Examination of the Long-range Effects of Aminofluorene-induced Conformational Heterogeneity and Its Relevance to the Mechanism of Translesional DNA Synthesis

Adduct-induced conformational heterogeneity complicates the understanding of how DNA adducts exert mutation. A case in point is the N-deacetylated AF lesion [N-(2′-deoxyguanosin-8-yl)-2-aminofluorene], the major adduct derived from the strong liver carcinogen N-acetyl-2-aminofluorene. Three conformational families have been previously characterized and are dependent on the positioning of the aminofluorene rings: B is in the “B-DNA” major groove, S is “stacked” into the helix with base-displacement, and W is “wedged” into the minor groove. Here, we conducted 19F NMR, CD, Tm, and modeling experiments at various primer positions with respect to a template modified by a fluorine tagged AF-adduct (FAF). In the first set, the FAF-G was paired with C and in the second set it was paired with A. The FAF-G:C oligonucleotides were found to preferentially adopt the B or S-conformers while the FAF-G:A mismatch ones preferred the B and W-conformers. The conformational preferences of both series were dependent on temperature and complementary strand length; the largest differences in conformation were displayed at lower temperatures. The CD and Tm results are in general agreement with the NMR data. Molecular modeling indicated that the aminofluorene moiety in the minor groove of the W-conformer would impose a steric clash with the tight-packing amino acid residues on the DNA binding area of the Bacillus fragment (BF), a replicative DNA polymerase. In the case of the B-type conformer, the carcinogenic moiety resides in the solvent-exposed major groove throughout the replication/translocation process. The present dynamic NMR results, combined with previous primer extension kinetic data by Miller & Grollman, support a model in which adduct-induced conformational heterogeneities at positions remote from the replication fork affect polymerase function through a long-range DNA–protein interaction

Insights into the Conformation of Aminofluorene-Deoxyguanine Adduct in a DNA Polymerase Active Site

The active site conformation of the mutagenic fluoroaminofluorene-deoxyguanine adduct (dG-FAF, N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) has been investigated in the presence of Klenow fragment of Escherichia coli DNA polymerase I (Kfexo−) and DNA polymerase β (pol β) using 19F NMR, insertion assay, and surface plasmon resonance. In a single nucleotide gap, the dG-FAF adduct adopts both a major-groove- oriented and base-displaced stacked conformation, and this heterogeneity is retained upon binding pol β. The addition of a non-hydrolysable 2′-deoxycytosine-5′-[(α,β)-methyleno]triphosphate (dCMPcPP) nucleotide analog to the binary complex results in an increase of the major groove conformation of the adduct at the expense of the stacked conformation. Similar results were obtained with the addition of an incorrect dAMPcPP analog but with formation of the minor groove binding conformer. In contrast, dG-FAF adduct at the replication fork for the Kfexo− complex adopts a mix of the major and minor groove conformers with minimal effect upon the addition of non-hydrolysable nucleotides. For pol β, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with 19F NMR data. Surface plasmon resonance binding kinetics revealed that pol β binds tightly with DNA in the presence of correct dCTP, but the adduct weakens binding with no nucleotide specificity. These results provide molecular insights into the DNA binding characteristics of FAF in the active site of DNA polymerases and the role of DNA structure and sequence on its coding potential

Unusual Sequence Effects on Nucleotide Excision Repair of Arylamine Lesions: DNA Bending/Distortion as a Primary Recognition Factor

The environmental arylamine mutagens are implicated in the etiology of various sporadic human cancers. Arylamine-modified dG lesions were studied in two fully paired 11-mer duplexes with a -G*C N- sequence context, in which G* is a C8-substituted dG adduct derived from fluorinated analogs of 4-aminobiphenyl (FABP), 2-aminofluorene (FAF) or 2-acetylaminofluorene (FAAF), and N is either dA or dT. The FABP and FAF lesions exist in a simple mixture of ‘stacked’ (S) and ‘B-type’ (B) conformers, whereas the N-acetylated FAAF also samples a ‘wedge’ (W) conformer. FAAF is repaired three to four times more efficiently than FABP and FAF. A simple A- to -T polarity swap in the G*C A/G*CT transition produced a dramatic increase in syn-conformation and resulted in 2- to 3-fold lower nucleotide excision repair (NER) efficiencies in Escherichia coli. These results indicate that lesion-induced DNA bending/thermodynamic destabilization is an important DNA damage recognition factor, more so than the local S/B-conformational heterogeneity that was observed previously for FAF and FAAF in certain sequence contexts. This work represents a novel 3\u27-next flanking sequence effect as a unique NER factor for bulky arylamine lesions in E. coli

Microbial and Metabolite Signatures of Stress Reactivity in Ulcerative Colitis Patients in Clinical Remission Predict Clinical Flare Risk.

