Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The relatively low fidelity of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) was implicated as a major factor that contributes to the genetic variability of the virus. Extension of mismatched 3' termini of the primer DNA was shown to be a major determinant of the infidelity of HIV-1 RT. Human immunodeficiency virus type 2 (HIV-2) also shows extensive genetic variations. Therefore, we have analyzed the fidelity of the DNA-dependent DNA polymerase activity of HIV-2 RT and compared it with those of RTs of HIV-1 and murine leukemia virus (MLV). Like other retroviral RTs, the HIV-2 RT was shown to lack a 3'----5' exonuclease activity. The ability of HIV-2 RT to extend preformed 3'-terminal A:A, A:C and A:G mispairs was examined by quantitating the amount and length of extended primers. The results demonstrate a relatively efficient mispair extension by HIV-2 RT with a specificity of A:C much greater than A:A greater than A:G. The mispair extension appears to be affected mainly by the increase of apparent Km values rather than by the change in Vmax values. The relative extension frequencies from all mispairs with HIV-1 and HIV-2 RTs was 6- to 9-fold greater than that of MLV RT, suggesting that the HIV enzymes are substantially more error-prone than MLV RT.
...
PMID:Fidelity of the reverse transcriptase of human immunodeficiency virus type 2. 137 91

Human immunodeficiency virus type 1 (HIV-1) replicates efficiently in nonproliferating monocytes and macrophages but not in resting primary T lymphocytes. To determine the contribution of cell division to the HIV-1 replicative cycle in T cells, we evaluated HIV-1 expression, integration of proviral DNA, and production of infectious progeny virus in C8166 T-lymphoid cells blocked in cell division by treatment with either mitomycin, a DNA cross-linker, or aphidicolin, a DNA polymerase alpha inhibitor. The arrest of cell division was confirmed by assay of [3H]thymidine uptake; the nondividing cells remained viable for at least 3 days after treatment. HIV-1 was expressed and replicated equally well in nondividing and dividing C8166 cells, as judged by the comparison of the levels of p24 core antigens in culture supernatants, the proportion of cells expressing HIV-1 specific antigens, the pattern and quantity of HIV-1 DNA present in the extrachromosomal and total cellular DNA fractions, and the biological activity of progeny viruses. A polymerase chain reaction-based viral DNA integration assay indicated that HIV-1 provirus was integrated in C8166 cells treated with either of the two inhibitors of cell division. Similar results were obtained by using growth-arrested Jurkat T-lymphoid cells. We conclude that cell division and cellular DNA synthesis are not required for efficient HIV-1 expression in T cells.
...
PMID:Human immunodeficiency virus type 1 DNA synthesis, integration, and efficient viral replication in growth-arrested T cells. 809 28

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) initiates reverse transcription from tRNA(Lys3). HIV-1 RT is a heterodimer consisting of two polypeptides, p66 and p51. In this work, the possible role of each subunit of RT in the interaction with its natural primer tRNA(Lys3) was studied. Two recombinant forms of HIV-1 RT, heterodimer p66/p51 and homodimer p51/p51, were used. Previously we have expressed and purified recombinant RT p51/p51 which possesses DNA polymerase activity [El Dirani-Diab, R., Andreola, M. L., Nevinsky, G., Tharaud, D., Barr, P. J., Litvak, S. & Tarrago-Litvak, L. (1992) FEBS Lett. 301, 23-28]. Here we show that HIV-1 RT p51/p51 displays certain properties very similar to the p66/p51 recombinant enzyme. The homodimer was able to anneal tRNA(Lys3) to the primer-binding site of the HIV-1 RNA template leading to a functional complex capable of synthesizing cDNA. Further, the p51/p51 enzyme behaved like RT p66/p51 concerning the strong inhibition produced by a non-nucleoside RT inhibitor. These data show that for RT p51/p51, one of the subunits of the homodimer adopts a conformation similar to the catalytic subunit (p66) present in the heterodimeric form. Part of this work was devoted to the study of the complex between the recombinant forms of HIV-1 RT and its primer tRNA. Each enzymatic form was cross-linked to tRNA(Lys3) in the presence of a platinum derivative, giving different ribonucleoprotein complexes of molecular masses higher than 100 kDa, suggesting that primer tRNA may interact with both subunits in the heterodimeric enzyme. After RNase A treatment of the complex RT p66/p51 x tRNA, the label was mainly found to migrate with the p66 subunit, although some cross-linking was also found associated to the p51 subunit. These results show that the p66 and p51 subunits of RT interact with tRNA(Lys3). Moreover, cross-linking of tRNA(Lys3) with HIV-1 RT p66/p51 in the presence of a DNA template containing the primer-binding-site sequence yielded an enzymatically active complex.
...
PMID:p66/p51 and p51/p51 recombinant forms of reverse transcriptase from human immunodeficiency virus type 1--interactions with primer tRNA(Lys3), initiation of cDNA synthesis, and effect of inhibitors. 949 22

