title: Binding kinetics and affinities of heterodimeric versus homodimeric HIV-1 reverse transcriptase on DNA-DNA substrates at the single-molecule level.
abstract: During viral replication, HIV-1 reverse transcriptase (RT) plays a pivotal role in converting genomic RNA into proviral DNA. While the biologically relevant form of RT is the p66-p51 heterodimer, two recombinant homodimer forms of RT, p66-p66 and p51-p51, are also catalytically active. Here we investigate the binding of the three RT isoforms to a fluorescently labeled 19/50-nucleotide primer/template DNA duplex by exploiting single-molecule protein-induced fluorescence enhancement (SM-PIFE). PIFE, which does not require labeling of the protein, allows us to directly visualize the binding/unbinding of RT to a double-stranded DNA substrate. We provide values for the association and dissociation rate constants of the RT homodimers p66-p66 and p51-p51 with a double-stranded DNA substrate and compare those to the values recorded for the RT heterodimer p66-p51. We also report values for the equilibrium dissociation constant for the three isoforms. Our data reveal great similarities in the intrinsic binding affinities of p66-p51 and p66-p66, with characteristic Kd values in the nanomolar range, much smaller (50-100-fold) than that of p51-p51. Our data also show discrepancies in the association/dissociation dynamics among the three dimeric RT isoforms. Our results further show that the apparent binding affinity of p51-p51 for its DNA substrate is to a great extent time-dependent when compared to that of p66-p66 and p66-p51, and is more likely determined by the dimer dissociation into its constituent monomers rather than the intrinsic binding affinity of dimeric RT.
authors.affiliation: Department of Chemistry and Center for Self Assembled Chemical Structures (CSACS/CRMAA), McGill University, 801 Sherbrooke Street West, Montreal, QC, H3A 0B8, Canada.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Marko
authors.forename: Hsiao-Wei
authors.initials: HW
authors.lastname: Liu
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Ablenas
authors.forename: Maryam
authors.initials: M
authors.lastname: Ehteshami
authors.forename: Matthias
authors.initials: M
authors.lastname: Götte
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Cosa
mesh_terms: D002232:Carbocyanines / Q000737:chemistry; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D002232:Carbocyanines; D017931:DNA Primers; D005456:Fluorescent Dyes; D020033:Protein Isoforms; D011994:Recombinant Proteins; C089187:cyanine dye 3; D012313:RNA; D004247:DNA; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase