Genetic evidence for the requirement of RecA loading activity in SOS induction after UV irradiation in Escherichia coli

Abstract

The SOS response in Escherichia coli results in the coordinately induced expression of more than 40 genes which occurs when cells are treated with DNA-damaging agents. This response is dependent on RecA (coprotease), LexA (repressor), and the presence of single-stranded DNA (ssDNA). A prerequisite for SOS induction is the formation of a RecA-ssDNA filament. Depending on the DNA substrate, the RecA-ssDNA filament is produced by either RecBCD, RecFOR, or a hybrid recombination mechanism with specific enzyme activities, including helicase, exonuclease, and RecA loading. In this study we examined the role of RecA loading activity in SOS induction after UV irradiation. We performed a genetic analysis of SOS induction in strains with a mutation which eliminates RecA loading activity in the RecBCD enzyme (recB1080 allele). We found that RecA loading activity is essential for SOS induction. In the recB1080 mutant RecQ helicase is not important, whereas RecJ nuclease slightly decreases SOS induction after UV irradiation. In addition, we found that the recB1080 mutant exhibited constitutive expression of the SOS regulon. Surprisingly, this constitutive SOS expression was dependent on the RecJ protein but not on RecFOR, implying that there is a different mechanism of RecA loading for constitutive SOS expression.

FIG. 1.

(A) Induced levels of SOS expression (in β-galactosidase units) for the wt strain (strain IIB386) (▪) and recO (strain IIB392) (⧫), recB1080 (strain IIB388) (▴), and recB268 (strain IIB420) (•) mutants after UV irradiation during a 180-min incubation and basal levels of SOS expression (in β-galactosidase units) for the wt strain (strain IIB386) (□) and recO (strain IIB392) (⋄), recB1080 (strain IIB388) (▵), and recB268 (strain IIB420) (○) mutants during a 180-min incubation. (B) IFs (see Materials and Methods) for the wt strain (strain IIB386) (▪) and recO (strain IIB392) (▴), recB1080 (strain IIB388) (▴), and recB268 (strain IIB420) (•) mutants after UV irradiation during a 180-min incubation. The symbols indicate means of at least three independent experiments, and the error bars indicate standard deviations.

FIG. 2.

(A) Induced levels of SOS expression (in β-galactosidase units) for recB1080 (strain IIB388) (▴), recB1080 recO (strain IIB401) (*), and recO (strain IIB392) (▪) mutants after UV irradiation during a 180-min incubation and basal levels of SOS expression (in β-galactosidase units) for recB1080 (strain IIB388) (▴), recB1080 recO (strain IIB401) (×), and recO (strain IIB392) (□) mutants during a 180-min incubation. (B) Induced levels of SOS expression (in β-galactosidase units) for recD (strain IIB395) (▪), recB1080 recD (strain IIB390) (▴), and recB1080 recD recO (strain IIB393) (•) mutants after UV irradiation during a 180-min incubation and basal levels of SOS expression (in β-galactosidase units) for recD (strain IIB395) (□), recB1080 recD (strain IIB390) (⋄), and recB1080 recD recO (strain IIB393) (○) mutants during a 180-min incubation. (C) IFs (see Materials and Methods) for recO (strain IIB392) (○), recB1080 (strain IIB388) (▪), recB1080 recO (strain IIB401) (▴), recB1080 recD (strain IIB390) (×), recB1080 recD recO (strain IIB393) (*), and recD (strain IIB395) (⋄) mutants after UV irradiation during a 180-min incubation. The symbols indicate means of at least three independent experiments, and the error bars indicate standard deviations.

FIG. 3.

(A) Induced levels of SOS expression (in β-galactosidase units) for recB268 (strain IIB420) (▴) and recB268 recO (strain IIB422) (▪) mutants after UV irradiation during a 180-min incubation and basal levels of SOS expression (in β-galactosidase units) for recB268 (strain IIB420) (▵) and recB268 recO (strain IIB422) (□) mutants during a 180-min incubation. (B) Induced levels of SOS expression (in β-galactosidase units) for recB268 recQ (strain IIB421) (⧫), recB268 (strain IIB420) (▴), and recB268 recJ (strain IIB423) (•) mutants after UV irradiation during a 180-min incubation and basal level of SOS expression (in β-galactosidase units) for recB268 recQ (strain IIB421) (⋄), recB268 (strain IIB420) (▵), and recB268 recJ (strain IIB423) (○) mutants during a 180-min incubation. (C) IFs (see Materials and Methods) for recB268 (strain IIB420) (▪), recB268 recO (strain IIB422) (*), recB268 recJ (strain IIB423) (×), and recB268 recQ (strain IIB421) (▴) mutants after UV irradiation during a 180-min incubation. The symbols indicate means of at least three independent experiments, and the error bars indicate standard deviations.

FIG. 4.

(A) Induced levels of SOS expression (in β-galactosidase units) for recB1080 (strain IIB388) (•), recB1080 recQ (strain IIB408) (▪), and recQ (strain IIB407) (▴) mutants after UV irradiation during a 180-min incubation and basal levels of SOS expression (in β-galactosidase units) for recB1080 (strain IIB388) (○), recB1080 recQ (strain IIB408) (□), and recQ (strain IIB407) (▵) mutants during a 180-min incubation. (B) Induced levels of SOS expression (in β-galactosidase units) for recB1080 (strain IIB388) (▪), recB1080 recJ (strain IIB410) (▴), and recJ (strain IIB409) (•) mutants after UV irradiation during a 180-min incubation and basal levels of SOS expression (in β-galactosidase units) for recB1080 (strain IIB388) (□), recB1080 recJ (strain IIB410) (▵), and recJ (strain IIB409) (○) mutants during a 180-min incubation. (C) IFs (see Materials and Methods) for recB1080 (strain IIB388) (▪), recB1080 recQ (strain IIB408) (•), recB1080 recJ (strain IIB410) (▴), recJ (strain IIB409) (○), and recQ (strain IIB407) (⋄) mutants after UV irradiation during a 180-min incubation. The symbols indicate means of at least three independent experiments, and the error bars indicate standard deviations.

FIG. 5.

(A) Basal levels of SOS expression (in β-galactosidase units) for recB1080 (strain IIB388) (•), wt (strain IIB386) (▪), recO (strain IIB392) (▴), recQ (strain IIB407) (×), recJ (strain IIB409) (*), recB268 (strain IIB420) (○), and recA (strain IIB424) (+) strains during a 180-min incubation. (B) Basal levels of SOS expression (in β-galactosidase units) for recB1080 (strain IIB388) (•), recB1080 recO (strain IIB401) (▪), recB1080 recJ (strain IIB410) (▴), recB1080 recQ (strain IIB408) (×), recD (strain IIB395) (⧫), and recB1080 recD (strain IIB390) (*) mutants during a180-min incubation. The symbols indicate means of at least three independent experiments, and the error bars indicate standard deviations.