Nitric oxide-induced homologous recombination in Escherichia coli is promoted by DNA glycosylases

Abstract

Nitric oxide (NO*) is involved in neurotransmission, inflammation, and many other biological processes. Exposure of cells to NO* leads to DNA damage, including formation of deaminated and oxidized bases. Apurinic/apyrimidinic (AP) endonuclease-deficient cells are sensitive to NO* toxicity, which indicates that base excision repair (BER) intermediates are being generated. Here, we show that AP endonuclease-deficient cells can be protected from NO* toxicity by inactivation of the uracil (Ung) or formamidopyrimidine (Fpg) DNA glycosylases but not by inactivation of a 3-methyladenine (AlkA) DNA glycosylase. These results suggest that Ung and Fpg remove nontoxic NO*-induced base damage to create BER intermediates that are toxic if they are not processed by AP endonucleases. Our next goal was to learn how Ung and Fpg affect susceptibility to homologous recombination. The RecBCD complex is critical for repair of double-strand breaks via homologous recombination. When both Ung and Fpg were inactivated in recBCD cells, survival was significantly enhanced. We infer that both Ung and Fpg create substrates for recombinational repair, which is consistent with the observation that disrupting ung and fpg suppressed NO*-induced recombination. Taken together, a picture emerges in which the action of DNA glycosylases on NO*-induced base damage results in the accumulation of BER intermediates, which in turn can induce homologous recombination. These studies shed light on the underlying mechanism of NO*-induced homologous recombination.

FIG. 1.

Relative NO^. sensitivities of E. coli strains with varied levels of expression of BER genes. Percent survival is shown on the y axis in all panels. (A) Survival of WT (×), xth nfo (▪), ung (○), and xth nfo ung (•) E. coli strains. (B) Survival of WT cells expressing ung from pTr-Ung (▴), xth nfo E. coli carrying control pTr vector (▵), xth nfo ung E. coli expressing ung from pTr-Ung (•), and xth nfo ung E. coli carrying control pTr vector (○). (C) Survival of WT (×), xth nfo (▪), fpg (⋄), and xth nfo fpg (♦) E. coli strains. (D) Survival of WT cells expressing fpg from pSL-Fpg (▪), xth nfo cells carrying control pSL (□), xth nfo fpg cells expressing fpg from pSL-Fpg (♦), and xth nfo fpg cells carrying control pSL (⋄). (E) Relative survival of WT (×), alkA tag (▵), xth nfo (▪), and xth nfo alkA (▴) E. coli strains. Data shown in panels A and C are the averages of three or more experiments. Experiments represented in panels B and D were repeated at least two times. Data shown in panel E are the averages of two or more experiments.

FIG. 2.

Relative NO^. sensitivities of WT E. coli and strains deficient in recombinational repair and DNA glycosylases. All strains were derived from GM7330. The figure shows survival of WT (×), recBCD (▴), recBCD ung (♦), recBCD fpg (•), and recBCD fpg ung (▪) E. coli strains. The data are the averages of at least three independent experiments.

FIG. 3.

Relative NO^. sensitivities of WT E. coli and strains deficient in recombinational repair and translesion DNA synthesis. The figure shows survival of WT (×), recBCD (▴), and recBCD umuDC (•) E. coli strains. The data are the averages of at least three independent experiments.

FIG. 4.

NO^.-induced recombination in WT E. coli and strains deficient in DNA glycosylases. All strains are derived from GM7330. Recombination frequencies (Lac⁺ E. coli per 10⁵ viable cells) were determined for control (open bars) and cells exposed to NO^. for 2 h (gray bars). Recombination frequencies at equitoxic doses are shown (survival is between 20 and 40%). The error bars denote 95% confidence intervals. The asterisk indicates that the difference between GM7330 and the fpg ung double mutant is statistically significant (Student's t test).

FIG. 5.

Model for the promotion of genetic changes upon exposure of cells to NO^.. Genetic changes can be avoided by successful repair of base damage by BER enzymes. However, NO^.-induced base damage can lead to mutations if encountered by the replication fork prior to BER. Recombinational repair is required when repair intermediates are converted into DSBs, possibly during DNA replication.