Human immunodeficiency virus 1 reverse transcriptase. Template binding, processivity, strand displacement synthesis, and template switching.
We have analyzed the kinetics of DNA synthesis catalyzed by reverse transcriptase from human immunodeficiency virus 1 (HIV-1). Reverse transcriptase, overproduced in Escherichia coli and purified to homogeneity, has polymerase and RNase H activity. Reverse transcriptase forms a stable complex with poly(rA).oligo(dT) primer-templates in the absence of Mg2+ and dTTP with an equilibrium dissociation constant of 3 nM. Synthesis from these preformed complexes can be initiated, and restricted to a single processive cycle, by the simultaneous addition of Mg2+, dTTP, and excess competitor RNA. Preformed complexes decay with a maximal half-life of 2-3 min. Synthesis on poly(rA) templates is processive with an incorporation rate of 10-15 nucleotides/s at 37 degrees C. Processivity varies widely with the template used, increasing from a few to greater than 300 nucleotides in the order: poly(dA) less than double-stranded DNA less than single-stranded DNA less than single-stranded RNA less than poly(rA). On double-stranded DNA reverse transcriptase catalyzes limited strand-displacement synthesis of up to 50 nucleotides. On RNA-DNA hybrids significant DNA synthesis is observed only after degradation of the RNA strand by the RNase H activity of reverse transcriptase. Intermolecular strand switching occurs with poly(rA) templates. At low ionic strength reverse transcriptase can use multiple templates with a single primer, leading to products of greater than template length. Reverse transcriptase and primer do not have to dissociate during the exchange of template strands, thus allowing processive DNA synthesis across template borders.
Huber
H E
HE
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
McCoy
J M
JM
Seehra
J S
JS
Richardson
C C
CC
D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis*; D006678:HIV / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000494:pharmacology
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D013942:Thymine Nucleotides; D011061:Poly A; C015465:poly(dA); C018368:poly(rA).oligo(dT); D012313:RNA; D012194:RNA-Directed DNA Polymerase; D008274:Magnesium; C024157:thymidine 5'-triphosphate