Human immunodeficiency virus type 1 reverse transcriptase (RT) has limited homology with DNA and RNA polymerases. The conserved Lys-220 of motif D is a signature of RNA-dependent polymerases. Motif D is located in the "palm" domain and forms a small loop from Thr-215 to Lys-223. This loop is absent from the polymerase I family of DNA-dependent polymerases. Analysis of RT structures in comparison with other polymerases reveals that the motif D loop has the potential to undergo a conformational change upon binding a nucleotide. We find that amino acid changes in motif D affect the interaction of RT with the incoming nucleotide. A chimeric RT in which the loop of motif D is substituted by the corresponding amino acid segment from Taq DNA polymerase lacking this loop has a decreased affinity for incoming nucleotides. We have also constructed a mutant RT where the conserved lysine at position 220 within the motif D is substituted with glutamine. Both RT(K220Q) and the chimeric RT are resistant in vitro to 3'-deoxy 3'-azidothymidine 5'-triphosphate (AZTTP). These results suggest that motif D is interacting with the incoming nucleotide and a determinant of the sensitivity of reverse transcriptases to AZTTP. We do not observe any interaction of motif D with the template primer.
...
PMID:The motif D loop of human immunodeficiency virus type 1 reverse transcriptase is critical for nucleoside 5'-triphosphate selectivity. 1058 59

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a putative source of the genomic hypermutation that promotes rapid evolution of HIV-1. To understand the molecular strategies that create a highly mutagenic DNA polymerase active site in HIV-1 RT, we investigated the roles of four residues in the beta 8-alpha E loop. Gln151, which interacts with the sugar of the incoming dNTP, and Lys154, which interacts with the template, yielded site-directed mutants with increased fidelity, suggesting that these residues are directly involved in the mutagenic architecture of the active site. Mutations at Gln151 and Lys154 also reduced processivity. Q151N RT showed enhanced ability to discriminate between TTP and AZT triphosphate, consistent with the observation that the Q151M mutation confers AZT resistance in vivo. Mutations at Gly152 greatly decreased RT activity; molecular modeling suggests that Gly152 is critical for the proper geometric alignment that permits base-pairing of the incoming dNTP with the template. Mutations at Trp153 reduced the expression level, and presumably the stability, of RT proteins in bacteria. These observations support the conclusion that interactions of active site residues in the beta 8-alpha E loop with incoming dNTPs and the template are determinants of the accuracy, processivity, and substrate selectivity of HIV-1 RT.
...
PMID:Molecular architecture of the mutagenic active site of human immunodeficiency virus type 1 reverse transcriptase: roles of the beta 8-alpha E loop in fidelity, processivity, and substrate interactions. 1097 52

Human immunodeficiency virus type 1 reverse transcriptase (RT) is an error-prone DNA polymerase. Structural determinants of its fidelity are incompletely understood. RT/template primer contacts have been shown to influence its fidelity and sensitivity to nucleoside analog inhibitors. The Phe(61) residue, located within the beta 3 sheet of the finger subdomain, is highly conserved among retroviral RTs. The crystal structure of a ternary complex revealed that Phe(61) contacts the first and second bases of the 5'-template overhang. To determine whether such contacts influence the dNTP-binding pocket, we performed a limited vertical scanning mutagenesis (Phe --> Ala, Leu, Trp, or Tyr) at Phe(61). The F61A mutant displayed the highest increase in fidelity, followed by the F61L and F61W variants, which had intermediate phenotypes. F61Y RT had a minimal effect. The increase in fidelity of the F61A mutant was corroborated by a 12-fold decrease in its forward mutation rate. The Phe(61) mutant RTs also displayed large reductions in sensitivity to 2',3'-dideoxythymidine triphosphate and 2',3'-dideoxy,2'3'-didehydrothymidine triphosphate. Mutants displaying the largest increase in fidelity (F61A and F61L) were also the most resistant. These results suggest that contacts between the finger subdomain of human immunodeficiency virus type 1 RT and the template 5'-overhang are important determinants of the geometry of the dNTP-binding pocket.
...
PMID:Substitutions of Phe61 located in the vicinity of template 5'-overhang influence polymerase fidelity and nucleoside analog sensitivity of HIV-1 reverse transcriptase. 1194 82

