DNA Holliday Junction (HJ) formation and resolution is requisite for maintaining genomic stability in processes such as replication fork reversal and double-strand break repair. If HJs are not resolved, chromosome disjunction and aneuploidy result, hallmarks of tumor cells. To understand the structural features that lead to processing of these four-stranded joint molecule structures, we seek to identify structural and dynamic features unique to the central junction core. We incorporated the fluorescent guanine analog 6-methylisoxanthopterin (6-MI) at ten different locations throughout a model HJ structure to obtain site-specific information regarding the structure and dynamics of bases relative to those in a comparable sequence context in duplex DNA. These comparisons were accomplished through measuring fluorescence lifetime, relative brightness, fluorescence anisotropy, and quenching assays. These time-resolved and steady-state fluorescence measurements demonstrate that the structural distortions imposed by strand crossing result in increased solvent exposure, less stacking of bases and greater extrahelical nature of bases within the junction core. The 6-MI base analogs in the junction reflect these structural changes through an increase in intensity relative to those in the duplex. Molecular dynamics simulations performed using a model HJ indicate that the primary sources of deformation are in the shift and twist parameters of the bases at the central junction step. These results suggest that junction-binding proteins may use the unique structure and dynamics of the bases at the core for recognition.