Effects of reverse transcriptase mediated displacement synthesis on reverse transcription and recombination

2005 2005

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Abstract (summary)

Reverse transcriptase (RT) is a retrovirus encoded DNA polymerase with ribonuclease H (RNase H) activity that is essential for converting the single-stranded, positive-sense RNA genome into double-stranded DNA at the reverse transcription stage of the viral life cycle. Reverse transcription is the primary process for generating genome sequence variation either through RT mediated mutagenesis, due to its low fidelity, or RT facilitated recombination. While synthesizing DNA, RT must utilize RNA and DNA in all possible primer/template combinations and encounters template sequences involved in complex secondary structures (RNA) or extensive base-pairing (DNA) which require strand-displacement synthesis activity; that is, the polymerase must disrupt nucleic acid base pairing in order to gain access to the template strand. All retroviral RTs studied thus far can perform displacement synthesis but whether a base-paired template and, therefore, strand displacement synthesis, influences mutation rate or recombination is unknown. Preliminary experiments designed to compare human immunodeficiency virus RT (HIV-RT) fidelity during the process of DNA and RNA displacement synthesis suggested that fidelity was greater during DNA displacement than during DNA non-displacement synthesis; however, a similar analysis was not possible for RNA displacement synthesis since HIV-RT encountered multiple strong pause sites only a few base pairs into an RNA duplex. Further analysis revealed that introducing an unpaired nucleotide to disrupt RNA base pair contiguity enhances polymerization through double-stranded RNA. This effect was a result of duplex melting ahead of the nascent DNA 3 terminus combined with attenuation of branch migration after pause site induced RT dissociation. Capitalizing on the ability to create a strong or weak template pause site without actually changing the template sequence, an in vitro strand transfer assay and an in vivo murine leukemia virus (MLV) based, single-cycle infection assay were employed to demonstrate that strong pausing during RNA displacement synthesis leads to increased strand transfer and recombination frequencies. Together the data demonstrates that both DNA and RNA duplex structures encountered during reverse transcription have a significant impact on the generation of retroviral genomic sequence diversity.

Indexing (details)

Molecular biology
0307: Molecular biology
Identifier / keyword
Biological sciences; HIV; RNA; Retrovirus; Reverse transcriptase
Effects of reverse transcriptase mediated displacement synthesis on reverse transcription and recombination
Lanciault, Christian P.
Number of pages
Publication year
Degree date
School code
DAI-B 66/07, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
9780542246289, 0542246287
Champoux, James J.
University of Washington
University location
United States -- Washington
Source type
Dissertations & Theses
Document type
Dissertation/thesis number
ProQuest document ID
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
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