Fingers-closing and other rapid conformational changes in DNA polymerase I (Klenow fragment) and their role in nucleotide selectivity.

We have developed a FRET-based assay for the fingers-closing conformational transition that occurs when a binary complex of DNA polymerase I (Klenow fragment) with a primer-template binds a complementary dNTP and have used this and other fluorescence assays to place the fingers-closing step within the reaction pathway. Because the rate of fingers-closing was substantially faster than the rate of nucleotide incorporation measured in chemical quench experiments, fingers-closing cannot be the rate-limiting prechemistry step defined by earlier kinetic studies. Experiments using Ca (2+) instead of Mg (2+) as the metal cofactor suggest instead that the prechemistry step may involve a change in metal ion occupancy at the polymerase active site.

Strand transfer events during HIV-1 reverse transcription.

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses replicate through reverse transcription, a process in which the single stranded RNA of the viral genome is converted to a double stranded DNA. The virally encoded reverse transcriptase (RT) mediates reverse transcription through DNA polymerase and RNase H activities. Conversion of the plus strand RNA to plus/minus strand RNA/DNA hybrid involves a transfer of the growing DNA strand from one site on the genomic RNA to another.