TABLE 1.

Single-turnover rates of dNTP incorporation by wild-type Pol I(KF) and the D705A and D882A mutants

Values reported as mean ± S.D. were from two independent experiments; the others were from single determinations. The reactions were carried out under single-turnover conditions (see “Experimental Procedures”) in the presence of a 10 mm concentration of the indicated divalent metal ion for WT and D882A and 20 mm for D705A. The DNA substrate, L:unmod:3′OH (Fig. 3A), has A at the templating position.

Proteina	kobs (s−1) in Mg2+	kobs (s−1) in Mn2+
WTb	43	35
D705Ac	(0.21 ± 0.07) × 10−5	(2.5 ± 0.1) × 10−5
D882Ac	(2.4 ± 0.5) × 10−5	(1.1 ± 0.2) × 10−5

^a In addition to the listed mutations, all proteins in this study were N-His₆, D424A, L744C, and C907S.

^b The reaction with WT Pol I(KF) contained 40 μm dTTP.

^c Reactions with the mutant Pol I(KF) derivatives contained 500 μm dTTP. Because the reactions were extremely slow, the rates were calculated from the yield of product DNA after 4 h.