Crystal structures of an N-terminal fragment from moloney murine leukemia virus reverse transcriptase complexed with nucleic acid: functional implications for template-primer binding to the fingers domain 1 1Edited by D. C. Rees

Najmudin, Shabir; Coté, Marie L.; Sun, Dunming; Yohannan, Sarah; Montaño, Sherwin P.; Gu, Jun; Georgiadis, Millie M.

doi:10.1006/jmbi.1999.3477

Cited by 48 publications

(65 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As previously reported, K103A and R110A enzymes retain less than 10% of wild-type activity (2,3), while the remaining enzymes retain 40 to 70% of wild-type activity in the polyriboadenylate-oligodeoxyribosylthymine assay. However, despite their relatively high levels of activity on homopolymeric templates, all of the enzymes with substitutions for D114, R116, or N119 synthesized significantly less full-length product than the wild-type enzyme when assayed on an mRNA template, suggesting that they have processivity defects (25). In light of these findings regarding defects detectable in reconstituted reactions, it is not surprising that replication of viruses harboring these alterations was impaired.…”

Section: Discussionmentioning

confidence: 99%

“…K103, R110, D114, R116, and N119 are highly conserved. Predictions suggest that K103 and R110 form hydrogen bonds with the incoming nucleotide (2,3,18,25). D114, R116, and N119 are involved in critical interactions of the primer-template with the fingers domain in crystal structures of the N-terminal fragment of MLV RT complexed with DNA.…”

Section: Discussionmentioning

confidence: 99%

“…D114, R116, and N119 are involved in critical interactions of the primer-template with the fingers domain in crystal structures of the N-terminal fragment of MLV RT complexed with DNA. A mechanistic role in processive synthesis has been proposed for interactions of the primertemplate with the fingers domain binding site (25). Y64 was predicted to interact with the single-stranded template overhang.…”

Section: Discussionmentioning

confidence: 99%

“…Some of these same mutant enzymes have previously been purified and characterized in several assays, including those that use polyriboadenylate-oligodeoxyribosylthymine template-primers (25). The studied enzymes included K103A, R110A, D114N, R116K, D114N/R116K, R116L, and N119A, all of which failed to support replication or led to a delayed phenotype when tested in viruses in the experiments described here.…”

Section: Discussionmentioning

confidence: 99%

“…Spread of virus was monitored by assaying RT activity (37). Note that even noninfectious mutants (D114N, R116L, and N119A) yielded robust signals when examined as purified enzymes (25); hence this assay was an appropriate method for monitoring virus spread. To detect subtle differences in replication, virus was quantified by the amount of viral RNA by an RNase protection assay (35).…”

Section: Methodsmentioning

confidence: 99%

See 4 more Smart Citations

Structure-Based Moloney Murine Leukemia Virus Reverse Transcriptase Mutants with Altered Intracellular Direct-Repeat Deletion Frequencies

Pfeiffer

Georgiadis

Telesnitsky

2000

J Virol

Self Cite

View full text Add to dashboard Cite

Template switching rates of Moloney murine leukemia virus reverse transcriptase mutants were tested using a retroviral vector-based direct-repeat deletion assay. The reverse transcriptase mutants contained alterations in residues that modeling of substrates into the catalytic core had suggested might affect interactions with primer and/or template strands. As assessed by the frequency of functional lacZ gene generation from vectors in which lacZ was disrupted by insertion of a sequence duplication, the frequency of template switching varied more than threefold among fully replication-competent mutants. Some mutants displayed deletion rates that were lower and others displayed rates that were higher than that of wild-type virus. Replication for the mutants with the most significant alterations in template switching frequencies was similar to that of the wild type. These data suggest that reverse transcriptase template switching rates can be altered significantly without destroying normal replication functions.Reverse transcriptase (RT) must perform two template switches-the first and second strong-stop template switches-to complete integration-competent cDNA synthesis (13). It has been proposed that the requirement to perform these two replicative switches confers onto RT the tendency to make additional, nonrequired template switches that can result in genetic recombination (5, 41).Most DNA polymerases do not switch templates as frequently as RT does, which suggests that this process may require some structural or biochemical properties of RT. RT has been mutagenized extensively to identify features important for polymerization, RNase H activity, fidelity, drug resistance, and processivity (39). It is thought that RT pauses before switching templates; therefore, mutants with altered pausing and/or processivity may be affected in template switching (20,27,43,44).In this report, a panel of RT mutants was constructed based on the crystal structure of a catalytic fragment of Moloney murine leukemia virus (MLV) RT (12). Although the biochemical properties of the mutants were not tested here, the basis of these mutants' design was the hypothesis that by limiting interactions with the primer-template, RT template switching rates might be altered. These mutants contained alterations in residues proximal to the primer and/or template strands in a model of nucleic acid bound in the polymerase active site. Experiments presented in this report examined the replication of Moloney MLV mutants harboring these mutations and tested these mutants for effects on template switching during viral replication. MATERIALS AND METHODSPlasmids. RT mutations were introduced by PCR and standard cloning procedures into the infectious clone pMLV-neo (34) and pMLV ⌿Ϫ, a packagingdefective clone (30), and the entire PCR-generated region for each mutant was confirmed by dideoxynucleotide sequencing.Cells. NIH 3T3 cells and rat2 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum (Gibco). 293T-and ETbased...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Structure-Based Moloney Murine Leukemia Virus Reverse Transcriptase Mutants with Altered Intracellular Direct-Repeat Deletion Frequencies

