Ribonucleotide inclusion (rNMP) is the most prevalent form of DNA damage. The incorporation of a single RNP impacts the local structure and dynamics of the double-stranded DNA. Our previous work has shown that for an rG intrusion, the perturbation is strongly on the flanking nucleotide sequences. We have prepared a series of duplexed DNA oligonucleotides all containing a single rG or rC inclusion in different flanking sequence environments for enzymatic and structural studies. For the rG DNA substrates I have examined all possible nearest neighbor flanking sequence combinations. It was found that the reaction-efficiency-with RNase HII strongly depends on the flanking sequence by an up to 20-fold difference. NMR analysis was performed to determine the effects a ribonucleotide misincorporation has on a replication fork. The proximity of an RNP to a replication fork greatly distorts the fraction-south (fs) characteristics of the sugar residues and immediate phosphodiester backbone.