INFLUENCE OF NEIGHBORING BASE-PAIRS ON THE STABILITY OF SINGLE-BASE BULGES AND BASE-PAIRS IN A DNA FRAGMENT

Temperature-gradient gel electrophoresis (TGGE) was used to determine the relative thermal stabilities of 32 DNA fragments that differ by a single unpaired base (base bulge) and 17 DNAs differing by a base pair. Homologous 373 and 372 bp DNA fragments differing by a single base pair substitution or deletion were employed. Heteroduplexes containing a single base bulge were formed by melting and reannealing pairs of 372 and 373 bp DNAs. Product DNAs were separated on the basis of their thermal stability by parallel and perpendicular TGGE. The order of stability was determined for all single unpaired bases in four different nearest neighbor environments: (GXT).(AYC), (GXG).(CYC), (CXA).(TYG), and (TXT).(AYA) with X = A, T, G, or C, and Y = no base, or visa versa. DNA fragments containing a base bulge were destabilized by 2-3.6 degrees C (+/-0.2 OC) with respect to homologous DNAs with complete Watson-Crick base pairing. Both the identity of the unpaired base and the sequence oi. the flanking base pairs influenced the degree of destabilization. The range of temperature shift correspond to estimated unfavorable free energies from 2.5 to 4.6 kcal/mol. Purine base bulges were generally not as destabilizing as pyrimidine base bulges. An unpaired base which was identical to one of its adjacent bases generally caused less destabilization than an unpaired base with an identity differing from its nearest neighbors. This implies that positional degeneracy of an unpaired base within a run of two or more identical bases is an important factor effecting stability. The ability of TGGE to order the stabilities of DNA fragments differing by a single base pair was used to determine the relative stabilities of base pair stacking interactions. The results determined by TGGE were consistent with the relative stabilities determined from UV melting transitions.