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 p air substitution or deletion were employed. Heteroduplexes containing a single base bulge were formed by melting and reannealing pairs of 37 2 and 373 bp DNAs. Product DNAs were separated on the basis of their t hermal stability by parallel and perpendicular TGGE. The order of stab ility was determined for all single unpaired bases in four different n earest neighbor environments: (GXT).(AYC), (GXG).(CYC), (CXA).(TYG), a nd (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 degr ees C (+/-0.2 OC) with respect to homologous DNAs with complete Watson -Crick base pairing. Both the identity of the unpaired base and the se quence oi. the flanking base pairs influenced the degree of destabiliz ation. The range of temperature shift correspond to estimated unfavora ble free energies from 2.5 to 4.6 kcal/mol. Purine base bulges were ge nerally not as destabilizing as pyrimidine base bulges. An unpaired ba se which was identical to one of its adjacent bases generally caused l ess destabilization than an unpaired base with an identity differing f rom its nearest neighbors. This implies that positional degeneracy of an unpaired base within a run of two or more identical bases is an imp ortant factor effecting stability. The ability of TGGE to order the st abilities 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 sta bilities determined from UV melting transitions.