BACKGROUND: Stress reactivity (SR) is associated with increased risk of flares in ulcerative colitis (UC) patients. Because both preclinical and clinical data support that stress can influence gut microbiome composition and function, we investigated whether microbiome profiles of SR exist in UC. METHODS: Ninety-one UC subjects in clinical and biochemical remission were classified into high and low SR groups by questionnaires. Baseline and longitudinal characterization of the intestinal microbiome was performed by 16S rRNA gene sequencing and fecal and plasma global untargeted metabolomics. Microbe, fecal metabolite, and plasma metabolite abundances were analyzed separately to create random forest classifiers for high SR and biomarker-derived SR scores. RESULTS: High SR reactivity was characterized by altered abundance of fecal microbes, primarily in the Ruminococcaceae and Lachnospiraceae families; fecal metabolites including reduced levels of monoacylglycerols (endocannabinoid-related) and bile acids; and plasma metabolites including increased 4-ethyl phenyl sulfate, 1-arachidonoylglycerol (endocannabinoid), and sphingomyelin. Classifiers generated from baseline microbe, fecal metabolite, and plasma metabolite abundance distinguished high vs low SR with area under the receiver operating characteristic curve of 0.81, 0.83, and 0.91, respectively. Stress reactivity scores derived from these classifiers were significantly associated with flare risk during 6 to 24 months of follow-up, with odds ratios of 3.8, 4.1, and 4.9. Clinical flare and intestinal inflammation did not alter fecal microbial abundances but attenuated fecal and plasma metabolite differences between high and low SR. CONCLUSIONS: High SR in UC is characterized by microbial signatures that predict clinical flare risk, suggesting that the microbiome may contribute to stress-induced UC flares

GATA2 mutant variant allele frequency may reflect prognosis in Chinese adult patients with de novo cytogenetically normal acute myeloid leukemia

In this study, we analyzed GATA2 mutations (GATA2mut) and co-mutations in 166 Chinese patients with cytogenetically normal acute myeloid leukemia. This was done through targeted next-generation sequencing of 34 genes associated with myeloid leukemia. GATA2mut was identified in 17 (10%) patients being significantly correlated with co-mutations in CCAAT/enhancer-binding protein alpha (CEBPA) double mutation (P = 0.001). We observed that the N-terminal zinc finger domain (ZF1) was linked to CEBPA mutations, while the C-terminal zinc finger domain (ZF2) was associated with Wilms' tumor 1 (WT1) mutations. It was also noted that patients with GATA2mut had lower platelet counts at diagnosis (P = 0.032). In the entire cohort, GATA2mut had no significant prognostic impact on overall survival (OS) (P = 0.762) and relapse-free survival (RFS) (P = 0.369) compared to patients with GATA2wt. The OS (P = 0.737) and RFS (P = 0.894) of the ZF1 mutation were similar to those of the ZF2 mutation. Most patients with GATA2 mutations were classified in the ELN2022 favorable- and intermediate-risk groups. GATA2mut patients in the favorable-risk group were divided into GATA2High and GATA2Low groups using a median cutoff variant allele frequency (VAF) of 40.13%. GATA2High patients were associated with worse OS (P = 0.031) and RFS (P = 0.021) than GATA2Low patients. In the intermediate-risk group, the high median VAF of GATA2 (≥38.51%) had no significant effect in OS and RFS compared with the low median VAF (<38.51%). This study offers new insights on the prognosis of GATA2mut in the favorable-risk group, where VAF can be used as a guide

Probing the thermodynamics of aminofluorene-induced translesion DNA synthesis by differential scanning calorimetry

