Context <p>Aflatoxin (AFT) is a toxic carcinogen and mutagen produced mainly by <i>Aspergillus species</i>. Dietary intake is the main route of exposure to AFT in humans, and DNA damage caused by AFT exposure receives wide concern as an endogenous factor in the pathogenesis of liver cancer. The systematic exploration on the toxicity mechanism of AFB1—the most toxic AFT subtype—is of great significance for the prevention and control of liver cancer. In this work, three adduct models, gap-AFT (GA), mispairing-AFT (MA), and insertion-AFT (IA), have been defined based on all the available three-dimensional structures of AFT in complex with DNA. In addition, their molecular recognition and conformational change features were investigated via comparative molecular dynamics (MD) simulations. Subsequently, the effects of six adduct models resulted from both AFB1-N7-dG and AFB1-Fapy on the recognition by DNA polymerase IV (Dpo4) were analyzed, with the possible toxicity mechanism of the destruction against DNA replication being given. Specifically, AFT partially destroyed the conservative structure including dATP and Ca²⁺ ion in the catalytic center of Dpo4. Moreover, the extent of disruption from AFB1-Fapy was slightly higher than that from AFB1-N7-dG. This work enriches the mechanism of AFT toxicity and provides clinical theoretical guidance for the control of human injuries caused by AFT exposure, making a contribution in the field of food and environmental safety.</p> Methods <p>The experimental structure of 16 aflatoxin-containing DNA systems was obtained from the protein database (<a href="http://www.rcsb.org/">http://www.rcsb.org/</a>), and the relevant structural parameters of DNA were analyzed using the Curves program, revealing the effect of AFT binding on DNA structural deformability. The initial models of the AFB1-N7-dG and AFB1-Fapy adducts were structurally optimized at the B3LYP-D3/6-311G(d,p) level of theory using the Gaussian 09 software package. Molecular dynamics simulations were performed using the AMBER 20 software package. The following force-field combination was employed: the AMBER ff19SB force field for the protein, the OL15 force field for DNA, and specialized parameters for key system components. The catalytic Ca²⁺ ions were described using the 12-6 Lennard-Jones non-bonded parameters developed by Li and Merz. For the dATP substrate, parameters including the refined partial charges for the triphosphate moiety were adopted from the specialized nucleotide force-field set based on the work of Meagher, Redman, and Carlson. All-atom simulations were conducted, yielding stable and physically reasonable trajectories. RDG analysis of covalent binding regions of DNA and AFT using the Multiwfn 3.8 program to visualize the type, intensity, and variation of weak intermolecular interactions; the molecular mechanics/Poisson-Boltzmann solvent area (MM/PBSA) method was used to calculate the binding free energy between protein and DNA. Subsequently, the energy decomposition technology based on the MM/GBSA method was used to quantitatively analyze the key residues in dATP recognition in Dpo4.</p>