M. Paul Chiarelli

Conformational and thermodynamic properties modulate the nucleotide excision repair of 2-aminofluorene and 2-acetylaminofluorene dG adducts in the NarI sequence

Nucleotide excision repair (NER) is a major repair pathway that recognizes and corrects various lesions in cellular DNA. We hypothesize that damage recognition is an initial step in NER that senses conformational anomalies in the DNA caused by lesions. We prepared three DNA duplexes containing the carcinogen adduct N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) at G1, G2 or G3 of NarI sequence (5′-CCG1G2CG3CC-3′). Our 19F-NMR/ICD results showed that FAAF at G1 and G3 prefer syn S- and W-conformers, whereas anti B-conformer was predominant for G2. We found that the repair of FAAF occurs in a conformation-specific manner, i.e. the highly S/W-conformeric G3 and -G1 duplexes incised more efficiently than the B-type G2 duplex (G3∼G1 > G2). The melting and thermodynamic data indicate that the S- and W-conformers produce greater DNA distortion and thermodynamic destabilization. The N-deacetylated N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (FAF) adducts in the same NarI sequence are repaired 2- to 3-fold less than FAAF: however, the incision efficiency was in order of G2∼G1 > G3, a reverse trend of the FAAF case. We have envisioned the so-called N-acetyl factor as it could raise conformational barriers of FAAF versus FAF. The present results provide valuable conformational insight into the sequence-dependent UvrABC incisions of the bulky aminofluorene DNA adducts.

Sequence effects of aminofluorene-modified DNA duplexes: thermodynamic and circular dichroism properties

Circular dichroism (CD) and UV-melting experiments were conducted with 16 oligodeoxynucleotides modified by the carcinogen 2-aminofluorene, whose sequence around the lesion was varied systematically [d(CTTCTNG[AF]NCCTC), N = G, A, C, T], to gain insight into the factors that determine the equilibrium between base-displaced stacked (S) and external B-type (B) duplex conformers. Differing stabilities among the duplexes can be attributed to different populations of S and B conformers. The AF modification always resulted in sequence-dependent thermal (Tm) and thermodynamic (−ΔG°) destabilization. The population of B-type conformers derived from eight selected duplexes (i.e. -AG*N- and -CG*N-) was inversely proportional to the −ΔG° and Tm values, which highlights the importance of carcinogen/base stacking in duplex stabilization even in the face of disrupted Watson–Crick base pairing in S-conformation. CD studies showed that the extent of the adduct-induced negative ellipticities in the 290–350 nm range is correlated linearly with −ΔG° and Tm, but inversely with the population of B-type conformations. Taken together, these results revealed a unique interplay between the extent of carcinogenic interaction with neighboring base pairs and the thermodynamic properties of the AF-modified duplexes. The sequence-dependent S/B heterogeneities have important implications in understanding how arylamine–DNA adducts are recognized in nucleotide excision repair.

Sequence Verification of Oligonucleotides Containing Multiple Arylamine Modifications by Enzymatic Digestion and Liquid Chromatography Mass Spectrometry (LC/MS)

An analytical method for the structure differentiation of arylamine modified oligonucleotides (ODNs) using on-line LC/MS analysis of raw exonuclease digests is described. Six different dodeca ODNs derived from the reaction of N-acetoxy-N-(trifluoroacetyl)-2-aminofluorene with the dodeca oligonucleotide 5′-CTCGGCGCCATC-3′ are isolated and sequenced with this LC/MS method using 3′- and 5′-exonucleases. When the three products modified by a single aminofluorene (AF) are subjected to 3′-exonuclease digestion, the exonuclease will cleave a modified nucleotide but when di-AF modified ODNs are analyzed the 3′-exonuclease ceases to cleave nucleotides when the first modification is exposed at the 3′-terminus. Small abundances of ODN fragments formed by the cleavage of an AF-modified nucleotide were observed when two of the three di-AF modified ODNs were subjected to 5′-exonuclease digestion. The results of the 5′-exonuclease studies of the three di-AF modified ODNs suggest that as the number of unmodified bases between two modifications in an ODN sequence increases, the easier it becomes to sequence beyond the modification closest to the 5′-terminus. The results of this study indicate that the LC/MS method described here would be useful in sequencing ODNs modified by multiple arylamines to be used as templates for site-specific mutagenesis studies.

LOW ENERGY TANDEM MASS SPECTROMETRY OF DEOXYNUCLEOSIDE ADDUCTS OF POLYCYCLIC AROMATIC HYDROCARBON DIHYDRODIOL-EPOXIDES

The use of fast-atom bombardment ionization-tandem mass spectrometry approaches, with collision energies on the order of 30–50 eV, was developed for the analysis of low picomole quantities of polycyclic aromatic hydrocarbon dihydrodiol-epoxide deoxynucleoside adducts. This strategy combines three experimental techniques: (1) product ion scans, (2) constant neutral loss scans, and (3) precursor ion scans. Product ion scans of the protonated molecule or the BH2+ ion that results from loss of the deoxyribose were dominated by fragments associated with cleavage of the sigma bond between the dihydrodiol-epoxide moiety and the nucleobase. Constant neutral loss scans were based upon the loss of deoxyribose (116 u) or the combined loss of the deoxynucleoside, water, and carbon monoxide (313 u); precursor ion scans utilized the latter fragment. The formation of trimethylsilyl derivatives increased the sensitivity of analysis, which allowed the simultaneous detection of DNA adducts in a mixture.

