Table 5.

Dissociation rate constants (k_off) for binary complexes of RT and DNA/DNA template–primers

RTs	koff (s−1)
	31T/21Pa	D38/25PGA	Lac46T/21P1
O_WT	0.080 ± 0.010	0.103 ± 0.012	0.207 ± 0.014
O_V75I	0.051 ± 0.003 (0.6)	0.093 ± 0.009 (0.9)	0.210 ± 0.017 (1.0)
O_D443N	0.187 ± 0.010 (2.3)**	0.214 ± 0.011 (2.1)**	0.264 ± 0.010 (1.3)**
O_E478Q	0.123 ± 0.018 (1.5)*	0.133 ± 0.006 (1.3)**	0.311 ± 0.005 (1.5)**
O_V75I/D443N	0.256 ± 0.025 (3.2)**	0.232 ± 0.025 (2.3)**	0.315 ± 0.020 (1.5)**
O_V75I/E478Q	0.163 ± 0.001 (2.0)**	0.169 ± 0.004 (1.6)**	0.270 ± 0.018 (1.3)*

The k_off values were obtained with DNA/DNA duplexes of different sizes: 31/21mer (31T/21P), 38/25mer (D38/25PGA) and 46/21mer (Lac46T/21P1). Reported values are the averages ± standard deviations, obtained from at least three independent experiments. Numbers between parentheses represent the fold-increase relative to the k_off value for the WT enzyme. **P < 0.01 and *P < 0.05 compared with the corresponding O_WT or O_V75I RTs by unpaired Student’s t test.

^aDeterminations based on the incorporation of several nucleotides gave consistent results. Thus, the k_off values obtained with O_WT, O_E478Q and O_V75I/D443N RTs, after elongating the primer 21P in the presence of a mixture of dTTP and dATP, were 0.092 ± 0.009, 0.138 ± 0.014 and 0.212 ± 0.020 s⁻¹, respectively.