Journal: Nucleic Acids Research

Article Title: Division of labor between SOS and PafBC in mycobacterial DNA repair and mutagenesis

doi: 10.1093/nar/gkab1169

Figure Lengend Snippet: Gene- and clastogen-specific requirements for PafBC and LexA in the transcriptional DDR. Normalized adnA mRNA measured by RT-qPCR in the indicated strains of M. smegmatis (black = WT, orange = Δ recA , blue = Δ pafBC , purple = lexA-S167A and red = Δ pafBC / lexA-S167A ) after exposure to (A) UV (20 mJ/cm 2 ) ( n = 3 biological replicates), (B) ciprofloxacin (0.5 μg/ml) ( n = 5 biological replicates) or (C) MMC (80 ng/ml) ( n = 3 biological replicates). All values are normalized to WT untreated at 1. (D) Genetic complementation. Normalized adnA mRNA measured by RT-qPCR in M. smegmatis Δ pafBC complemented with M. tuberculosis pafBC , M. smegmatis pafBC or empty vector (EV) after exposure to UV (20 mJ/cm 2 ) ( n = 2 biological replicates). All values are normalized to M. smegmatis Δ pafBC complemented with M. smegmatis pafBC . Normalized dnaE2 mRNA measured by RT-qPCR in the same M. smegmatis strains as in panels (A)–(C) after exposure to (E) UV (20 mJ/cm 2 ) ( n = 3 biological replicates), (F) ciprofloxacin (0.5 μg/ml) ( n = 5 biological replicates) or (G) MMC (80 ng/ml) ( n = 3 biological replicates). All values are normalized to WT untreated at 1. (H) Normalized dnaE2 mRNA in M. smegmatis Δ pafBC strains complemented with M. tuberculosis pafBC, M. smegmatis pafBC or empty vector (EV) (same strain legend as in panel D) after exposure to UV (20 mJ/cm 2 ) ( n = 2 biological replicates). All values are normalized to M. smegmatis Δ pafBC complemented with M. smegmatis pafBC untreated at 1. Normalized mRNA measured by RT-qPCR for (I) adnA or (J) dnaE2 in M. tuberculosis H37Rv, H37Rv pafC :: tn and pafC :: tn + pafC . All values are normalized to WT untreated at 1. Significance is calculated as * P < 0.05 or ** P < 0.01 using two-way ANOVA compared to the WT strain (mc 2 155 or H37Rv) at a comparable time point/condition. ‡ P < 0.05 using two-way ANOVA compared to lexA-S167A strain at a comparable time point/condition. Error bars are SEM.

Article Snippet: The RT-qPCR reaction was made for a TaqMan assay using Thermo Scientific DyNAmo Flash Probe qPCR Kit (10 μl of master mix, 0.1 μl of each primer, 0.05 μl of each probe, 5 μl of cDNA sample and 4.5 μl of deionized H 2 O per reaction) and analyzed using the Applied Biosystems 7500 Real-Time System (cycling conditions: 95°C for 7 min, 45 cycles of 95°C for 5 s and 60°C for 30 s).

Techniques: Quantitative RT-PCR, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Division of labor between SOS and PafBC in mycobacterial DNA repair and mutagenesis

doi: 10.1093/nar/gkab1169

Figure Lengend Snippet: Mycobacterial mutagenesis requires both SOS and PafBC. (A) Frequency of rifampin-resistant mutants per 10 8 cells in M. smegmatis mc 2 155, Δ recA , Δ pafBC , lexA-S167A and Δ pafBC / lexA-S167A with or without DNA damage (20 mJ/cm 2 UV) ( n = 4 biological replicates). # indicates that no Rif R colonies were recovered. ( B ) Frequency of rifampin-resistant mutants per 10 8 cells in WT M. smegmatis , Δ pafBC or Δ pafBC complemented with M. smegmatis pafBC after exposure to UV (20 mJ/cm 2 ) ( n = 2 biological replicates). ( C ) pafBC is required for mutagenesis in M. tuberculosis . Frequency of rifampin resistance per 10 8 cells in M. tuberculosis strains of H37Rv, transposon insertion mutant of pafC ( pafC TN) and transposon insertion mutant of pafC complemented with pafC ( pafC comp) with or without DNA damage (20 mJ/cm 2 UV) ( n = 6 biological replicates). ( D ) Normalized imuB mRNA measured by RT-qPCR in the indicated strains of M. smegmatis after exposure to either UV (20 mJ/cm 2 ) ( n = 3 biological replicates) or ciprofloxacin (0.5 μg/ml) ( n = 3 biological replicates). All values are normalized to WT untreated at 1. ( E ) Normalized imuB mRNA measured by RT-qPCR in the indicated strains of M. tuberculosis after exposure to either UV (20 mJ/cm 2 ) ( n = 4 biological replicates) or ciprofloxacin (0.5 μg/ml) ( n = 4 biological replicates). All values are normalized to WT untreated at 1. Significance is calculated as * P < 0.05 using two-way ANOVA compared to WT untreated. Error bars are SEM.

Article Snippet: The RT-qPCR reaction was made for a TaqMan assay using Thermo Scientific DyNAmo Flash Probe qPCR Kit (10 μl of master mix, 0.1 μl of each primer, 0.05 μl of each probe, 5 μl of cDNA sample and 4.5 μl of deionized H 2 O per reaction) and analyzed using the Applied Biosystems 7500 Real-Time System (cycling conditions: 95°C for 7 min, 45 cycles of 95°C for 5 s and 60°C for 30 s).

Techniques: Mutagenesis, Quantitative RT-PCR