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Updated: Oct 5, 2025
13:07
Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells
Published on: January 30, 2019
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Separable structural requirements for cDNA synthesis, nontemplated extension, and template jumping by a non-LTR
Sydney C Pimentel1, Heather E Upton1, Kathleen Collins1
1Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California, USA.
The Journal of Biological Chemistry
|January 23, 2022
Summary
Nontemplated nucleotide addition (NTA) is required for template jumping in reverse transcriptases (RTs), but this can be overcome with specific primer overhangs. This clarifies RT activity relationships for biological functions and lab applications.
Area of Science:
- Molecular Biology
- Biochemistry
- Evolutionary Biology
Background:
- Polymerase families exhibit evolutionary expansion, specializing in cellular roles.
- Many polymerases perform nontemplated nucleotide addition (NTA) alongside template-complementary synthesis.
- Reverse transcriptases (RTs) can perform NTA and template jumping, with unclear structural requirements and interdependence.
Purpose of the Study:
- To characterize structural requirements for cDNA synthesis, NTA, template jumping, and terminal transferase activity in Bombyx mori R2 non-long terminal repeat retroelement RT.
- To dissect the relationship between NTA and template jumping in RTs.
Main Methods:
- Sequence alignments and structure modeling guided mutagenesis of RT enzyme variants.
- Generated enzyme variants across conserved and subgroup-specific motifs.
- Assayed activities including cDNA synthesis, NTA, template jumping, and terminal transferase activity under various conditions.
Main Results:
- Enzyme variants showed diverse NTA profiles, unlinked to other synthesis activities or template jumping.
- Template jumping was shown to require NTA.
- NTA-deficient enzymes could still perform template jumping when provided with a primer duplex featuring a specific 3' overhang.
Conclusions:
- NTA is a prerequisite for template jumping in non-long terminal repeat retroelement RTs.
- Specific primer configurations can rescue template jumping in NTA-deficient RTs.
- Findings clarify RT activity relationships, informing specialization of biological functions and laboratory applications.
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