Fig 5.

Excision/extension assays. As described in Materials and Methods, a 5′-end-labeled primer was annealed to a template. The 3′ end of the primer was then blocked by the addition of AZTMP. The excision/extension assays were done in the presence of 3.0 mM ATP as the pyrophosphate donor and 10.0 μM each dNTP. All experiments were done at least twice at different times. When experiments were repeated with different batches of enzymes, the results were reproducible. Typical results are shown. The ATP used for the experiments shown in Fig. 5C was obtained from Sigma. ATP for other reactions was obtained from ChemCyte. We have consistently seen more excision in assays that are performed with ChemCyte ATP; however, the rank order of the ability of the various mutant RTs to carry out excision is not affected. (A) Excision and extension of an AZTMP-blocked primer by Q151M-containing RT variants on a DNA template/DNA primer. (B) Excision and extension of an AZTMP-blocked primer by Q151M-containing RT variants on an RNA template-DNA primer. (C) Excision and extension of an AZTMP-blocked primer by S215Y-containing HIV-2 RT variants on a DNA template/DNA primer. AZT-R is an HIV-1 variant that contains the mutations M41L, D67N, K70R, T215Y, and K219Y and is known to be ATP-dependent excision competent.