Interaction of the Ty3 Reverse Transcriptase Thumb Subdomain with Template-Primer

Bibiłło, Arkadiusz; Lener, Daniela; Tewari, Alok K.; Grice, Stuart F. J. Le

doi:10.1074/jbc.m502457200

Cited by 11 publications

(16 citation statements)

References 60 publications

(47 reference statements)

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“…As might be expected, removing template nucleobase Ϫ1 was most deleterious for DNA polymerase function because this would most likely affect positioning of the primer 3Ј-OH in the active site. These data again support our recent mutagenesis studies on the Ty3 thumb subdomain showing that alteration of the counterpart of ␣-helix H severely influences the stability of the replication complex (42). Thus, when Ty3 RT adopts the orientation with its RNase H catalytic center positioned for cleavage at the PPT/U3 junction, it is not unreasonable to assume that abnormal nucleic acid geometry upstream of this junction is also sensed by its thumb domain.…”

Section: Resultssupporting

confidence: 75%

“…This effect continued as far as the Ϫ5/Ϫ4LNA sub- stitution, after which subsequent positions of LNA substitution presumably already lie beyond this important protein motif. Current HIV literature (37-41) and our recent work with Ty3 RT (42) suggest that this would be the thumb of the DNA polymerase domain, where ␣-helix H of the HIV-1 enzyme may function as a sensor of nucleic acid geometry (37)(38)(39)(40)(41). Another feature, although small, was pausing at multiple positions throughout the template for substrates containing LNA insertions between positions Ϫ11 and Ϫ4 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The DNA synthesis data of Fig. 9, combined with our recent mutagenesis studies on the Ty3 RT thumb subdomain (42), show that it, too, is responsive to subtle alterations to duplex architecture. Thus, the model we envisage proposes that each LNA-induced alteration to the sugar pucker between positions Ϫ11 and Ϫ8 can provide a transient interaction site for the Ty3 RT thumb subdomain, which would relocate the RNase H catalytic center over sites ahead of the PPT/U3 junction.…”

Section: Discussionmentioning

confidence: 99%

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Examining Ty3 Polypurine Tract Structure and Function by Nucleoside Analog Interference

Dash

Marino

Grice

2006

Journal of Biological Chemistry

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We have combined nucleoside analog interference with chemical footprinting, thermal denaturation, NMR spectroscopy, and biochemical studies to understand recognition of the polypurine tract (PPT) primer of the Saccharomyces cerevisiae long terminal repeatcontaining retrotransposon Ty3 by its cognate reverse transcriptase. Locked nucleic acid analogs, which constrain sugar ring geometry, were introduced pairwise throughout the PPT (؊)-DNA template, whereas abasic tetrahydrofuran linkages, which lack the nucleobase but preserve the sugar phosphate backbone, were introduced throughout the (؊)-strand DNA template and (؉)-strand RNA primer. Collectively, our data suggest that both the 5-and 3-portions of the PPT-containing RNA/DNA hybrid are sensitive to nucleoside analog substitution, whereas the intervening region can be modified without altering cleavage specificity. These two regions most likely correspond to portions of the PPT that make close contact with the Ty3 reverse transcriptase thumb subdomain and RNase H catalytic center, respectively. Achieving a similar phenotype with nucleoside analogs that have different effects on duplex geometry reveals structural features that are important mediators of Ty3 PPT recognition. Finally, the results from introducing tetrahydrofuran lesions around the scissile PPT/unique 3-sequence junction indicate that template nucleobase ؊1 is dispensable for catalysis, whereas a primer nucleobase on either side of the junction is necessary.Although reverse transcription in long terminal repeat (LTR) 3 -containing retrotransposons of Saccharomyces cerevisiae (Ty1 and Ty3) and Saccharomyces paradoxus (Ty5) can be likened to that in retroviruses, notable differences have recently been documented. These include (a) initiation of (Ϫ)-strand DNA synthesis from an internal site of the host-coded tRNA primer as opposed to its 3Ј terminus (1), (b) initiation of (Ϫ)-strand DNA synthesis via self-priming (2), (c) use of a bipartite primer-binding site derived from each end of the viral RNA genome to initiate (Ϫ)-strand DNA synthesis (3), and (d) divergence in size and sequence of the (ϩ)-strand polypurine tract (PPT) primers (see Fig. 1) (4, 5). A detailed biochemical study of these processes has been hampered by the lack of purified LTR-containing retrotransposon reverse transcriptases (RTs). However, the availability of active recombinant Ty1 and Ty3 enzymes (6, 7) now allows interactions with their cis-acting signals to be compared and contrasted with those of the retroviral counterparts.A focus of our recent studies (8 -13) has been understanding the mechanism that positions RT on the PPT-containing RNA/DNA hybrid for accurate cleavage at the PPT/unique 3Ј-sequence (U3) junction. Such specificity is required to create the (ϩ)-strand primer and later to remove it from nascent (ϩ)-DNA, thereby creating an integration-competent, double-stranded DNA provirus. Surprisingly, although a compilation of PPT sequences (14, 15) suggests a common "modular" make-up of homopolymeric rA:dT and rG:dC blo...

