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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 995-1006 |
| Number of pages | 12 |
| Journal | Journal of Molecular Biology |
| Volume | 395 |
| Issue number | 5 |
| DOIs | |
| State | Published - Feb 5 2010 |
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Keywords
- DNA flow-stretching assay
- DNA hairpin
- HIV-1 reverse transcriptase
- single molecule
- strand displacement synthesis
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology
Cite this
Single-Molecule Study of DNA Polymerization Activity of HIV-1 Reverse Transcriptase on DNA Templates. / Kim, Sangjin; Schroeder, Charles M.; Xie, X. Sunney.
In: Journal of Molecular Biology, Vol. 395, No. 5, 05.02.2010, p. 995-1006.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Single-Molecule Study of DNA Polymerization Activity of HIV-1 Reverse Transcriptase on DNA Templates
AU - Kim, Sangjin
AU - Schroeder, Charles M.
AU - Xie, X. Sunney
PY - 2010/2/5
Y1 - 2010/2/5
N2 - 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.
AB - 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.
KW - DNA flow-stretching assay
KW - DNA hairpin
KW - HIV-1 reverse transcriptase
KW - single molecule
KW - strand displacement synthesis
UR - http://www.scopus.com/inward/record.url?scp=73649092707&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=73649092707&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2009.11.072
DO - 10.1016/j.jmb.2009.11.072
M3 - Article
C2 - 19968999
AN - SCOPUS:73649092707
VL - 395
SP - 995
EP - 1006
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 5
ER -