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), like all retroviral RTs, is a versatile DNA polymerase that can copy both RNA and DNA templates. In spite of extensive investigations into the structure-function of this enzyme, the structural basis for this dual template specificity is poorly understood. Biochemical studies with two mutations in HIV-1 RT that affect residues contacting the template-primer now provide some insight into this specialized property. The mutations are N255D and N265D, both adjoining the minor groove-binding track, in the thumb region. The N265D substitution led to a loss of processive polymerization on DNA but not on RNA, whereas N255D drastically reduced processive synthesis on both templates. This differential template usage was accompanied by a rapid dissociation of the N265D variant on DNA but not RNA templates, whereas the N255D variant rapidly dissociated from both templates. Molecular dynamics modeling suggested that N265D leads to a loss of template strand-specific hydrogen bonding, indicating that this is a key determinant of the differential template affinity. The N255D substitution caused local changes in conformation and a consequent loss of interaction with the primer, leading to a loss of processive synthesis with both templates. We conclude that N265 is part of a subset of template-enzyme contacts that enable RT to utilize DNA templates in addition to RNA templates and that such residues play an important role in facilitating processive DNA synthesis on both RNA and DNA templates.
...
PMID:Mutations proximal to the minor groove-binding track of human immunodeficiency virus type 1 reverse transcriptase differentially affect utilization of RNA versus DNA as template. 1271 77

Human immunodeficiency virus type I reverse transcriptase (RT) possesses distinct DNA polymerase and RNase H sites, whereas integrase (IN) uses the same active site to perform 3'-end processing and strand transfer of the proviral DNA. These four enzymatic activities are essential for viral replication and require metal ions. Two Mg2+ ions are present in the RT polymerase site, and one or two Mg2+ ions are required for the catalytic activities of RNase H and IN. We tested the possibility of inhibition of the RT polymerase and RNase H as well as the IN 3'-end processing and transfer activities of purified enzymes by a series of 3,7-dihydroxytropolones designed to target two Mg2+ ions separated by approximately 3.7 angstroms. The RT polymerase and IN 3' processing and strand transfer activities were inhibited at submicromolar concentrations, while the RNase H activity was inhibited in the low micromolar range. In all cases, the lack of inhibition by tropolones and O-methylated 3,7-dihydroxytropolones was consistent with the active molecules binding the metal ions in the active site. In addition, inhibition of the DNA polymerase activity was shown to depend on the Mg2+ concentration. Furthermore, selective inhibitors were identified for several of the activities tested, leaving some potential for design of improved inhibitors. However, all tested compounds exhibited cellular toxicity that presently limits their applications.
...
PMID:Inhibition of human immunodeficiency virus type 1 reverse transcriptase, RNase H, and integrase activities by hydroxytropolones. 1630 49

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.
...
PMID:Strand transfer events during HIV-1 reverse transcription. 1827 92

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) catalyzes synthesis of integration-competent, double-stranded DNA from the single-stranded viral RNA genome, combining both polymerizing and hydrolytic functions to synthesize approximately 20,000 phosphodiester bonds. Despite a wealth of biochemical studies, the manner whereby the enzyme adopts different orientations to coordinate its DNA polymerase and ribonuclease (RNase) H activities has remained elusive. Likewise, the lower processivity of HIV-1 RT raises the issue of polymerization site targeting, should the enzyme re-engage its nucleic acid substrate several hundred nucleotides from the primer terminus. Although X-ray crystallography has clearly contributed to our understanding of RT-containing nucleoprotein complexes, it provides a static picture, revealing few details regarding motion of the enzyme on the substrate. Recent development of site-specific footprinting and the application of single molecule spectroscopy have allowed us to follow individual steps in the reverse transcription process with significantly greater precision. Progress in these areas and the implications for investigational and established inhibitors that interfere with RT motion on nucleic acid is reviewed here.
...
PMID:Reverse transcriptase in motion: conformational dynamics of enzyme-substrate interactions. 1966 97


1 2 Next >>

---