block in ΔdksA cells. The rate of thymidine incorporation (treated vs. untreated) 20 min. after SHX-treatment is plotted. (H) The rate of thymidine incorporation in cells with or without GreA/GreB/DksA, 40 min. after treatment with 1.5 mg/ml SHX. Strains were grown at 30°C. (I) The rate of thymidine incorporation in ΔdksA or dksA+ cells with plasmid-borne GreA (pGreA) or control (pBR322) is plotted against time after SHX-addition. Expression of GreA was induced by 0.1mM IPTG. (J) The rate of thymidine incorporation in strains with or without greA, greB and dksA is plotted as a function of time after addition of rifampicin. See also Figure S4.
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