Differential scanning calorimetry (DSC) was used to investigate the thermodynamic contribution to replication fidelity and specificity associated with translesion synthesis across FAF, N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene, a fluorine-tagged DNA adduct derived from the carcinogen 2-aminofluorene. As a control, insertion of matched nucleoside dC at the primer terminus (n) and subsequent extensions (n + 1 to n + 6) resulted in an incremental increase in thermodynamic parameters. In contrast, incorporation of dC opposite FAF-dG and subsequent extensions up to n + 2 showed little change in thermodynamics. A similar thermodynamic stalling was observed for a control template primer containing a G:A mismatch at n and subsequent Watson-Crick primer extensions. The thermodynamic paucity generated by either a lesion or a mismatch was not localized at the replication fork but extended to 5′-downstream n + 2 sites, thus providing an explanation for the short-term memory effects observed with replicative polymerases. Interestingly, FAF modification did not alter the overall DSC profiles of the G:A mismatch template primer and, in fact, resulted in thermal stabilization. Entropy around the lesion site appears to play a critical role in the adduct-induced increase in thermodynamic stability. While addition of matched nucleoside dC at n was thermodynamically favored over the presence of a mismatched dA (ΔΔG° = 1.7 kcal/mol, ΔΔH° = 9.1 kcal/mol), no such thermodynamic advantage was observed with the FAF lesion at n (ΔΔG° ∼0 kcal/mol). These equilibrium thermodynamic results provide insight into the most prevalent mismatch (i.e., G → T transversion mutations) induced by this lesion. However, kinetic effects undoubtedly play a key role in the processing of this bulky lesion, and the nature of the polymerase is likely to govern and to determine the balance between kinetic and thermodynamic effects. Copyright © 2007 American Chemical Society

Enthalpy - entropy contribution to carcinogen-induced DNA conformational heterogeneity

DNA damage by adduct formation is a critical step for the initiation of carcinogenesis. Aromatic amines are strong inducers of environmental carcinogenesis. Their DNA adducts are known to exist in an equilibrium between the major groove (B) and base-displaced stacked (S) conformations. However, the factors governing such heterogeneity remain unclear. Here we conducted extensive calorimetry/NMR/CD studies on the model DNA lesions caused by fluorinated 2-aminfluorene (FAF) and 4-aminobiphenyl (FABP) in order to gain thermodynamic and kinetic insights into the S/B conformational equilibrium.We demonstrate that there are large differences in enthalpy-entropy compensations for FABP and FAF. The small and flexible FABP exclusively adopts the less perturbed B conformer with small enthalpy (ΔΔH-2.7 kcal/mol)/entropy (ΔΔS-0.7 eu) change. In contrast, FAF stacks better and exists as a mixture of B and S conformers with large enthalpy (ΔΔH-13.4 kcal/mol)/entropy (ΔΔS-34.2 eu) compensation. van\u27t Hoff analysis of dynamic 19F NMR data indicated ΔHB↔S=4.1 kcal/mol in favor of the B conformer and ΔSB↔S=15.6 cal mol -1 K-1 in favor of the intercalated S conformer. These findings demonstrate that the favorable entropy of the S conformer over B conformer determines the S/B population ratios at physiological temperatures. ©2009 American Chemical Society

Conformational and thermodynamic impact of bulky aminofluorene adduction on simulated translesion DNA synthesis

We report a systematic spectroscopic investigation on the conformational evolution during primer extension of a bulky fluoroaminoflurene-modfied dG adduct (FAF-dG) in chemically simulated translesion synthesis. FAF-dG was paired either with dC or dA (dC-match and dA-mismatch series, respectively). Dynamic 19F NMR/CD results showed that the FAF-adduct exists in a syn/anti equilibrium and that its conformational characteristics are modulated by the identity of an inserted nucleotide at the lesion site and the extent of primer elongation. At the preinsertion site, the adduct adopted preferentially a syn conformation where FAF stacked with preceding bases. Insertion of the correct nucleotide dC at the lesion site and subsequent elongation resulted in a gradual transition to the anti conformation. By contrast, the syn conformer was persistent along with primer extension in the dA-mismatch series. In the dC-match series, FAF-induced thermal (Tm) and thermodynamic (-ΔG°37 °C) stabilities were significantly reduced relative to those of the controls. However, the corresponding Tm and -ΔG°37 °C values were increased in the FAF-modified mismatched dA series. The lesion impact persisted up to three 5′-nucleotides from the lesion. Occupation of the minor groove of the W-conformer with the bulky carcinogenic fluorene moiety not only would limit the DNA mobility but also would impose a serious difficulty for the active site of a polymerase throughout the replication process. Our spectroscopic results are consistent with reported data on AF, which showed dramatic (∼10 4-fold) differences in the nucleotide insertion rates between the dC-match and dA-mismatch series. The results emphasize the importance of adduct-induced steric constraints for determining the replication fidelity of a polymerase. © 2011 American Chemical Society