Induction of the SOS response increases the efficiency of global nucleotide excision repair of cyclobutane pyrimidine dimers, but not 6-4 photoproducts, in UV-irradiated Escherichia coli

Abstract

Nucleotide excision repair (NER) is responsible for the removal of a variety of lesions from damaged DNA and proceeds through two subpathways, global repair and transcription-coupled repair. In Escherichia coli, both subpathways require UvrA and UvrB, which are induced following DNA damage as part of the SOS response. We found that elimination of the SOS response either genetically or by treatment with the transcription inhibitor rifampin reduced the efficiency of global repair of the major UV-induced lesion, the cyclobutane pyrimidine dimer (CPD), but had no effect on the global repair of 6-4 photoproducts. Mutants in which the SOS response was constitutively derepressed repaired CPDs more rapidly than did wild-type cells, and this rate was not affected by rifampin. Transcription-coupled repair of CPDs occurred in the absence of SOS induction but was undetectable when the response was expressed constitutively. These results suggest that damage-inducible synthesis of UvrA and UvrB is necessary for efficient repair of CPDs and that the levels of these proteins determine the rate of NER of UV photoproducts. We compare our findings with recent data from eukaryotic systems and suggest that damage-inducible stress responses are generally critical for efficient global repair of certain types of genomic damage.

FIG. 1

Rifampin inhibits transcription-coupled repair and reduces the efficiency of repair of CPDs in the nontranscribed strand of the lac operon. Quantitative Southern hybridization of strand-specific RNA probes to the IPTG-induced lac operon was performed on DNA isolated from wild-type strain HL108 not treated (A) or treated with 50 μg of rifampin (RIF) per ml (B). The average number of CPDs was measured in each strand of the 6.6-kb restriction fragment in DNA isolated at the indicated times after UV irradiation with 40 J/m². Each point represents the average repair calculated from three independent experiments. Δ, transcribed strand; ○, nontranscribed strand.

FIG. 2

Rifampin inhibits global repair of CPDs but not 6-4PPs. Monoclonal antibodies specific for CPDs (A) and 6-4PPs (B) were used in an immunoassay with DNA isolated at the indicated times after UV irradiation with 40 J/m². □, HL108 cells; ◊, HL108 cells treated with 50 μg of rifampin per ml. Points represent the average repair calculated from at least two immunoslot blots of samples from each of three independent biological experiments. Each error bar represents 1 standard deviation calculated from the averages of three independent experiments. Error bars not shown are obscured by the datum points.

FIG. 3

Constitutive expression of the SOS response results in rapid repair of CPDs and 6-4PPs and eliminates rifampin inhibition of genomic CPD repair. Monoclonal antibodies specific for CPDs (A) and 6-4PPs (B) were used in an immunoassay with DNA isolated from DM1187 lexA51(Def) cells at the indicated times after UV irradiation with 40 J/m². □, untreated cells; ◊, cells treated with 50 μg of rifampin per ml. Points represent the average repair calculated from at least two immunoslot blots of samples from each of two independent biological experiments. Each error bar represents 1 standard deviation calculated from the averages of two independent experiments. Error bars not shown are obscured by the datum points.

FIG. 4

Constitutive expression of the SOS response leads to rapid repair of CPDs in both strands of the lactose operon regardless of rifampin treatment. Transcription-coupled repair assays were performed on DNA isolated from DM1187 lexA51(Def) cells not treated (A) or treated with 50 μg of rifampin (RIF) per ml (B). For experimental details, see Materials and Methods and the legend to Fig. 1. Points represent the average repair calculated from two independent experiments. Δ, transcribed strand; ○, nontranscribed strand.

FIG. 5

Global repair of CPDs, but not 6-4PPs, is attenuated in cells unable to induce the SOS response. Monoclonal antibodies specific for CPDs (A) and 6-4PPs (B) were used in an immunoassay with DNA isolated from HL942 lexA3(Ind⁻) cells at the indicated times after UV irradiation with 40 J/m². □, untreated cells; ◊, cells treated with 50 μg of rifampin per ml. Points represent the average repair calculated from at least two immunoslot blots of samples from three independent biological experiments. Each error bar represents 1 standard deviation calculated from the averages of three independent experiments. Error bars not shown are obscured by the datum points.

FIG. 6

Cells unable to induce the SOS response perform transcription-coupled repair of CPDs but exhibit a reduced rate of CPD repair in the nontranscribed strand in the presence or absence of rifampin. Transcription-coupled repair assays were performed on DNA isolated from HL942 lexA3(Ind⁻) cells not treated (A) or treated with 50 μg of rifampin (RIF) per ml (B). For experimental details, see Materials and Methods and the legend to Fig. 1. Points represent the average repair calculated from two independent experiments. Δ, transcribed strand; ○ and ⋄, nontranscribed strand.

FIG. 7

Western blot analyses of UvrA protein levels in HL108 lexA⁺ (A), DM1187 lexA51(Def) (B), and HL942 lexA3(Ind⁻) (C) in the presence or absence of rifampin (RIF). Cultures were sampled prior to UV irradiation (No UV) and at the indicated times after UV irradiation with 40 J/m². Cell extracts were prepared as described in Materials and Methods. Known amounts of purified UvrA protein were loaded onto each gel to generate a standard curve for quantitation. The Western blots shown were generated from phosphorimager scans and associated Molecular Analyst software. Associated graphs depict the average levels of UvrA/cell equivalent prior to irradiation and at each time point as determined from at least three independent experiments. These levels were calculated by using the UvrA standard curve and by measuring the amount of DNA in each cell extract to determine approximate cell number (assuming 2.1 chromosomes/cell). □, UV-irradiated cells (no rifampin); ○, UV-irradiated cells treated with rifampin; ◊, unirradiated cells (no rifampin); Δ, unirradiated cells treated with rifampin.

FIG. 8

Western blot analyses of UvrB protein levels in HL108 lexA⁺ (A), DM1187 lexA51(Def) (B), and HL942 lexA3(Ind⁻) (C) in the presence or absence of rifampin (RIF). Experiments were performed as described in the legend to Fig. 7. □, UV-irradiated cells (no rifampin); ○, UV-irradiated cells treated with rifampin; ◊, unirradiated cells (no rifampin); Δ, unirradiated cells treated with rifampin.