DNA-STACKING INTERACTIONS DETERMINE THE SEQUENCE SPECIFICITY OF THE DEOXYRIBONUCLEASE ACTIVITY OF 1,10-PHENANTHROLINE-COPPER ION

Bis(1,10-phenanthroline)-copper(I) ion (OP2Cu+) binds reversibly to B- DNA and makes single-stranded cuts by oxidative attack on the deoxyrib ose moiety. The deoxyribonuclease activity is sequence-dependent yet n ot nucleotide-specific at the cutting site. OP2Cu+ sequence specificit y was analysed in terms of local variations of DNA stability. Kinetic constants of strand cleavage were measured at sequence positions on th e two strands and converted into activation free energies of the cleav age reaction. DNA unwinding free energies were calculated from the bas e sequence using B-DNA stacking parameters for calculations. The two f ree-energy variations were statistically compared for a series of DNA restriction fragments bearing the binding sites of regulatory proteins and representing a total of 345 DNA base positions. This study shows that the mean activation free energy of strand cleavage at a pair of o pposing sugars across the DNA minor groove varies like the unwinding f ree energy of the DNA sequence delimited by opposing sugars (3 to 4 bp ). A statistical equality between the two free-energy variations is de monstrated when considering the sum of the two cleavage events at the opposing sugars. Systematic deviations between the two free-energy dis tributions were observed at specific sequences, including polypurine-p olypyrimidine tracts (A(n)T(m)/A(m)T(n), CnTmCp/G(p)A(m)G(n)), alterna ting purine-pyrimidine tracts ((TA)(n)/(TA)(n), (TG)(n)/(CA)(n)) and a t certain Gi+C-rich triplets (GGC, GCC and CGC). The physical signific ance of these observations is discussed and a model of OP2Cu+ binding and cleavage specificity based on the free-energy equality is proposed . (C) 1996 Academic Press Limited