Structure and Thermodynamic Insights on Acetylaminofluorene-Modified Deletion DNA Duplexes as Models for Frameshift Mutagenesis

Abstract

2-Acetylaminofluorene (AAF) is a prototype arylamine carcinogen that forms C8-substituted dG-AAF and dG-AF as the major DNA lesions. The bulky N-acetylated dG-AAF lesion can induce various frameshift mutations depending on the base sequence around the lesion. We hypothesized that the thermodynamic stability of bulged-out slipped mutagenic intermediates (SMIs) is directly related to deletion mutations. The objective of the present study was to probe the structural/conformational basis of various dG-AAF–induced SMIs formed during a translesion synthesis. We performed spectroscopic, thermodynamic, and molecular dynamics studies of several AAF-modified 16-mer model DNA duplexes, including fully paired and −1, −2, and −3 deletion duplexes of the 5′-CTCTCGATG[FAAF]CCATCAC-3′ sequence and an additional −1 deletion duplex of the 5′-CTCTCGGCG[FAAF]CCATCAC-3′ NarI sequence. Modified deletion duplexes existed in a mixture of external B and stacked S conformers, with the population of the S conformer being ‘GC’ −1 (73%) > ‘AT’ −1 (72%) > full (60%) > −2 (55%) > −3 (37%). Thermodynamic stability was in the order of −1 deletion > −2 deletion > fully paired > −3 deletion duplexes. These results indicate that the stacked S-type conformer of SMIs are thermodynamically more stable than the conformationally flexible external B conformer. Results from the molecular dynamics simulations indicate perturbation of base stacking dominate the relative stability along with contributions from bending, duplex dynamics, solvation effects that are important in specific cases. Taken together, these results support a hypothesis that the conformational and thermodynamic stabilities of the SMIs are critical determinants for the induction of frameshift mutations.

DNA base sequence effects on bulky lesion-induced conformational heterogeneity during DNA replication

“…FABP- and FAF-modified 16mer oligos were characterized by exonuclease enzyme digestion followed by MALDI-TOF mass spectrometry, according to published procedures ( 15 , 28 , 29 ). Supplementary Figure S1B shows the MALDI-TOF MS spectra of the 3′-5′ SVP exonuclease digestions of peaks 1 and 2 from FABP-HPLC profile ( Supplementary Figure S1A ) at different time points (0, 0.5, 2 and 5 min).…”

“…The predominant B-conformer of TG 1 G 2 *T highly affects the bending of DNA helix around the adduct site. G 1 and G 2 FABP-modified duplexes displayed slight blue shifts (∼3 nm) relative to the unmodified controls, indicating adduct-induced DNA bending ( 28 ). Similar results were obtained for FAF-modified series ( Supplementary Figure S8 ).…”

“…The FABP- and FAF-modified mono-adducted 16mer oligos above were digested by 3′–5′ exonuclease enzyme digestion and characterized by MALDI-TOF mass spectrometry following published procedures ( 28 ). Briefly, 1 μL (0.1 unit) of snake venom phosphodiesterase (SVP) was added to a solution of 1 μL of modified oligo (∼100–200 pmol), 6 μl of ammonium citrate (100 mM, pH 9.4), and 6 μl of water for 3′–5′digestion.…”

Characterization of Byproducts from Chemical Syntheses of Oligonucleotides Containing 1-Methyladenine and 3-Methylcytosine

“…To further confirm the location of the modified base, we performed enzyme digestion coupled with MALDI-TOF MS analysis. 11 − 13 , 40 , 41 The procedure has been widely used to confirm the location of lesion positions by determining the mass changes after partial digestion from the 3′ end by the exonuclease snake venom phosphodiesterase (SVP). 42 − 44 Figure 5 shows the positive ion MALDI-TOF MS spectra of the 3′ to 5′ SVP exonuclease digestion of the product oligonucleotide at different time intervals.…”

“…Samples were analyzed by a Shimadzu Axima Performance MALDI-TOF mass spectrometer using a 50 Hz laser with a power setting of 90 in a positive ion reflection mode with 500 shots collected. 13 …”