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. 2003 Aug;77(16):8621–8632. doi: 10.1128/JVI.77.16.8621-8632.2003

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Copyright © 2003, American Society for Microbiology

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FIG. 3. — Synthesis of full-length DNA using WT and mutant enzymes. (A) Graphic representation of the cell-free system used for the synthesis of full-length (−) ssDNA. The RNA template (pHIV-PBS) used in this system consists of 258 nucleotides (nt) at the 5′ end of the HIV-1 genome, which contains the R, U5, and PBS regions. (B) Synthesis of (−) ssDNA from the pHIV-PBS RNA template by each of the WT and mutant RTs. A time course experiment was performed by heat annealing tRNA₃^Lys to the RNA template to yield a complex which was incubated together with each RT studied at 37°C for 5 min. Reactions were initiated by the addition of dNTPs and monitored by incorporation of [α-³²P]dCTP into the growing DNA chain. The reactions were stopped at different times during a period of 60 min (lanes 1 to 12). Lane 13 exceptionally shows one of these reactions after 90 min. (C) Synthesis of (−) ssDNA from the pHIV-PBS RNA template by WT, M184V, and M184I RTs. Reaction conditions were as described for panel B.

FIG. 3. — Synthesis of full-length DNA using WT and mutant enzymes. (A) Graphic representation of the cell-free system used for the synthesis of full-length (−) ssDNA. The RNA template (pHIV-PBS) used in this system consists of 258 nucleotides (nt) at the 5′ end of the HIV-1 genome, which contains the R, U5, and PBS regions. (B) Synthesis of (−) ssDNA from the pHIV-PBS RNA template by each of the WT and mutant RTs. A time course experiment was performed by heat annealing tRNA₃^Lys to the RNA template to yield a complex which was incubated together with each RT studied at 37°C for 5 min. Reactions were initiated by the addition of dNTPs and monitored by incorporation of [α-³²P]dCTP into the growing DNA chain. The reactions were stopped at different times during a period of 60 min (lanes 1 to 12). Lane 13 exceptionally shows one of these reactions after 90 min. (C) Synthesis of (−) ssDNA from the pHIV-PBS RNA template by WT, M184V, and M184I RTs. Reaction conditions were as described for panel B.