title: Human immunodeficiency virus reverse transcriptase. Effect of primer length on template-primer binding.
abstract: Poly(rA).oligo(dT)n binding to human immunodeficiency virus type-1 reverse transcriptase heterodimer (p66-p51) was primer length-dependent. The estimated Kd for (n = 10-14) was 20-30 nM and for (n = 16-20) was 0.11-0.14 nM. Gel electrophoretic analysis of the patterns of primer extension was consistent with an abrupt change in the Kd between a primer length of 14 and 16 nucleotides. Further, the rate constant for dissociation of the reverse transcriptase-template-primer complex was determined from steady state kinetics and enzyme-template-primer trapping experiments to be independent of primer length. Thus, the abrupt change in Kd was most likely due to a change in the rate constant for formation of the reverse transcriptase-template-primer complex. A similar shift in the Kd for template-primer binding was observed with poly(dA).oligo(dT)n. Reverse transcriptase homodimer (p66) catalyzed the incorporation of dTMP into poly(rA).oligo(dT)n with the same primer length dependence observed for the heterodimer. In contrast, binding of the p51 homodimer to poly(rA).oligo(dT)n was independent of primer length. Thus, the RNase H domain may contribute to reverse transcriptase heterodimer or p66 homodimer binding to template-primers in which the primer length is greater than 14 nucleotides.
authors.affiliation: Division of Experimental Therapy, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
authors.forename: J E
authors.initials: JE
authors.lastname: Reardon
authors.forename: E S
authors.initials: ES
authors.lastname: Furfine
authors.forename: N
authors.initials: N
authors.lastname: Cheng
mesh_terms: D015497:HIV-1 / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D015394:Molecular Structure; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013698:Templates, Genetic
chemical_list: D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; C027903:oligo (dT); D012194:RNA-Directed DNA Polymerase