Figure 6.

C-terminal domain mutations do not inhibit oligonucleotide cleavage and religation. The oligonucleotide substrate (O) was labeled at the 5′-end with ³²P. (A) Cleavage products accumulated by the indicated amount of wild-type and mutant EcTOP1 in the absence of Mg(II). The labeled cleavage products are P1: 5′-GATTATGCAATGCGCT; P2: 5′-GATTATGCAAT; P3: 5′-GATTATGCAATGCGCTTT. (B) Rapid religation of cleavage products from the addition of Mg(II). Following the addition of 2 mM MgCl₂ and 1 M NaCl to the cleavage reactions containing 100 ng of enzyme, the reactions were left on ice for the indicated time before quenching with loading buffer for sequencing gel analysis.