Investigating the Conformational Dynamics of DNA with Lesions through Fluorescence-Based Technniques and Computer Simulations

Abstract

Accurate propagation of genetic material is essential for life. Yet, DNA is constantly being bombarded by various external factors resulting in millions of lesions per day. Lesions include modified bases, backbone nicks, and single stranded gaps. The presence of such lesions could lead to differences in the conformational dynamics of DNA, suggesting a potential mechanism for DNA repair enzymes to locate damages. To investigate how the presence of lesions affects the conformational dynamics of DNA, Forster resonance energy transfer (FRET) was used. The DNA samples contained internally modified Cy3/Cy5 dyes. Each DNA sample was designed to have a different lesion such as a nick or abasic site. Higher FRET efficiencies were expected if the DNA was more dynamic. However, only small differences in the FRET efficiency were found. Therefore, time-resolved anisotropy was used in combination with FRET, where faster acceptor emission depolarization is indicative of more dynamic DNA. To gain an understanding of the atomistic details, molecular dynamics (MD) simulations were performed and MD trajectories analyzed in terms of global and local structural quantities such as bending angle and helicoidal parameters. Both experiments and simulations showed that nicked DNA behaves similarly to intact DNA. However, the introduction of a single stranded gap of nucleotides increases the dynamic behavior of the DNA. While a nick alone did not change the conformational dynamics of the DNA, the introduction of a mismatch base or abasic site increased conformational dynamics by weakening the hydrogen bonding and stacking interactions around the nick. The combination of experimental and computational results suggests that the disruption of local structural stability leads to global conformational changes.