Volume 19, Issue 2, Pages 247-258 (July 2005) RNA Polymerase Modulators and DNA Repair Activities Resolve Conflicts between DNA Replication and Transcription  Brigitte W. Trautinger, Razieh P. Jaktaji, Ekaterina Rusakova, Robert G. Lloyd  Molecular Cell  Volume 19, Issue 2, Pages 247-258 (July 2005) DOI: 10.1016/j.molcel.2005.06.004 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Transcription Complexes Formed by Wild-Type and Mutant Polymerases (A) Complexes formed on λcro +24. Lane 1, DNA substrate; lanes 2 and 9, open complexes; lanes 3–8 and 10–15, time courses showing accumulation of stalled elongation complexes (sEC) and a second complex (sC) at 1, 2, 5, 10, 15, and 20 min with wild-type and RNAP*35 enzymes, as indicated. (B) Loading multiple transcription complexes (mC) on λcro +84 DNA. Reactions were as in (A), except the time courses in lanes 3–8 and 10–15 were at 2, 5, 10, 15, 20, and 40 min, respectively. (C) Quantification of complexes. Free DNA and complexes containing four or more polymerases formed by wild-type RNAP (triangles), RNAP788 (squares), RNAP*35 (diamonds), and RNAP*551 (circles) were quantified in at least three assays. Error bars indicate standard deviations. (D) Formation of RNA transcripts by wt RNAP, RNAP788, and RNAP*35 on the indicated λcro template under nucleotide deprivation (A+U+G) or run-off (A+U+G+C) conditions, as indicated. RNA1, RNA2, RNA3, and RNA5 most likely represent transcripts generated by stable RNAP complexes that have escaped the promoter. RNA4 probably reflects formation of abortive transcripts as the enzymes attempt to escape. Molecular Cell 2005 19, 247-258DOI: (10.1016/j.molcel.2005.06.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 ppGpp Reduces Arrays of RNAP (A) Effect of ppGpp on wild-type RNAP. Lane 1, λcro +84 substrate; lanes 2–6 and 7–11, time courses showing complexes at 2, 5, 10, 15, and 20 min in the absence or presence of ppGpp, respectively. (B) Effect of UV on promoter activity. Reactions were incubated for 3 min without nucleotides to detect open complex formation (lanes 2 and 3) or for 20 min with the three nucleotides indicated to detect elongation complexes stalled at +84 (lanes 5 and 6). Lanes 1 and 4 contained only the DNA substrate. (C) Complexes formed by wild-type RNAP on UV-damaged DNA. Lane 1, irradiated λcro +24; lanes 2–6 and 7–11, time courses of run-off transcriptions at 5, 10, 15, 20, and 40 min in the absence or presence of ppGpp, respectively. (D) Quantification of complexes on UV-irradiated DNA. Transcription assays were as in C, and the amount of free DNA (squares) or DNA with one (circles) or multiple (triangles) stalled complexes was measured in at least three independent assays. Error bars indicate standard deviations. (E) AFM image of complexes formed during run-off transcription on UV-irradiated λ cro +24. Molecular Cell 2005 19, 247-258DOI: (10.1016/j.molcel.2005.06.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Effect of Stringent Mutations on Accumulation of Transcription Arrays (A) Comparison of arrays made by RNAP788, RNAP*35, and RNAP*551. Lane 1, UV-irradiated λcro +24 substrate; lanes 2–5, 6–9, and 10–13, time courses of run-off transcriptions at 1, 2, 5, and 10 min with the indicated enzyme. (B) Quantification of complexes formed on UV-irradiated λcro +24. Free DNA (squares) or DNA with one (circles) or multiple (triangles) complexes was measured in at least three run-off transcription assays. Error bars indicate standard deviations. Molecular Cell 2005 19, 247-258DOI: (10.1016/j.molcel.2005.06.