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The exocyclic lesion 1,N2-propano-2'-deoxyguanosine (PdG) was incorporated into 5'-d[ATCGC(PdG)CGGCATG]-3', derived from the hisD3052 gene of Salmonella typhimurium. The modified oligodeoxynucleotide was annealed with the complementary strand 5'-d[CATGCCGCGAT]-3' which contained a CpG deletion. The resulting duplex 5'-d[ATCGC(PdG)CGGCATG]-3'.5'-d[CATGCCGCGAT]-3' required PdG and one adjacent cytosine to be unpaired. Four thymine imino 1H NMR resonances were observed at temperatures below 25 degrees C, which demonstrated formation of a stable duplex with a two-base bulge. PdG was accommodated within the DNA helix, whereas the 3'-neighbor cytosine was poorly stacked and appeared to be extrahelical. The sequential nuclear Overhauser enhancement connectivities between aromatic and H1' protons along the modified strand were interrupted between PdG and the 3'-neighboring unpaired cytosine. On the complementary strand no interruptions were observed. An NOE was observed between the PdG methylene protons H8a,b and the imino proton of the 5'-neighbor base pair. Weaker NOEs were observed between the PdG H8a,b protons and the imino proton from guanine two nucleotides removed in the 3'-direction, and to the amino proton of cytosine located in the complementary strand two nucleotides removed in the 3'-direction. Chemical shift perturbations were also observed for the latter cytosine as compared to the corresponding cytosine in the unmodified fully complementary duplex. These observations provided evidence for a poorly stacked or an extrahelical conformation of this unpaired cytosine. The amino proton resonances of the 3'-neighbor cytosine were not observed, presumably due to increased exchange with solvent. The methylene protons from PdG were shifted upfield relative to the monomer PdG, probably as a result of aromatic ring current shielding, consistent with an intrahelical location. Multimeric derivative oligonucleotides containing the PdG bulge migrated anomalously on nondenaturing polyacrylamide gels, consistent with a structure in which the unpaired nucleotides induced a bend in the DNA.