ISOTOPE EFFECTS ON THE SEQUENCE-SPECIFIC CLEAVAGE OF DNA BY NEOCARZINOSTATIN - KINETIC PARTITIONING BETWEEN 4'-HYDROGEN AND 5'-HYDROGEN ABSTRACTION AT UNIQUE THYMIDINE SITES

In this article it is shown that in certain sequences cleavage of DNA at thymidine residues by activated neocarzinostatin occurs via a rate-limiting abstraction of either a 4'- or 5'-hydrogen from the deoxyribose moiety and that this partitioning can be modulated by deuteriation at either position. An analysis by gel electrophoresis of the HindIII-BamHI restriction fragment (346 bp) of pBR322 revealed that in the first 45 nucleotides of the (+)-strand T14, T17, T27, and T44 are strongly sensitive to deuterium substitution at C-4' (k(H)/k(D) approximately 2.4-5.5) and are less sensitive to deuteriation at C-5' (k(H)/k(D) approximately 1.0-2.6). All four of these T sites are located in GT steps. Other T residues are cleaved and exhibit variable sensitivity to 5'-deuteriation; however, they show no evidence of 4'-chemistry. The 5'-radical intermediate yields 3'-phosphate termini. The 4'-radical intermediate is demonstrated to partition between a modified abasic carbohydrate terminus and a 3'-phosphoglycolate terminus. The relative ratio of these two termini is, in turn, modulated by the structure of the thiol activator/reductant.