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Section: Resultssupporting

confidence: 75%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Examining Ty3 Polypurine Tract Structure and Function by Nucleoside Analog Interference

Dash

Marino

Grice

2006

Journal of Biological Chemistry

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“…Examples of such motifs include the "translocation track" (8,9) and primer grip of the p66 thumb subdomain (2), the template grip (10), and a second primer grip in the vicinity of the ribonuclease H catalytic center (6). Roles for these motifs have been investigated in vitro by site-directed mutagenesis (8,9,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and complemented by studying the effects of strategic placement of nucleoside analogs into either the template or primer (21)(22)(23)(24)(25)(26)(27)(28). In parallel, replication of viruses harboring mutations in these motifs have been extensively examined in vivo (17, 29 -31).…”

mentioning

confidence: 99%

Examining Interactions of HIV-1 Reverse Transcriptase with Single-stranded Template Nucleotides by Nucleoside Analog Interference

Dash

Fisher

Prasad

et al. 2006

Journal of Biological Chemistry

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Crystallographic studies have implicated several residues of the p66 fingers subdomain of human immunodeficiency virus type-1 reverse transcriptase in contacting the single-stranded template overhang immediately ahead of the DNA polymerase catalytic center. This interaction presumably assists in inducing the appropriate geometry on the template base for efficient and accurate incorporation of the incoming dNTP. To investigate this, we introduced nucleoside analogs either individually or in tandem into the DNA template ahead of the catalytic center and investigated whether they induce pausing of the replication machinery before serving as the template base. Analogs included abasic tetrahydrofuran linkages, neutralizing methylphosphonate linkages, and conformationally locked nucleosides. In addition, several Phe-61 mutants were included in our analysis, based on previous data indicating that altering this residue affects both strand displacement synthesis and the fidelity of DNA synthesis. We demonstrate here that altering the topology of the template strand two nucleotides ahead of the catalytic center can interrupt DNA synthesis. Mutating Phe-61 to either Ala or Leu accentuates this defect, whereas replacement with an aromatic residue (Trp) allows the mutant enzyme to bypass the template analogs with relative ease.The structures of unliganded, inhibitor-bound, and nucleic acid-containing human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) 4 have been instrumental in dissecting the multifunctional properties of this key retroviral enzyme and exploiting it as a target for antiviral therapy (1-6). In particular, co-crystals of RT with duplex DNA (2, 7) and an RNA/DNA hybrid (6) have highlighted critical contacts mediated by protein motifs of its N-terminal DNA polymerase and C-terminal ribonuclease H domains. Examples of such motifs include the "translocation track" (8, 9) and primer grip of the p66 thumb subdomain (2), the template grip (10), and a second primer grip in the vicinity of the ribonuclease H catalytic center (6). Roles for these motifs have been investigated in vitro by site-directed mutagenesis (8,9,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and complemented by studying the effects of strategic placement of nucleoside analogs into either the template or primer (21-28). In parallel, replication of viruses harboring mutations in these motifs have been extensively examined in vivo (17, 29 -31).Although a number of studies have investigated the geometry of duplex nucleic acid positioned between the catalytic centers of HIV-1 RT, the manner in which the single-stranded template traverses the p66 fingers subdomain before accessing the DNA polymerase catalytic center is less well understood. Stated differently, when is the relatively disordered, singlestranded nucleic acid oriented to serve as the template base for accurate and efficient incorporation of the incoming dNTP? Although less precise in the resolution they provide, enzymatic footprinting and photocross-linking studies (32, 33) sugg...