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Effect of ppGpp, RNAP Mutations, and DNA Repair Factors on Survival of UV-Irradiated ruv Mutants (A) Survival of exponential and stationary phase (SP) cells. Strains were AB1157 (wt) and its derivatives N4146, N4155, N4161, N4735, and N4753. (B) Effect of uspA. Strains were MG1655 derivatives N5641, N5643, N5644, and N5647. (C) Effect of lexA3. Strains were MG1655 (wt) and its derivatives N4583, N5120, and N5123 (left) and N4849, N4862, RJ1248, and RJ1270 (right). Note the reduced sensitivity of the ruv single mutant in the MG1655 background relative to the AB1157 background (A), which is true for all ruv alleles. We do not know the reason for the difference. (D) Effect of lexA(Def). Strains were AB1157 derivatives N5217, N5234, and N5259. (E) Effect of recF and recB. Strains were MG1655 and its derivatives N4680, N5540, and N5620 (left) and N4849, N4863, N4864, RJ1251, and RJ1262 (right). Molecular Cell 2005 19, 247-258DOI: (10.1016/j.molcel.2005.06.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Effect of RNAP*35 on SOS Induction (A and C) Time course of induction after irradiation with UV (A) or addition of nalidixic acid (C). Strains were N5170 and N5185. (B, D, and E) SOS induction after exposure to UV light or nalidixic acid. Enzyme levels with or without treatment were measured after 1 hr. Levels in untreated cells are shown in black and in treated cells in gray (rpo wild-type) or cyan (rpo*35). Strains used are identified beneath each panel, with the strain encoding wild-type RNAP on the left and the RNAP*35 strain on the right. Repair genotype is indicated above each panel; wt = lex+ ruv+ rec+ uvr+. Molecular Cell 2005 19, 247-258DOI: (10.1016/j.molcel.2005.06.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Effect of Transcription Modulators on Cell Viability (A) Phase contrast images of ruvC and dksA greA ruvC cells with or without rpo*35 and also of dksA greA cells. Two fields of dksA greA ruvC are shown to represent the range observed. The strains were N5794, N5844, N5848, N5867, and N5881. (B) Sensitivity to mitomycin C. The strains were, from left to right, MG1655, N5753, N5301, N5794, N4849, and N5759. (C) Segregation of plasmid-free cells in cultures of ruv+, ruvC, and dksA greA ruvC strains N5752, N5747, N5831, and N5838, respectively. Plates with similar numbers of well-separated colonies were photographed after 50 hr at 37°C. (D) Sensitivity to UV. Strains were MG1655, N5794, N5848, and N5881 (left), and N4849, N5867, N5796, and N5881 (right). (E) Effect of DksA on survival of ruv strains lacking ppGpp. Strains were N4235, N4304, N4307, and N5892 (left), and N5755, N5784, N5890, and N5893 (right). Molecular Cell 2005 19, 247-258DOI: (10.1016/j.molcel.2005.06.004) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Models of How Transcription Regulators Might Promote Replication (A) With low levels of ppGpp, DksA, GreA, and Mfd activity, replication may stall at an array of transcription complexes backed-up by a lesion in the template. Fork reversal generates a Holliday junction. Subsequent fork rescue occurs via RecBCD nuclease or recombination, the latter being mandatory if RuvABC breaks the fork. Productive replication resumes provided the initial block has been removed. (B) With high levels of activity, RNAP arrays are less prevalent. A fork encounters the lesion, and ssDNA is exposed on which RecA is loaded, inducing SOS repair proteins (RecQ, UvrA, UvrB, UvrD, UmuCD, and DinB). Fork reversal may occur via helicase (RecQ, UvrD) and strand-specific exonuclease (RecJ) activities or by DNA branch migration (RecG), providing many ways to rescue the fork without recombination (Courcelle et al., 2003; McGlynn and Lloyd, 2000, 2002; Rangarajan et al., 2002; Seigneur et al., 1998). If RuvABC is present and breaks the fork, replication then resumes via recombination. Red triangles = noncoding lesions; thick blue lines = new DNA; green ovals = RNAP; cyan ovals = RecA monomers; gray ovals = replication proteins; blue arrowhead = RecBCD enzyme. Molecular Cell 2005 19, 247-258DOI: (10.1016/j.molcel.2005.06.004) Copyright © 2005 Elsevier Inc. Terms and Conditions