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Science 338 (6112): 1344-1348

Copyright © 2012 by the American Association for the Advancement of Science

Identity and Function of a Large Gene Network Underlying Mutagenic Repair of DNA Breaks

Abu Amar M. Al Mamun1, Mary-Jane Lombardo1,*,{dagger}, Chandan Shee1,*, Andreas M. Lisewski1, Caleb Gonzalez1,{ddagger}, Dongxu Lin1,§, Ralf B. Nehring1, Claude Saint-Ruf2,||, Janet L. Gibson1, Ryan L. Frisch1, Olivier Lichtarge1,3, P. J. Hastings1, and Susan M. Rosenberg1,3,4,5,

1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030–3411, USA.
2 U1001 INSERM, Université Paris, Descartes, Sorbonne Paris cité, site Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France.
3 Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
4 Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.
5 The Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.


Figure 1
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Fig. 1. (A) Roles of stress responses in mutagenic repair of DNA DSBs by homologous recombination (HR) [reviewed, (7)]. (B) Primary screen for DSB-dependent SIM-deficient mutants. Blue papillae in the white colonies are Lac+ mutant clones formed after prolonged starvation stress (8). (C) Identities of 93 SIM-network genes and results of secondary screens. 1Previously known, found in this screen. 2Previously known, not found in this screen. 3Identified on the basis of genes discovered in this screen. (p), transposon inserted in the promoter (table S12). Superscripts S, E, and SOS indicate decreased {sigma}S activity, {sigma}E activity, and spontaneous SOS induction, respectively (table S7).

 

Figure 2
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Fig. 2. (A to F) Validation of mutants in chromosomal Tet frameshift and Nal base substitution SIM assays. *Significantly SIM-deficient (P values in table S5, two-tailed Student’s t test). Relative mutant frequencies, mutant frequency divided by that of the WT-DSB (I-Sce I–positive) controls assayed in parallel. Means ± SEM (n ≥ 3 experiments each), for this and all figures.

 

Figure 3
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Fig. 3. The stress-induced mutation network. (A) Protein-protein interactions: CytoScape 2.8.3 software, "unweighted force-directed layout" (28), links from STRING 9.0 (12). Proteins that promote {sigma}S, {sigma}E, and SOS activation (Fig. 4), as green, black circle, and red circle, constitute 54% of the network. Downstream of SOS (7), solid red. (B) Coexpression and protein-protein interaction are significantly more clustered than random controls. Gene expression data (13). The 93 SIM genes, (92 x 93)/2 = 4278 pairs, show correlation coefficient distributions (top): bars, entire range; boxes 25th and 75th percentile; red bars, mean. Of 4278 pairs, 3350 show positive correlation coefficient; 928 lie below the zero threshold level. High statistical significance for the strong phenotype (S) genes is increased by addition of moderate (M) and weak (W) (table S3). (Bottom) Significantly more protein-protein interactions for SIM than random genes. Of 4278 pairs, 1320 show positive interaction scores; 2958 pairs do not. P values: sign test of the probability of failure to reject the null hypothesis "number of positively correlated pairs is the same as in the random control." (C) Allocation of network genes upstream of stress responses (data summarized in tables S1 and S7).

 

Figure 4
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Fig. 4. Identification of upstream activators of the {sigma}S, {sigma}E, and SOS stress responses. Results summarized in table S1. (A) Sample of ETC mutants showing decreased {sigma}S activity. See table S8 and fig. S4 for 26 others. (B) No change in transcription from the lac promoter. (C) Mutants enter stationary phase normally (also fig. S5). (D to F) ETC mutants are partially suppressed by up-regulation of {sigma}S via deletion of (D) arcB, (E) arcA, or (F) rssB (table S9 and fig. S7). Ratio of mutation rate (bars) and percent mutation restored relative to wild type (WT). (G to I) {triangleup}rpoS is epistatic to ETC mutations in SIM. Double-mutant analyses without (G) or with (H and I) I-Sce I–induced DSBs, showing action in the same pathway. (J) Sample of SIM genes upstream of {sigma}E activity [β-galactosidase (β-gal) expression from a {sigma}E-regulated promoter, fig. S9, and table S11 for the rest]. (K) Spontaneous SOS induction (21) is reduced in recB, recC, pgi, and uvrY mutants (P = 0.00013, 0.017, 0.0013, and 0.00011, two-tailed Student’s t test). (L) Model: ETC-mediated stress-sensing from starvation to mutation. Described in supplementary text S4. Products of genes identified in screens are in red.

 


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