Utilization of a deoxynucleoside diphosphate substrate by HIV reverse transcriptase

Background: Deoxynucleoside triphosphates (dNTPs) are the normal substrates for DNA sysnthesis is catalyzed by polymerases such as HIV-1 reverse transcriptase (RT). However, substantial amounts of deoxynucleoside diphosphates (dNDPs) are also present in the cell. Use of dNDPs in HIV-1 DNA sysnthesis could have significant implications for the efficacy of nucleoside RT inhibitors such as AZT which are first line therapeutics fro treatment of HIV infection. Our earlier work on HIV-1 reverse transcriptase (RT) suggested that the interaction between the γ phosphate of the incoming dNTP and RT residue K65 in the active site is not essential for dNTP insertion, implying that this polymerase may be able to insert dNPs in addition to dNTPs. Methodology/Principal Findings: We examined the ability of recombinant wild type (wt) and mutant RTs with substitutions at residue K65 to utilize a dNDP substrate in primer extension reactions. We found that wild type HIV-1 RT indeed catalyzes incorporation of dNDP substrates whereas RT with mutations of residue K645 were unable to catalyze this reaction. Wild type HIV-1 RT also catalyzed the reverse reaction, inorganic phosphate-dependent phosphorolysis. Nucleotide-mediated phosphorolytic removal of chain-terminating 3′-terminal nucleoside inhibitors such as AZT forms the basis of HIV-1 resistance to such drugs, and this removal is enhanced by thymidine analog mutations (TAMs). We found that both wt and TAM-containing RTs were able to catalyze Pi-mediated phosphorolysis of 3′-terminal AZT at physiological levels of Pi with an efficacy similar to that for ATP-dependent AZT-excision. Conclusion: We have identified two new catalytic function of HIV-1 RT, the use of dNDPs as substrates for DNA synthesis, and the use of Pi as substrate for phosphorolytic removal of primer 3′-terminal nucleotides. The ability to insert dNDPs has been documented for only one other DNA polymerase The RB69 DNA polymerase and the reverse reaction employing inorganic phosphate has not been documented for any DNA polymerase. Importantly, our results show that Pi-mediated phosphorolysis can contribute to AZT resistance and indicates that factors that influence HIV resistance to AZT are more complex than previously appreciated. © 2008 Garforth et al

A model for transition of 5 '-nuclease domain of DNA polymerase I from inert to active modes

Bacteria contain DNA polymerase I (PolI), a single polypeptide chain consisting of similar to 930 residues, possessing DNA-dependent DNA polymerase, 3'-5' proofreading and 5'-3' exonuclease (also known as flap endonuclease) activities. PolI is particularly important in the processing of Okazaki fragments generated during lagging strand replication and must ultimately produce a double-stranded substrate with a nick suitable for DNA ligase to seal. PolI's activities must be highly coordinated both temporally and spatially otherwise uncontrolled 5'-nuclease activity could attack a nick and produce extended gaps leading to potentially lethal double-strand breaks. To investigate the mechanism of how PolI efficiently produces these nicks, we present theoretical studies on the dynamics of two possible scenarios or models. In one the flap DNA substrate can transit from the polymerase active site to the 5'-nuclease active site, with the relative position of the two active sites being kept fixed; while the other is that the 5'-nuclease domain can transit from the inactive mode, with the 5'-nuclease active site distant from the cleavage site on the DNA substrate, to the active mode, where the active site and substrate cleavage site are juxtaposed. The theoretical results based on the former scenario are inconsistent with the available experimental data that indicated that the majority of 5'-nucleolytic processing events are carried out by the same PolI molecule that has just extended the upstream primer terminus. By contrast, the theoretical results on the latter model, which is constructed based on available structural studies, are consistent with the experimental data. We thus conclude that the latter model rather than the former one is reasonable to describe the cooperation of the PolI's polymerase and 5'-3' exonuclease activities. Moreover, predicted results for the latter model are presented

Farmer-Scientist Knowledge Exchange

The last 25 years has seen a paradigm shift in the understanding of the nature of knowledge and how it is exchanged in the agricultural context. A changing backdrop, with the move towards multi-functional land management, persistent environmental problems and the search for sustainable agricultural approaches, has brought new challenges. At the same time the research agenda on knowledge has changed as an era of positivism, during which science and scientific experts were given unrivalled authority, was challenged by social studies of science that began to question the superiority of scientific knowledge, and value alternative forms of knowledge such as those held by farmers. Theory and practice of knowledge exchange in agriculture has evolved in line with this, shifting from a linear model of knowledge transfer to a perspective that integrates knowledge from multiple actors through facilitation and participation and emphasises learning in a social context

Direct observation of DNA threading in flap endonuclease complexes

Maintenance of genome integrity requires that branched nucleic acid molecules are accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates, and products, at resolutions of 1.9–2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme enclosed by an inverted Vshaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate’s single-stranded branch approaches, threads through, and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual “flycasting, thread, bend and barb” mechanis

K70Q Adds High-Level Tenofovir Resistance to “Q151M Complex” HIV Reverse Transcriptase through the Enhanced Discrimination Mechanism

HIV-1 carrying the “Q151M complex” reverse transcriptase (RT) mutations (A62V/V75I/F77L/F116Y/Q151M, or Q151Mc) is resistant to many FDA-approved nucleoside RT inhibitors (NRTIs), but has been considered susceptible to tenofovir disoproxil fumarate (TFV-DF or TDF). We have isolated from a TFV-DF-treated HIV patient a Q151Mc-containing clinical isolate with high phenotypic resistance to TFV-DF. Analysis of the genotypic and phenotypic testing over the course of this patient's therapy lead us to hypothesize that TFV-DF resistance emerged upon appearance of the previously unreported K70Q mutation in the Q151Mc background. Virological analysis showed that HIV with only K70Q was not significantly resistant to TFV-DF. However, addition of K70Q to the Q151Mc background significantly enhanced resistance to several approved NRTIs, and also resulted in high-level (10-fold) resistance to TFV-DF. Biochemical experiments established that the increased resistance to tenofovir is not the result of enhanced excision, as K70Q/Q151Mc RT exhibited diminished, rather than enhanced ATP-based primer unblocking activity. Pre-steady state kinetic analysis of the recombinant enzymes demonstrated that addition of the K70Q mutation selectively decreases the binding of tenofovir-diphosphate (TFV-DP), resulting in reduced incorporation of TFV into the nascent DNA chain. Molecular dynamics simulations suggest that changes in the hydrogen bonding pattern in the polymerase active site of K70Q/Q151Mc RT may contribute to the observed changes in binding and incorporation of TFV-DP. The novel pattern of TFV-resistance may help adjust therapeutic strategies for NRTI-experienced patients with multi-drug resistant (MDR) mutations