Binding Kinetics and Affinities of Heterodimeric versus Homodimeric HIV-1 Reverse Transcriptase on DNA鈥揇NA Substrates at the Single-Molecule Level

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• 作者：Ryan A. Marko ; Hsiao-Wei Liu ; Christopher J. Ablenas ; Maryam Ehteshami ; Matthias G枚tte ; Gonzalo Cosa
• 刊名：The Journal of Physical Chemistry B
• 出版年：2013
• 出版时间：April 25, 2013
• 年：2013
• 卷：117
• 期：16
• 页码：4560-4567
• 全文大小：398K
• 年卷期：v.117,no.16(April 25, 2013)
• ISSN：1520-5207

文摘

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鈥損51 heterodimer, two recombinant homodimer forms of RT, p66鈥損66 and p51鈥損51, 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鈥損66 and p51鈥損51 with a double-stranded DNA substrate and compare those to the values recorded for the RT heterodimer p66鈥損51. 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鈥損51 and p66鈥損66, with characteristic K_d values in the nanomolar range, much smaller (50鈥?00-fold) than that of p51鈥損51. 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鈥損51 for its DNA substrate is to a great extent time-dependent when compared to that of p66鈥損66 and p66鈥損51, and is more likely determined by the dimer dissociation into its constituent monomers rather than the intrinsic binding affinity of dimeric RT.