The pyrophosphate (PPi) analogue phosphonoformic acid (PFA or foscarnet) inhibits the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1); however, the mechanisms of drug action and resistance remain elusive. Here we studied the effects of the translocational status of HIV-1 RT on drug binding and inhibition of DNA synthesis. We identified "hot spots" for inhibition during active elongation. Site-specific footprinting analyses revealed that the corresponding complexes exist predominantly in the pre-translocational state. The sensitivity to PFA is significantly reduced with sequences that show a bias toward the post-translocational state. Binding studies showed that PFA stabilizes selectively the complex in the pre-translocated configuration. These findings are consistent with a Brownian ratchet model of polymerase translocation. The enzyme can rapidly shuttle between pre- and post-translocated states. The bound inhibitor acts like a pawl of a ratchet and prevents the forward motion of HIV-1 RT, whereas the bound nucleotide binds to the post-translocated complex and prevents the reverse motion. The proposed mechanisms of RT translocation and drug action are consistent with the PFA-resistant phenotypes. We show that certain sequences and the PFA-resistant E89K mutant diminishes the stability of the pre-translocated complex. In these cases, the enzyme is seen at multiple positions around the 3' end of the primer, which provides a novel mechanism for resistance. These findings validate the pre-translocated complex as a target for the development of novel, perhaps less toxic and more potent inhibitors that block HIV-1 RT translocation.
"The higher fluorescence intensity of polymerase complexes formed with the L412M-DNA polymerase indicates that the L412M-DNA polymerase favors formation of the highly fluorescent Pre-T complexes over Post-T complexes in the absence of PPi and dNTPs (Hariharan and Reha-Krantz, 2005). The role of the Pre-T complex in drug sensitivity is further substantiated by studies that show that PFA inhibits the HIV-1 reverse transcriptase by trapping a Pre-T complex (Marchand et al., 2007). We extend these findings by linking PAA-sensitivity of the L412M-DNA polymerase to increased intrinsic processivity, which suggests that Pre-T complexes are more stable, and less subject to enzyme dissociation than Post-T complexes (see further discussion by Li et al., 2010). "
[Show abstract][Hide abstract] ABSTRACT: DNA polymerases need to be engineered to achieve optimal performance for biotechnological applications, which often require high fidelity replication when using modified nucleotides and when replicating difficult DNA sequences. These tasks are achieved for the bacteriophage T4 DNA polymerase by replacing leucine with methionine in the highly conserved Motif A sequence (L412M). The costs are minimal. Although base substitution errors increase moderately, accuracy is maintained for templates with mono- and dinucleotide repeats while replication efficiency is enhanced. The L412M substitution increases intrinsic processivity and addition of phage T4 clamp and single-stranded DNA binding proteins further enhance the ability of the phage T4 L412M-DNA polymerase to replicate all types of difficult DNA sequences. Increased pyrophosphorolysis is a drawback of increased processivity, but pyrophosphorolysis is curbed by adding an inorganic pyrophosphatase or divalent metal cations, Mn(2+) or Ca(2+). In the absence of pyrophosphorolysis inhibitors, the T4 L412M-DNA polymerase catalyzed sequence-dependent pyrophosphorolysis under DNA sequencing conditions. The sequence specificity of the pyrophosphorolysis reaction provides insights into how the T4 DNA polymerase switches between nucleotide incorporation, pyrophosphorolysis and proofreading pathways. The L-to-M substitution was also tested in the yeast DNA polymerases delta and alpha. Because the mutant DNA polymerases displayed similar characteristics, we propose that amino acid substitutions in Motif A have the potential to increase processivity and to enhance performance in biotechnological applications. An underlying theme in this chapter is the use of genetic methods to identify mutant DNA polymerases with potential for use in current and future biotechnological applications.
Frontiers in Microbiology 08/2014; 5:380. DOI:10.3389/fmicb.2014.00380 · 3.99 Impact Factor
"When assayed against HIV-1 RT, it competitively blocks pyrophosphorolysis and PPi exchange reactions, suggesting that foscarnet and PPi share overlapping binding sites . It has been shown that foscarnet traps the RT pretranslocated complex preventing the binding of the next nucleotide, and, thus, the pretranslocated complex has been proposed as a target for drug discovery . In vivo and in vitro foscarnet-resistant HIV-1 variants have been shown to carry mutations in the RT gene at several positions, including W88G/S, E89K/G, L92I, A114S, S156A, Q161L, and H208Y [122–125]. "
[Show abstract][Hide abstract] ABSTRACT: During the retrotranscription process, characteristic of all retroviruses, the viral ssRNA genome is converted into integration-competent dsDNA. This process is accomplished by the virus-coded reverse transcriptase (RT) protein, which is a primary target in the current treatments for HIV-1 infection. In particular, in the approved therapeutic regimens two classes of drugs target RT, namely, nucleoside RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs). Both classes inhibit the RT-associated polymerase activity: the NRTIs compete with the natural dNTP substrate and act as chain terminators, while the NNRTIs bind to an allosteric pocket and inhibit polymerization noncompetitively. In addition to these two classes, other RT inhibitors (RTIs) that target RT by distinct mechanisms have been identified and are currently under development. These include translocation-defective RTIs, delayed chain terminators RTIs, lethal mutagenesis RTIs, dinucleotide tetraphosphates, nucleotide-competing RTIs, pyrophosphate analogs, RT-associated RNase H function inhibitors, and dual activities inhibitors. This paper describes the HIV-1 RT function and molecular structure, illustrates the currently approved RTIs, and focuses on the mechanisms of action of the newer classes of RTIs.
"However, our results collectively suggest that TGT action is best explained by formation of a pre-translocated TGT–TEC complex with natively folded TL. Specifically, we propose that TGT is a high-affinity pyrophosphate analog that acts in a mechanistically similar manner to phosphonoformic acid, a clinically used antiviral drug that targets B-family nucleic acid polymerases (43,44). Both drugs likely employ adjacent phosphate and carboxylate groups to mimic PPi and function by inducing backward translocation via active site closure, but are specific for non-homologous classes of enzymes. "
[Show abstract][Hide abstract] ABSTRACT: Multisubunit RNA polymerase (RNAP) is the central information-processing enzyme in all cellular life forms, yet its mechanism of translocation along the DNA molecule remains conjectural. Here, we report direct monitoring of bacterial RNAP translocation following the addition of a single nucleotide. Time-resolved measurements demonstrated that translocation is delayed relative to nucleotide incorporation and occurs shortly after or concurrently with pyrophosphate release. An investigation of translocation equilibrium suggested that the strength of interactions between RNA 3' nucleotide and nucleophilic and substrate sites determines the translocation state of transcription elongation complexes, whereas active site opening and closure modulate the affinity of the substrate site, thereby favoring the post- and pre-translocated states, respectively. The RNAP translocation mechanism is exploited by the antibiotic tagetitoxin, which mimics pyrophosphate and induces backward translocation by closing the active site.
Nucleic Acids Research 05/2012; 40(15):7442-51. DOI:10.1093/nar/gks383 · 9.11 Impact Factor
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