Decoding H2A/H3-DNA Interactions through Experimental and Computational Studies

Histone-mediated DNA condensation is a critical process for packaging DNA into the nucleus of eukaryotic cells. This process involves a complex interaction between DNA and histone proteins. In this work, we want to investigate the DNA-binding properties and associated binding modes of histone proteins (H2A, H3) using experimental and computational methods. Based on experimental results it was found that the interaction between H3 and ctDNA is stronger than that between H2A and ctDNA, likely due to the higher number of arginine residues in H3. TEM and CD results indicate that histone proteins can induce structural changes and condensation of ctDNA, resulting in a more compact and aggregated morphology, also supported by gel studies. The alteration observed in the phosphate and base pair peaks in FTIR suggest that electrostatic interaction could be one of the driving forces for the formation of complex between ctDNA and the histone proteins. H3 prefers to bind GC-rich sequence compared to AT-rich sequence. Based on MD analysis, H3 was found to be more dynamic in its interactions with DNA compared to H2A. The fluorescence displacement assay (FDA) suggests that H2A and H3 most probably bind in the DNA major groove. Moreover, H3 demonstrates higher drug displacement efficiency compared to H2A due to its stronger DNA binding capability. FDA assay further gives valuable insights into how condensed DNA restricts EtBr’s ability to access all intercalation sites, whereas DAPI is able to bind efficiently to condensed DNA in the minor groove.