Article Figures & Data
Figures
- FIG. 1.
Distributions of cells with different levels of constitutive SOS expression (detected as GFP fluorescence) expressed as the percentage of cells in the population. The graphs truncate the percentage of cells at 16%. The strains are in order from top of the graph to the bottom, with the relevant part of the genotype in parentheses. All strains were grown in minimal medium at 37°C with aeration. All strains except SS996 have additionally the genotype recAo1403 recA4142. The other relevant genotypes are shown in parentheses, and full genotypes are shown in Table 1. The strains are SS996 (wild type, including recA+), SS4796 (wild type), SS7413 (sbcC), SS7162 (ruvC), SS7143 (recG), SS7174 (recQ), SS7160 (ruvAB), SS7161 (sbcB or xonA), SS4696 (recF), SS6023 (recBCD), SS5305 (recJ), SS5312 (recX), and SS7184 (recX recJ).
- FIG. 2.
The three strains used in this figure have the same background: SS7160 (recAo1403 recA4142 ruvAB6203::tet) with the three different plasmids as indicated. Panel A is the same as Fig. 1. Panel B shows a UV survival curve of the three strains. Strains were grown in minimal media, UV irradiated at a rate of 0.5 J m2/s, and then serially diluted on to minimal plates. Plates were counted after 36 to 48 h of incubation at 37°C.
- FIG. 3.
Same as Fig. 1. The strains are SS7404 (recBCD sbcBC), SS7423 (recBCD sbcBC ruvAB), SS7421 (recBCD sbcBC recF), SS7413 (sbcC), SS7198 (sbcC sbcB), SS7419 (sbcC recF), SS7420 (sbcC recJ), SS7417 (sbcC recBCD), and SS7418 (sbcC ruvAB). All strains also have the genotype recAo1403 recA4142.
- FIG. 4.
Model for how RecA4142 can interact with a reversed replication fork with the help of RuvAB, RecJ, SbcB, and RecBCD. The red and blue strands of DNA show the nascent lagging and leading strands of DNA, respectively. The black strands are the parental DNA. The image suggests that RuvAB binds to a stopped fork and reverses it (annealing of the nascent leading and lagging strands [red and blue]). These ends are then processed by the exonuclease activities of RecJ (5′→3′) and SbcB (XonA) (3′→5′). RecBCD can then load onto the DNA, and, depending if there is a Chi site, it will either just degrade newly annealed strands, resetting the fork and allowing for the replication restart proteins to reload the fork, or it can the load RecA4142. Presumably once RecA4142 is loaded, it can start to interact with LexA and help accelerate its auto-cleavage. Since RecA4142 is recombination proficient, it could strand invade the homologous duplex, creating a structure that again is a substrate for the replication restart proteins. If so, RecA4142 may stably persist on the dsDNA and continue to accelerate LexA cleavage until removed.
Tables
- TABLE 1.
Strains used in this work
Strain recA recBCD Other relevant genotype Source or reference CAG18642 + + zfj-3131::Tn10 41 JC18923 + + recJ284::Tn10 Lab stock JJC296 + + ruvA60::tet B. Michel JJC754 + + ruvABC::cat B. Michel JJC783 + + ruvC::cat B. Michel KM78 + cat K. Murphy SMR839 + + xonA::cat S. Rosenberg TP538 + + recG6200::tet T. Poteete TP540 + + ruvAB6203::tet T. Poteete TP640 + + recQ6218::tet T. Poteete SS996a + + 31 SS4639 730 + recF4115 tnaA::miniTn5 cam 28 SS4696 4142 b + recF4115 tnaA300::Tn10 28 SS4976 4142 + 28 SS5179 + + del(sbcC)100::kan 2 SS5303 4142 + recX::cat g 28 SS5305 4142 + recJ264::Tn10 JC18923→SS4976 SS5312 4142 + recX::cat f 28 SS6021 + cat KM78→SS996d SS6023 4142 cat 28 SS6156 4142 + zfj-3131::Tn10 CAG18642→SS4976c SS7143 4142 + recG6200::tet TP538→SS4976c SS7144 4142 + ruvABC::cat JJC754→SS4976d SS7160 4142 + ruvAB6203::tet TP540→SS4976c SS7161 4142 + xonA::cat SMR839→SS4976d SS7162 4142 + ruvC::cat JJC783→SS4976d SS7165 + + ruvC::cat JJC783→SS996d SS7174 4142 + recQ6218::tet TP640→SS4976c SS7178 4142 + del(sbcC)100::kan SS5179→SS996a SS7184 4142 + recX::cat recJ284::Tn10 SS5303→SS5305d SS7186 + + xonA::cat SMR839→SS996d SS7187 4142 + ruvA60::tet JJC296→SS4976c SS7188 + + ruvA60::tet JJC296→SS996c SS7189 4142 + ruvAB6203::tet pRuvAB→SS7160 SS7190 4142 + ruvAB6203::tet pGB2→SS7160 SS7191 4142 + ruvAB6203::tet pRuvAB H198→SS7160 SS7194 + + recJ284::Tn10 JC18923→SS996c SS7195 + + recQ6218::tet TP640→SS996c SS7196 + + ruvAB6203::tet TP540→SS996c SS7197 + + del(sbcB)200::frt del(sbcC)200::frt SS5162→SS7178e SS7198 4142 + del(sbcB)200::frt del(sbcC)200::frt SS4976→SS7197b SS7404 4142 cat del(sbcB)200::frt del(sbcC)200::frt KM78→SS7198d SS7411 + + del(sbcC)100::kan SS5179→SS996b SS7413 4142 + del(sbcC)200::frt SS4976→SS7411b SS7417 4142 cat del(sbcC)200::frt KM78→SS7413d SS7418 4142 cat ruvAB6203::tet del(sbcC)200::frt TP540→SS7413c SS7419 4142 + del(sbcC)200::frt recF4115 tnaA300::Tn10 SS5394→SS7413b SS7420 4142 + recJ284::Tn10 del(sbcC)200::frt JC18923→SS7413c SS7421 4142 cat recF4115 tnaA300::Tn10 SS5394→SS7404b SS7423 4142 cat ruvAB6203::tet TP540→SS7404c SS7424 + + del(sbcD)100::kan SS5180→SS996a SS7425 4142 + del(sbcD)100::kan zfj-3131::Tn10 SS6156→SS7424c SS7429 4142 + xonA::cat del(sbcD)100::kan zfj-3131::Tn10 SMR839→SS7425d SS7430 4142 cat del(sbcD)100::kan zfj-3131::Tn10 KM78→SS7425d SS7431 4142 + del(sbcD)100::kan zfj-3131::Tn10 recF4115 tnaA::miniTn5 cam SS4639→SS7425d SS7432 4142 + ruvABC::cat del(sbcD)100::kan zfj-3131::Tn10 JJC754→SS7425d SS7433 4142 + del(sbcD)100::kan zfj-3131::Tn10 recJ284::Tn10(cat) SS4907→SS7425d ↵ a The genotype for this strain is sulB103Δattλ::sulApΩgfp-mut2 lacMS286 φ80dIIlacBK1 argE3 hi-4 thi-1 xyl-5 mtl-1 rpsL31 tsx. The lacMS286 φ80dIIlacBK1 genes code for two partial nonoverlapping deletions of the lac operon (21, 46), and Δattλ::sulApΩgfp-mut2 is the sulAp-gfp reporter gene inserted into the attλ site (31). All recA4142 strains used in this work also are ygaD1::kan recAo1403 (28).
↵ b Select for Kanr and then screen for other marker phenotypically or by PCR.
↵ c Select for Tetr and then screen for other marker phenotypically or by PCR.
↵ d Select for Catr and then screen for other marker phenotypically or by PCR.
↵ e This deletion allele was created by first transducing the Kanr allele from the Kieo collection into the strain as indicated in the reference column. pLH29, carrying the flp gene, was then introduced, and Kan-sensitive derivatives were screened (20).
↵ f The Tn10 tetA::cat insertion deletion mutation was amplified with prSJS690,691 using pACYC184 as a template. The Tn10 tetA::cat insertion deletion mutation was transferred to the chromosome by the exo-bet method (13) into a strain containing recJ284::Tn10. This original combination of mutants was named and saved as the strain indicated as the donor in this cross.
↵ g The full notation for the recX mutation is del(recX)4166::cat. The full notation for the recBCD mutation is del(recBCD)::cat. The full notation for the Ωgfp mutation is Δattλ::sulApΩgfp-mut2.
