. 2002 Jun 18;3:18. doi: 10.1186/1471-2091-3-18

Table 2.

DNA and dNTP binding affinities of the mutant HIV-1 RT carrying insertion in the β7–β8 loop of either or both the subunits

Enzyme Species	K_{d [DNA]} (nM)	App. K_{d [dNTP]} (μM)
p66^WT/p51^WT	2.0	6.0
p66^WT/p66^WT	2.6	5.8
p66^INS/p51^INS	48.0	8.0
p66^INS/p66^INS	34.0	ND
p66^INS/p51^WT	4.0	9.0
p66^WT/p51^INS	3.0	5.0

The dissociation constant of E-TP binary complex (K_{d [DNA]}) and the apparent dNTP binding affinity (K_{d [dNTP]}) for the mutant HIV-1 RT was determined by gel mobility shift assay. For K_{d [DNA]} determination, 5' ³²P-labeled dideoxy terminated 33/21-mer DNA template-primer was incubated with varying concentrations of the individual WT or mutant enzyme and resolved on a native polyacrylamide gel at 4°C. The slower migrating E-TP complexes were detected by phosphorImaging and quantified using ImageQuant software. For determining the apparent K_{d [dNTP]}, the concentration of the individual enzyme used was such that retarded 100% of the labeled TP. The extent of stable E-DNA-dNTP ternary complex formed in the presence of varying concentrations of the next correct nucleotide (dGTP) was determined following the addition of the DNA trap. This was estimated by calculating the percent of the un-dissociated template primer in the ternary complex using ImageQuant software. The values shown are the average of two independent experiments. ND indicates that the apparent K_{d [dNTP]} in case of p66^INS/p66^INS could not be determined by this analysis.