FIGURE 3.

Processive DNA synthesis catalyzed by the Y226F mutant. A, DNA synthesis catalyzed by the wild type Φ29 DNAP (lanes 1–6) or the Y226F mutant (lanes 7–12) as a function of enzyme concentration. A series of 2-fold serial dilutions of each enzyme was tested, in which the highest concentration (lanes 1 and 7, indicated by enzyme dilution = 1) was 30 pm; the reactions were conducted at 30 °C for 30 min. B, DNA synthesis as a function of time for reactions catalyzed by the wild type Φ29 DNAP (lanes 1–5) or the Y226F mutant (lanes 6–10). Reactions were conducted using an enzyme concentration of 60 pm at 30 °C for the indicated times. In both A and B, the replication substrate was oligonucleotide-primed bacteriophage M13 single-stranded DNA (∼3.35 pm). The reaction products were resolved by electrophoresis in alkaline agarose gels.