Pfeiffer

Georgiadis

Telesnitsky

2000

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

Triazole‐Linked DNA as a Primer Surrogate in the Synthesis of First‐Strand cDNA

Fujino

Yasumoto

Yamazaki

et al. 2011

Chemistry An Asian Journal

View full text Add to dashboard Cite

A phosphate-eliminated nonnatural oligonucleotide serves as a primer surrogate in reverse transcription reaction of mRNA. Despite of the nonnatural triazole linkages in the surrogate, the reverse transcriptase effectively elongated cDNA sequences on the 3'-downstream of the primer by transcription of the complementary sequence of mRNA. A structure-activity comparison with the reference natural oligonucleotides shows the superior priming activity of the surrogate containing triazole-linkages. The nonnatural linkages also protect the transcribed cDNA from digestion reactions with 5'-exonuclease and enable us to remove noise transcripts of unknown origins.

show abstract

Structural and energetic characterization of nucleic acid‐binding to the fingers domain of Moloney murine leukemia virus reverse transcriptase

et al. 2004

Self Cite

View full text Add to dashboard Cite

Reverse transcriptase is an essential retroviral enzyme that replicates the single-stranded RNA genome of the retrovirus producing a double-stranded DNA copy, which is subsequently integrated into the host's genome. We have previously reported that processive DNA synthesis of Moloney murine leukemia virus reverse transcriptase (MMLV RT) is severely compromised by substitution of an Ala for the fingers domain residue Arg 116. In order to further investigate the role of Arg 116 in interactions of MMLV RT with nucleic acids, we have determined the crystal structure of the R116A N-terminal fragment and characterized the binding of two self-complementary DNA duplexes [d(CATGCATG)2 and d(CGCGCGCG)2] to both the wild-type and R116A fragments by isothermal titration calorimetry. The resultant thermodynamic profiles extrapolated to 25 degrees C reveal that binding of the wild-type N-terminal fragment to both DNA duplexes is enthalpy-driven and characterized by an unfavorable entropy. Although the temperature dependence of the respective protein-DNA binding enthalpies is markedly different reflecting distinct heat capacity changes, the binding free energies are nearly identical and relatively invariant to temperature (DeltaG approximately -6.0 kcal x mol(-1)). In contrast to the wild-type fragment, the R116A fragment exhibits no measurable affinity for either DNA duplex, yet its crystal structure reveals no significant changes when compared to the wild-type structures. We suggest that hydrogen-bonding interactions involving the fingers domain residue Arg 116 are critical for DNA binding as well as processive DNA synthesis by MMLV RT.

show abstract

Crystal structures of an N-terminal fragment from moloney murine leukemia virus reverse transcriptase complexed with nucleic acid: functional implications for template-primer binding to the fingers domain 1 1Edited by D. C. Rees

Cited by 48 publications

References 37 publications

Structure-Based Moloney Murine Leukemia Virus Reverse Transcriptase Mutants with Altered Intracellular Direct-Repeat Deletion Frequencies

Structure-Based Moloney Murine Leukemia Virus Reverse Transcriptase Mutants with Altered Intracellular Direct-Repeat Deletion Frequencies

Triazole‐Linked DNA as a Primer Surrogate in the Synthesis of First‐Strand cDNA

Structural and energetic characterization of nucleic acid‐binding to the fingers domain of Moloney murine leukemia virus reverse transcriptase

Contact Info

Product

Resources

About