Efficient repair of hydrogen peroxide-induced DNA damage by Escherichia coli requires SOS induction of RecA and RuvA proteins

doi:10.1016/S0921-8777(99)00073-7

Mutation Research/DNA Repair

Volume 459, Issue 3, 28 April 2000, Pages 187-194

Mutation Research/DN...

https://doi.org/10.1016/S0921-8777(99)00073-7 Get rights and content

Abstract

The survival of Escherichia coli following treatment with a low dose (1–3 mM) of hydrogen peroxide (H₂O₂) that causes extensive mode-one killing of DNA repair mutants is stimulated by the induction of the SOS regulon. Results for various mutants indicate that induction of recA and RecA protein-mediated recombination are critical factors contributing to the repair of H₂O₂-induced oxidative DNA damage. However, because DNA damage activates RecA protein's coprotease activity essential to cleavage of LexA repressor protein and derepression of all SOS genes, it is unclear to what extent induction of RecA protein stimulates this repair. To make this determination, we examined mode-one killing of ΔrecA cells carrying plasmid-borne recA (P_tac-recA⁺) and constitutively expressing a fully induced level of wild-type RecA protein when SOS genes other than recA are non-inducible in a lexA3 (Ind⁻) genetic background or inducible in a lexA⁺ background. At a H₂O₂ dose resulting in maximal killing, ΔrecA lexA3 (Ind⁻) cells with P_tac-recA⁺ show 40-fold greater survival than lexA3 (Ind⁻) cells with chromosomal recA having a low, non-induced level of RecA protein. However, they still show 10- to 15-fold lower survival than wild-type cells and ΔrecA lexA⁺ cells with P_tac-recA⁺. To determine if the inducible RuvA protein stimulates survival, we examined a ruvA60 mutant that is defective for the repair of UV-induced DNA damage. This mutant also shows 10- to 15-fold lower survival than wild-type cells. We conclude that while induction of RecA protein has a pronounced stimulatory effect on the recombinational repair of H₂O₂-induced oxidative DNA damage, the induction of other SOS proteins such as RuvA is essential for wild-type repair.

Introduction

Normal aerobic metabolism results in the formation of reactive oxygen species that can damage cellular lipids, proteins and DNA. Among the oxidative DNA lesions that occur are damaged bases, damaged sugar groups, abasic sites and strand breaks (reviewed in Refs. 1, 2, and references therein). Some of these lesions are refractory or miscoding with regard to replicative DNA synthesis and therefore they can result in mutagenesis and lethality. Organisms have evolved highly efficient enzymatic mechanisms that function in the repair of oxidative DNA damage. For example, damaged bases are repaired through a process of base excision repair (BER) while strand breaks are repaired by enzymes involved with homologous genetic recombination.

In Escherichia coli, DNA damage invokes recombinational repair because this damage, together with processes such as DNA replication, produces single-stranded DNA (ssDNA) to which RecA protein binds and forms a recombination-proficient RecA-ATP-ssDNA ternary complex 3, 4, 5, 6, 7. Biochemical data for purified mutant RecA proteins indicate that defects in RecA protein's ability to bind ssDNA and/or metabolize ATP are the cause of defective recombinational repair in vivo 6, 7, 8, 9. That RecA-mediated recombinational repair is relevant to the repair of oxidative DNA damage is evidenced by the findings that hydrogen peroxide (H₂O₂) causes DNA strand breaks and recA mutants are very sensitive to killing by H₂O₂ 10, 11, 12, 13, 14, 15, 16.

In addition to its role in recombinational repair, a RecA-ATP-ssDNA ternary complex is activated for cleavage of LexA repressor (referred to as RecA's coprotease activity; Ref. [17]). Coproteolytic inactivation of LexA repressor results in the induction of the SOS regulon comprised of at least 27 genes including recA, many of which are relevant to various DNA repair and DNA damage tolerance processes 18, 19. A low concentration of H₂O₂ (1–3 mM) results in SOS gene induction in wild-type cells 14, 20and extensive killing of polA, recA and xthA mutants that are defective for DNA repair (referred to as mode-one killing; Refs. 13, 14). In addition, cells with a lexA3 (Ind⁻) mutation, which renders LexA resistant to cleavage and prevents SOS gene induction [21], are very sensitive to mode-one killing by H₂O₂ [14]. Mutations of LexA-regulated genes such as uvrA or uvrB that inactivate nucleotide excision repair (NER), or umuD or umuC that inactivate SOS mutagenesis and DNA damage tolerance, or various din genes, have little effect on mode-one killing by H₂O₂ [14]. However, mutation of the LexA-regulated recN gene and a lack of RecN protein function 22, 23, 24renders cells about five-fold more sensitive to mode-one killing than wild-type cells [14]. In addition, mutational inactivation of the inducible DNA polymerase II (Pol II) protein 25, 26results in similar sensitivity [27]. Therefore, it appears that the induction of RecA, RecN and Pol II proteins is of particular relevance to the repair and/or tolerance of oxidative DNA damage.

