Photochemical cross‐linking of Escherichia coli Fpg protein to DNA duplexes containing phenyl(trifluoromethyl)diazirine groups

Abstract

Formamidopyrimidine-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that excises oxidized purine bases, most notably the mutagenic 7-hydro-8-oxoguanine, from damaged DNA. In order to identify specific contacts between nucleobases of DNA and amino acids from the E. coli Fpg protein, photochemical crosslinking was employed using new reactive DNA duplexes containing 5-[4-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]-2¢-deoxyuridine dU* residues near the 7-hydro-8-oxoguanosine (oxoG) lesion. The Fpg protein was found to bind specifically and tightly to the modified DNA duplexes and to incise them. The nicking efficiency of the DNA duplex containing a dU* residue 5¢ to the oxoG was higher as compared to oxidized native DNA. The conditions for the photochemical cross-linking of the reactive DNA duplexes and the Fpg protein have been optimized to yield as high as 10% of the cross-linked product. Our results suggest that the Fpg protein forms contacts with two nucleosides, one 5¢ adjacent to oxoG and the other 5¢ adjacent to the cytidine residue pairing with oxoG in the other strand. The approaches developed may be applicable to pro-and eukaryotic homologues of the E. coli Fpg protein as well as to other repair enzymes.Keywords: formamidopyrimidine-DNA glycosylase; modified DNA duplexes; 7-hydro-8-oxoguanosine;Derivatives of nucleic acids containing photolabile carbene-generating aryl(trifluoromethyl)diazirine groups are conveniently used to identify specific nucleic acidAEnucleic acid and nucleic acidAEprotein interactions [1][2][3][4][5]. These derivatives have a number of essential merits. First, they produce highly reactive carbene, which breaks even aliphatic C-H bonds. Second, the lifetime of carbene is on a nanosecond timescale. Third, photolysis proceeds at a relatively high light wavelength (350-360 nm) that does not cause damage to biological molecules. Finally, these derivatives may be handled under moderate laboratory illumination. These reagents have been successfully employed to investigate RNAAERNA and RNAAEprotein interactions in ribosomes [1], and to ascertain 2 specific contacts between DNA and some DNA-recognizing proteins, such as the restriction-modification enzymes Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg protein) is a DNA repair enzyme that catalyzes the removal of oxidized purine bases from damaged DNA and cleaves the DNA strand [6]. 7-Hydro-8-oxoguanine is the major mutagenic base produced in DNA by reactive oxygen species that are generated by cellular metabolism, cell injury and exposure to physical and chemical oxygen radical-forming agents [7]. It is a miscoding lesion because it pairs preferentially with adenine rather than cytosine and induces GC fi TA transversions in vivo and in vitro [8]. The physiological function of the Fpg protein is to prevent the mutagenic action 3 of oxoG residues in DNA and to maintain genetic integrity. Three-dimensional structures of the complexes formed by Lactococcus lactis, Bacillus stearothermophilus and E. coli Fpg...

Theoretical Characterization of the H-Bonding and Stacking Potential of Two Nonstandard Nucleobases Expanding the Genetic Alphabet

“…The first two functional groups that we studied were the cyano (−CN) and trimethyl fluoride (−CF 3 ) groups, resulting in the 5-CN-Z:P and 5-CF 3 -Z:P base pairs. The cyano and trifluoromethyl functional groups exhibit electronic properties similar to those of the nitro group (i.e., strongly electron-withdrawing) and have been extensively studied for different applications in the context of nucleic acid structures. − As expected, the geometries of the 5-CN-Z:P and 5-CF 3 -Z:P base pairs are very similar to that of the Z:P base pair, with H-bond differences within 0.04 Å. In terms of the interaction energies, the 5-CN-Z:P and 5-CF 3 -Z:P base pairs have comparable stabilities, with E mod values of −0.21 and +0.21 kcal/mol, respectively, in the gas phase and +0.14 and +0.25 kcal/mol, respectively, in water, as compared to the Z:P base pair.…”

“…Furthermore, to gain a better understanding of the dependence of the H-bonding capability of the Z:P base pair on the nature of the substituent at the C5 position, we also analyzed modified Z bases bearing functional groups with different electronic properties, as quantified by the values of their Hammett constants, σ P . The first two functional groups that we studied were the cyano (−CN) and trimethyl fluoride (−CF 3 ) groups, which are strongly electron-withdrawing like the nitro group and have been studied extensively for different applications in the context of nucleic acid structures. − In particular, the 5-CN-Z:P base pair was designed to study the influence of a different electron-withdrawing group (cyano versus nitro) on the epimerization and enzymatic incorporation of the substituted Z:P pair . Next, we focused on the formyl (−CHO) and carboxyl (−COOH) groups, which are much less electron-withdrawing and also preserve the intramolecular H-bond, similar to that observed between the amino and nitro groups in the parent Z base (see Figure ).…”

Cytotoxic activity of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide is underlain by DNA interchain cross-linking

“…3A). The retardation of the latter could be caused either by DNAprotein cross-linking or by cross-linking within DNA [17][18][19][20]. Further experiments on purified plasmid and genomic DNA revealed that EDC induced interchain crosslinking within the double-stranded DNA ( fig.…”

Development and application of diazirines in biological and synthetic macromolecular systems

“…Photoactivated cross-linking experiments revealed that the duplex formed covalent bonds at specific locations with restriction-modification enzymes (EcoRII). 81 DNA duplexes that incorporated 20 have also been used to identify specific contacts between nucleotides of DNA and Fpg protein 82,83 or HIV-1 intergrase. 84 The uridine analogue, 5-(N-((3-(trifluoromethyl)diazirine-3-yl)benzoyl)-3-aminoallyl)-29-deoxyuridine-59-triphosphate 85 was used as a substrate for different DNA polymerases to obtain a series of photoactivatable fragments of duplexes which form unusual complexes with DNA recognising proteins.…”