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. The use of ribonucleotide substrates shows there is a base discriminating step that precedes fingers-closing. This earlier step, detected by 2-AP fluorescence, is promoted by complementary nucleotides (ribo- as well as deoxyribo-) but is blocked by mismatches. The complementary rNTP blocks the subsequent fingers-closing step. Thus, discrimination against rNTPs occurs during the transition from open to closed conformations, whereas selection against mismatched bases is initiated earlier in the pathway, in the open complex. Mismatched dNTPs accelerate DNA release from the polymerase, suggesting the existence of an early intermediate in which DNA binding is destabilized relative to the binary complex; this could correspond to a conformation that allows an incoming dNTP to preview the template base. The early kinetic checkpoints identified by this study provide an efficient mechanism for the rejection of mismatched bases and ribose sugars and thus enhance polymerase throughput.

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. This is called minus strong-stop DNA transfer. Later synthesis of the second or plus DNA strand involves a second strand transfer, involving a similar mechanism as the minus strand transfer. A basic feature of the strand transfer mechanism is the use of the RT RNase H to remove segments of the RNA template strand from the growing DNA strand, freeing a single stranded region to anneal to the second site. Viral nucleocapsid protein (NC) functions to promote transfer by facilitating this strand exchange process. Two copies of the RNA genomes, sometimes non-identical, are co-packaged in the genomes of retroviruses. The properties of the reverse transcriptase allow a transfer of the growing DNA strand between these genomes to occur occasionally at any point during reverse transcription, producing recombinant viral progeny. Recombination promotes structural diversity of the virus that helps it to survive host immunity and drug therapy. Recombination strand transfer can be forced by a break in the template, or can occur at sites where folding structure of the template pauses the RT, allowing a concentration of RNase H cleavages that promote transfers. Transfer can be a simple one-step process, or can proceed by a complex multi-step invasion mechanism. In this latter process, the second RNA template interacts with the growing DNA strand well behind the DNA 3'-terminus. The newly formed RNA-DNA hybrid expands by branch migration and eventually catches the elongating DNA primer 3'-terminus to complete the transfer. Transfers are also promoted by interactions between the two RNA templates, which accelerate transfer by a proximity effect. Other details of the role of strand transfers in reverse transcription and the biochemical features of the transfer reaction are discussed.