In the present study, we have sought to determine the extent to which the induction of recA and the resultant increase in RecA protein level stimulates the repair of oxidative DNA damage. To do so, we examined H₂O₂-induced mode-one killing of ΔrecA lexA3 (Ind⁻) cells carrying plasmid-borne recA and constitutively expressing a high steady-state level of RecA protein when other SOS proteins are at low repressed levels. The high level of survival for these cells indicates that induction of recA alone has a remarkably potent stimulatory effect on the repair of oxidative DNA damage. Nevertheless, the induction of additional SOS genes by wild-type cells allows 10- to 15-fold greater survival indicating that the induction of recN and polB, and perhaps other SOS genes, contributes to this repair. Since the ruvAB operon is induced as part of the SOS response 28, 29, 30and a ruvA60 mutation renders cells sensitive to UV radiation [31], we examined the effects of this mutation on mode-one killing by H₂O₂. The sensitivity of these cells indicates that induction of ruvA and increased capacity for RuvA protein stimulation of branch migration increases the efficiency of oxidative DNA damage repair.

Section snippets

Bacterial strains, plasmids and growth conditions

The E. coli K-12 derivative bacterial strains used in this study are shown in Table 1. Bacteria were grown at 37°C using LB broth or LB agar (both from Bio-101, Vista, CA) supplemented with 100 μg/ml ampicillin, 50 μg/ml kanamycin, 20 μg/ml chloramphenicol or 10 μg/ml tetracycline, where appropriate. Strains GW2771 [32], GW2749, GW8505 and GW8506 were kindly provided by G. Walker (MIT) and DB1318 [33]was supplied by M. Volkert (University of Massachusetts Medical School, Worcester, MA).

Effects of SOS gene induction and RecA protein level on mode-one killing by H₂O₂

Low concentrations of H₂O₂ cause extensive mode-one killing of recA mutants lacking recombination function and lexA3 (Ind⁻) mutants lacking SOS gene induction. This effect can be attributed to the decreased ability of these mutants to repair H₂O₂-induced DNA damage 13, 14. To assess the extent to which recA induction alone can protect cells from mode-one killing, we compared the survival of ΔrecA lexA3 (Ind⁻) cells carrying plasmid-borne P_tac-recA⁺ and constitutively expressing a high level of

Discussion

E. coli lexA3 (Ind⁻) mutants defective for SOS gene induction are highly susceptible to mode-one killing by H₂O₂ (Ref. [14], this study). Given that the extent of mode-one killing correlates with DNA repair proficiency, this finding implies that the SOS response provides substantial protection against oxidative DNA damage. Among the various physiological responses that make up the SOS response, recombinational repair appears to have the greatest protective effect in that recA mutants lacking

Acknowledgements

We are grateful to G. Walker and M. Volkert for supplying bacterial strains and M. Marinus for providing P1 vir stock used in strain construction. This study was supported by grant CA68290 from the National Institutes of Health.

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Efficient repair of hydrogen peroxide-induced DNA damage by Escherichia coli requires SOS induction of RecA and RuvA proteins

Abstract

Introduction

Section snippets

Bacterial strains, plasmids and growth conditions

Effects of SOS gene induction and RecA protein level on mode-one killing by H2O2

Discussion

Acknowledgements

J. Mol. Biol.

Prog. Nucleic Acid Res. Mol. Biol.

Gene

J. Mol. Biol.

J. Mol. Biol.

Gene

Cell

Oxidative stress responses in Escherichia coli and Salmonella typhimurium

Microbiol. Rev.

Repair of oxidative damage to DNA: enzymology and biology

Annu. Rev. Biochem.

recA-Dependent DNA repair processes

BioEssays

Enzymes and molecular mechanisms of genetic recombination

Annu. Rev. Biochem.

Homologous pairing and DNA strand-exchange proteins

Annu. Rev. Biochem.

The RecA protein: structure and function

Crit. Rev. Biochem. Mol. Biol.

Biochemistry of genetic recombination: energetics and mechanism of DNA strand exchange

Annu. Rev. Biophys. Biophys. Chem.

Repair of hydrogen peroxide-induced single-strand breaks in Escherichia coli deoxyribonucleic acid

J. Bacteriol.

The recA+ gene product is more important than catalase and superoxide dismutase in protecting Escherichia coli against hydrogen peroxide toxicity

J. Bacteriol.

Repair response of Escherichia coli to hydrogen peroxide DNA damage

J. Bacteriol.

Bimodal pattern of killing of DNA-repair-defective or anoxically grown Escherichia coli by hydrogen peroxide

J. Bacteriol.

Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide

J. Bacteriol.

Synergistic killing of Escherichia coli K-12 by UV (254 nm) and H2O2

Int. J. Radiat. Biol.

Effects of metal ion chelators on DNA strand breaks and inactivation produced by hydrogen peroxide in Escherichia coli: detection of iron-independent lesions

J. Bacteriol.

Mechanism of specific LexA cleavage: autodigestion and the role of RecA coprotease

Biochimie

Effects of SOS gene induction and RecA protein level on mode-one killing by H₂O₂

The recA⁺ gene product is more important than catalase and superoxide dismutase in protecting Escherichia coli against hydrogen peroxide toxicity

Synergistic killing of Escherichia coli K-12 by UV (254 nm) and H₂O₂