Parameters that influence processive synthesis and site-specific termination by human immunodeficiency virus reverse transcriptase on RNA and DNA templates.
ABSTRACT We have examined the parameters that determine the length and distribution of products synthesized processively by the human immunodeficiency virus reverse transcriptase (HIV-RT). On native or homopolymer templates, the overall length distribution of processively synthesized products is increased by increased temperature or deoxynucleoside triphosphate concentration, or decreased ionic strength. Specific terminations of processive synthesis on either native DNA or RNA templates occur most frequently at positions where the reverse transcriptase (RT) pauses during synthesis. These sites correlate with the template sequence 3'-(A/U)(A/U)(G/C)-5', particularly when this sequence is predicted to be base paired with another region of the template in a secondary structure. Many positions of termination are in similar positions on DNA or RNA templates. Notable exceptions are runs of A residues, which promote termination on DNA but not RNA templates. Termination intensities vary when different RTs are used demonstrating an influence of RT structure.
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ABSTRACT: HIV-1 RT (human immunodeficiency virus-1 reverse transcriptase) is a multifunctional polymerase responsible for reverse transcription of the HIV genome, including DNA replication on both RNA and DNA templates. During reverse transcription in vivo, HIV-1 RT replicates through various secondary structures on RNA and single-stranded DNA (ssDNA) templates without the need for a nucleic acid unwinding protein, such as a helicase. In order to understand the mechanism of polymerization through secondary structures, we investigated the DNA polymerization activity of HIV-1 RT on long ssDNA templates using a multiplexed single-molecule DNA flow-stretching assay. We observed that HIV-1 RT performs fast primer extension DNA synthesis on single-stranded regions of DNA (18.7 nt/s) and switches its activity to slow strand displacement synthesis at DNA hairpin locations (2.3 nt/s). Furthermore, we found that the rate of strand displacement synthesis is dependent on the GC content in hairpin stems and template stretching force. This indicates that the strand displacement synthesis occurs through a mechanism that is neither completely active nor passive: that is, the opening of the DNA hairpin is driven by a combination of free energy released during dNTP (deoxyribonucleotide triphosphate) hydrolysis and thermal fraying of base pairs. Our experimental observations provide new insight into the interchanging modes of DNA replication by HIV-1 RT on long ssDNA templates.Journal of Molecular Biology 02/2010; · 3.91 Impact Factor
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