Escherichia coli Fpg protein and UvrABC endonuclease repair DNA damage induced by methylene blue plus visible light in vivo and in vitro

Abstract

pBR322 plasmid DNA was treated with methylene blue plus visible light (MB-light) and tested for transformation efficiency in Escherichia coli mutants defective in either formamidopyrimidine-DNA glycosylase (Fpg protein) and/or UvrABC endonuclease. The survival of pBR322 DNA treated with MB-light was not significantly reduced when transformed into either fpg-1 or uvrA single mutants compared with that in the wild-type strain. In contrast, the survival of MB-light-treated pBR322 DNA was greatly reduced in the fpg-1 uvrA double mutant. The synergistic effect of these two mutations was not observed in transformation experiments using pBR322 DNA treated with methyl methanesulfonate, UV light at 254 nm, or ionizing radiation. In vitro experiments showed that MB-light-treated pBR322 DNA is a substrate for the Fpg protein and UvrABC endonuclease. The number of sites sensitive to cleavage by either Fpg protein or UvrABC endonuclease was 10-fold greater than the number of apurinic-apyrimidinic sites indicated as Nfo protein (endonuclease IV)-sensitive sites. Seven Fpg protein-sensitive sites per pBR322 molecule were required to produce a lethal hit when transformed into the uvrA fpg-1 mutant. These results suggest that MB-light induces DNA base modifications which are lethal and that these modifications are repaired by Fpg protein and UvrABC endonuclease in vivo and in vitro. Therefore, one of the physiological functions of Fpg protein might be to repair DNA base damage induced by photosensitizers and light.The Fpg protein of Escherichia coli was initially identified as a DNA glycosylase which excised the imidazole ringopened form of N7-methylguanine (2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine or Fapy) residues in DNA (2, 9, 13). This enzyme was shown also to liberate the imidazole ring-opened form of adenine residues from DNA treated with ionizing radiation (10) and of guanine residues modified at the N-7 position with bulky alkylating agents (12). The molecular cloning of the fpg+ gene of E. coli (8), the purification of the Fpg protein (9), and the isolation of a bacterial mutant defective in Fpg protein (4) were critical steps for studies of the biological significance of this protein.The Fpg protein exhibits a large substrate specificity, including imidazole ring-opened purines modified at the C-8 position by N-hydroxy-2-aminofluorene (3). The Fpg protein is a metalloprotein containing one zinc atom per monomer (9) and is endowed with a nicking activity which incises DNA at apurinic-apyrimidinic (AP) sites (38). The biological importance of this enzyme is suggested by the fact that Fapy-DNA glycosylase activities have been conserved in prokaryotes (7) and eukaryotes (29,33) and that the imidazole ringopened form of N7-methylguanine residues in DNA is a potentially lethal lesion (6, 37). However, the lack of sensitivity of the E. coli mutant defective in Fpg protein to methylating agents suggested that imidazole ring-opened N7-methylguanine is not a biological substrate and/or that these residues are excised ...

Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases

“…Fpg primarily removes oxidized purines, whereas Nei and Nth primarily remove oxidized pyrimidines. We generated a substrate for Fpg by treating λ DNA with methylene blue (MB) plus visible light (24)(25)(26). Treatment of DNA with MB results not only in the formation of 8-oxoG but primarily its further oxidation products such as spiroiminodihydantoin (27,28), which are also recognized by Fpg (29).…”

“…Single-molecule experiments were performed using λ DNA (New England BioLabs). λ DNA containing Tg or oxidized purine was generated by exposure to osmium tetroxide (OsO 4 ) plus heat and MB plus visible light, respectively, as previously described (24)(25)(26)(30)(31)(32). Briefly, 100 μg/mL λ DNA was treated with 0.2 and 1 μg/mL MB and then illuminated for 60 min with one 100-W lamp (Reveal; 100 W; 1,352 Lumens; A-19 Shape; General Electric) that was placed at a distance of 20 cm from the surface of a thinly coated 10-cm plate containing the DNA solution placed on ice.…”

8-Oxo-7,8-Dihydroguanine Is Removed by a Nucleotide Excision Repair-Like Mechanism in Porphyromonas gingivalis W83

“…Guanines are especially susceptible to modification by the reactive oxygen species, yielding mainly 8-oxoguanine, which results in GC-to-TA transversion mutagenesis (9). Several reports have indicated that the primary repair of 8-oxoG is performed by BER (6,10,35,40). Because oxidative stress represents a significant challenge to anaerobes such as P. gin- givalis, it is imperative that this organism develop mechanisms of oxidative stress resistance that will allow for persistence in the inflammatory condition of periodontitis.…”

Important role of the nucleotide excision repair pathway in Mycobacterium smegmatis in conferring protection against commonly encountered DNA-damaging agents

“…The other strains showed no visible growth defect even at the higher dose of 60 J m 22 . Based on earlier studies in other organisms (Czeczot et al, 1991;Scott et al, 1999;Huang et al, 2006), it was not unexpected that the deficiency of Ung and Fpg did not result in any increased sensitivity of M. smegmatis to UV C radiation. However, these observations highlight that the NER pathway provides the major means to protect M. smegmatis against DNA damage caused by UV radiation.…”