Fig. 2.

Assays of 3′-5′ exonucleolytic activity of mutants in residue Asp¹²¹ of ϕ29 DNAP, using as templates a perfectly matched dsDNA (A), a 3′-mismatched dsDNA molecule (B), and an ssDNA molecule (C). The assays were performed as described in Materials and Methods. After incubation for the indicated times at 25 °C, degradation of the labeled DNA was analyzed by electrophoresis in 7 M urea/20% polyacrylamide gels and autoradiography. The position of the 4mer degradation intermediate is indicated. (D) ssDNA binding of wild-type or mutant ϕ29 DNAPs. The EMSA assay was carried out as described in Materials and Methods, using the 5′-labeled sp1 (15mer) as substrate, in the presence of the indicated amounts of either wild-type or mutant ϕ29 DNAPs. After nondenaturing gel electrophoresis, the bands corresponding to free ssDNA and to the DNAP/DNA complex were detected by autoradiography. c, control lane without enzyme. Asterisks indicate the 5′-³²P-labeled end of the primer strand.