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“…It differs from that of retroviral PPT in being slightly shorter and lacking homopolymeric tracts. Based on the sensitivity of RNaseH activities to mutation and the crystal structure, Ty3 RT thumb residues Q290-F292-G294-N297-Y298 (corresponding to HIV-1 RT Q258-L260-G262-N265-W266) interact with the DNA backbone and position the PPT into the RNaseH active site (106,110). Nuclear magnetic resonance of unbound Ty3 PPT heteroduplex and the crystal structure of the RT-bound PPT heteroduplex agree on mostly base-paired and A-form duplex lacking major structural distortions.…”

Section: Ty3 Cdna Primersmentioning

confidence: 99%

Ty3, a Position-specific Retrotransposon in Budding Yeast

Sandmeyer

Patterson²,

Bilanchone³

2015

Microbiol Spectr

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Long terminal repeat (LTR) retrotransposons constitute significant fractions of many eukaryotic genomes. Two ancient families are Ty1/Copia (Pseudoviridae) and Ty3/Gypsy (Metaviridae). The Ty3/Gypsy family probably gave rise to retroviruses based on the domain order, similarity of sequences, and the envelopes encoded by some members. The Ty3 element of Saccharomyces cerevisiae is one of the most completely characterized elements at the molecular level. Ty3 is induced in mating cells by pheromone stimulation of the mitogen-activated protein kinase pathway as cells accumulate in G1. The two Ty3 open reading frames are translated into Gag3 and Gag3-Pol3 polyprotein precursors. In haploid mating cells Gag3 and Gag3-Pol3 are assembled together with Ty3 genomic RNA into immature virus-like particles in cellular foci containing RNA processing body proteins. Virus-like particle Gag3 is then processed by Ty3 protease into capsid, spacer, and nucleocapsid, and Gag3-Pol3 into those proteins and additionally, protease, reverse transcriptase, and integrase. After haploid cells mate and become diploid, genomic RNA is reverse transcribed into cDNA. Ty3 integration complexes interact with components of the RNA polymerase III transcription complex resulting in Ty3 integration precisely at the transcription start site. Ty3 activation during mating enables proliferation of Ty3 between genomes and has intriguing parallels with metazoan retrotransposon activation in germ cell lineages. Identification of nuclear pore, DNA replication, transcription, and repair host factors that affect retrotransposition has provided insights into how hosts and retrotransposons interact to balance genome stability and plasticity.

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Interaction of the Ty3 Reverse Transcriptase Thumb Subdomain with Template-Primer

Cited by 11 publications

References 60 publications

Examining Ty3 Polypurine Tract Structure and Function by Nucleoside Analog Interference

Examining Ty3 Polypurine Tract Structure and Function by Nucleoside Analog Interference

Examining Interactions of HIV-1 Reverse Transcriptase with Single-stranded Template Nucleotides by Nucleoside Analog Interference

Ty3, a Position-specific Retrotransposon in Budding Yeast

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