Isolation and characterization of SOS constitutive mutations in Escherichia coli.

Abstract

Early events occurring during induction of the SOS response in Escherichia coli are poorly understood. In order to understand the early steps in SOS induction more fully, we have isolated several mutations which constitutively express the SOS regulon. Using a Mud(Apᴿ,lac) fusion to the SOS regulated gene sulA, we isolated Lac⁺ colonies as mutants in which RecA protein is constitutively activated for repressor cleavage. The mutations map to four loci: dam, lig, uvrD and recA. The extent of constitutive SOS induction in these mutants varied greatly, indicating different levels or types of signal in the cell. The mutations isolated demonstrate two early steps in SOS induction. The first step in SOS induction is signal generation and includes mutations found in dam, lig and uvrD genes. The mutant gene products presumably alter DNA metabolism to produce an inducing signal. These non-lethal mutations lead to sub-induction and probably generate very specific signals, such as abnormally unwound DNA in the case of DNA helicase II mutants or unsealed DNA nicks that result from deficient ligation in lig mutants. Greater induction may require quantitatively more signal or different types of signal generated by severe defects leading to cell death. These mutations also show that signal is a variable quantity, allowing the cell to fine tune the levels of SOS repair activity according to the amount or type of signal (damage) perceived. In some cases (such as dam mutations), blocking the SOS response by lexA(Ind⁻) alleles leads to cell death. In this type of constitutively activated strain, the increased level of repair from SOS induction is required to allow the cell to tolerate potentially lethal DNA structures generated by the mutant gene product. The second step in induction is the interaction of signal with RecA protein and is shown by isolating 8 recA mutants. Mutant recA alleles caused the strongest SOS induction in any mutants obtained, similar to the level found in strains lacking repressor (lexA(Def) mutants). This full induction in the absence of lethal DNA damage underscores the pivotal role of RecA protein in regulating the SOS response.