Product ion studies of diastereomeric benzo[ghi]fluoranthene tetraols by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and post-source decay

The product ion formation characteristics of the four diastereomeric tetrahydroxy benzo[ghi] fluoranthene compounds formed by hydrolysis of the syn and anti diastereomers of trans-3,4-dihydroxy-5,5a-epoxy-3,4,5,5a-tetrahydrobenzo[ghi]fluoranthene are studied using matrix-assisted laser desorption/ionization and post-source decay (PSD) to determine a correlation between the fragmentation characteristics of these tetraols and the structures of the diolepoxide diastereomers from which they are hydrolyzed. The tetraols formed by the trans ring opening of the diol epoxides during hydrolysis yield product ion spectra specific for the syn and anti configurations of their precursor diol epoxides. All four diastereomeric tetraols form product ions by the losses of one and/or two water molecules in varying proportions when lithium-cationized molecule ions (m/z 301) are selected for PSD product ion analysis. The differences in the PSD spectra of these four Li+-cationized molecules are rationalized in terms of a water loss mechanism that involves the 1,2 elimination of a hydrogen atom and hydroxyl group that are cis with respect to each other on adjacent carbons.

Differentiation of isomeric C8- and N2-deoxyguanosine adducts of 2-acetylaminofluorene by fast-atom bombardment and tandem mass spectrometry

Product-ion studies of source-produced ions corresponding to acetylated and nonacetylated N2- and C8-substituted aminofluorene adducts of deoxyguanosine were conducted to identify specific fragmentation pathways differentiating the isomers and to determine the influence of the acetyl group on the fragmentation of the arylamide modified deoxyguanosine adducts. The collision-induced dissociation spectra of the BHZ2+ ion and other significant source-produced ions showed no evidence to suggest that ketene loss (deacetylation) resulted in significant alteration of the structure of the adducts. However, other significant ion formation processes, particularly loss of water from the N2-substituted arylamide did appear to require rearrangement, likely involving bond formation between the carcinogen moiety (acetyl group) and the N1 or N2 position of the guanine base. The combined loss of ketene and water constitute a fragmentation pattern specific for the N2-arylamide, 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene.

PRODUCT ION STUDIES OF SOME NOVEL ARYLAMINE ADDUCTS OF DEOXYGUANOSINE BY MATRIX-ASSISTED LASER DESORPTION/IONIZATION AND POST-SOURCE DECAY

The product ions of the BH(2)(+) ions formed by the glycosidic cleavage of N-(deoxyguanosin-O(6)-yl)-2-methylaniline, 4-(deoxyguanosin-8-yl)-2-methylaniline, and N-(deoxyguanosin-1-yl)-2-methylaniline have been studied using matrix-assisted laser desorption/ionization (MALDI) and post-source decay (PSD) to identify fragment ions and pathways that may be used to differentiate their structures. All three isomers may be distinguished based on their PSD product ion spectra using only femtomole quantities of sample. N-(Deoxyguanosin-O(6)-yl)-2-methylaniline produces product ions at m/z 107 and 134 that are diagnostic for 2-methylaniline attachment to the O(6) position of guanine. The BH(2)(+) ion from 4-(deoxyguanosin-8-yl)-2-methylaniline yields a product ion formed by the consecutive losses of 17 and 42 u neutral fragments that may be regarded as specific for guanine-arylamine adducts that possess two primary amine groups. The BH(2)(+) ion from 4-(deoxyguanosin-8-yl)-2-methylaniline yields no product ions that correlate with specificity for guanine N1 substitution. However, the product ion abundance ratio of the protonated arylamine to that of the ammonia loss ion may be used to differentiate an adduct formed by N1 substitution from other arylamine adducts of guanine studied thus far.

Post-source decay production studies of aniline and methylaniline adducts of deoxyguanosine

Abstract Matrix-assisted laser desorption ionization (MALDI) and post-source decay (PSD) are used to study the product ion formation processes of the BH2+ ions of three different arylamine adducts of deoxyguanosine; N-(deoxyguanosin-8-yl)-aniline, N-(deoxyguanosin-8-yl)-2,4-dimethylaniline, and 6-(deoxyguanosin-N2-yl)-2,4-dimethylaniline. Both C8-substituted adducts fragment to lose the guanine C8 carbon and the aniline group to give an ion at m/z 140, whose formation is specific for C8-substituted adducts of guanine. The BH2+ ion derived from N-(deoxyguanosin-8-yl)-2,4-dimethylaniline fragments to form an m/z 165 ion, composed of the guanine base and the nitrogen atom of 2,4-dimethylaniline, specific for guanine attachment to the amine nitrogen. The BH2+ ion derived from N-(deoxyguanosin-8-yl)-aniline does not form m/z 165 because it is not energetically favorable. 6-(deoxyguanosin-N2-yl)-2,4-dimethylaniline fragments to lose the guanine-N2 nitrogen and the 2,4-dimethylaniline group to give an ion at m/z 135, whose formation is a signature for aniline substitution at the N2 position of guanine. Isomers are differentiated with approximately 200 femtomoles of analyte introduced into the TOFMS. The product ion formation processes of these aniline and methylaniline adducts are contrasted with those of other C8- and N2-substituted arylamine adducts of guanine acquired with different MS methods as well.