Figure 1.

Enhancement of top1 cleavage complexes by ara-C incorporation at the +1 position of a top1 cleavage site. (A) Structures of deoxycytidine (C) and ara-C. (B) Modified Tetrahymena hexadecameric rDNA sequence with a strong top1 cleavage site (10, 31) indicated by the arrowhead was labeled with [³²P]cordycepin (*A) at the 3′ terminus of the scissile (upper) strand. Top1 cleavage yields a 23-mer product. Oligonucleotides were synthesized with either C or ara-C at the indicated positions. (C) Enhancement of top1 cleavage by ara-C at the +1 but not at the +2 position of the nonscissile strand. Oligonucleotides with the indicated base pair at the +1 or +2 position relative to the top1 cleavage site are shown above lanes. (D) Enhancement of top1 cleavage by ara-C at the +1 position of the scissile strand. For each oligonucleotide used in C and D: Lanes A, DNA alone; lanes B and C, + top1; lanes D and E, + top1 + 10 μM CPT. Reactions were performed at 25°C for 15 min and stopped either immediately with 0.5% SDS (lanes B and D) or were first incubated with 0.5 M NaCl (final concentration) for an additional 30 min at 25°C before addition of 0.5% SDS (lanes C and E).