doi:10.1016/j.jmb.2006.09.057 
Copyright © 2006 Elsevier Ltd All rights reserved.
Site-directed Mutagenesis in the Fingers Subdomain of HIV-1 Reverse Transcriptase Reveals a Specific Role for the β3–β4 Hairpin Loop in dNTP Selection
Scott J. Garforth1, Tae Woo Kim2, Michael A. Parniak3, Eric T. Kool2 and Vinayaka R. Prasad1, 
, 
1Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY10461, USA
2Department of Chemistry, Stanford University, Stanford, CA 94305, USA
3Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
Received 29 June 2006;
revised 15 September 2006;
accepted 19 September 2006.
Edited by J. Karn.
Available online 27 September 2006.
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Abstract
HIV-1 reverse transcriptase shares the key features of high fidelity polymerases, such as a closed architecture of the active site, but displays a level of fidelity that is intermediate to that of high fidelity, replicative polymerases and low fidelity translesion synthesis (TLS) polymerases. The β3–β4 loop of the HIV-1 RT fingers subdomain makes transient contacts with the dNTP and template base. To investigate the role of active site architecture in HIV-1 RT fidelity, we truncated the β3–β4 loop, eliminating contact between Lys65 and the γ-phosphate of dNTP. The mutant, in a manner reminiscent of TLS polymerases, was only able to incorporate a nucleotide that was capable of base-pairing with the template nucleotide, but not a nucleotide shape-analog incapable of Watson–Crick hydrogen bonding. Unexpectedly, however, the deletion mutant differed from the TLS polymerases in that it displayed an increased fidelity. The increased fidelity was associated with reduced dNTP binding affinity as measured using the dead end complex formation. In an effort to delineate the specific amino acid residue in the deleted segment responsible for this phenotype, we examined the K65 residue. Two substitution mutants, K65R and K65A were studied. The K65A mutant behaved similarly to the deletion mutant displaying dependence on Watson–Crick hydrogen bonding, increased fidelity and reduced dNTP-binding, while the K65R was more akin to wild-type enzyme. These results underscore the key role of the K65 residue in the phenotype observed in the deletion mutant. Based on the well-known electrostatic interaction between K65 and the γ-phosphate moiety of incoming dNTP substrate in the ternary complex structure of HIV-1 RT, we conclude that non-discriminatory interactions between β3–β4 loop and the dNTP in wild-type HIV-1 RT help lower dNTP selectivity. Our results show that the fidelity of dNTP insertion is influenced by protein interactions with the triphosphate moiety.
Keywords: RT fidelity; K65R; DNA replication; dNTP selection; steric effects
Abbreviations: HIV-1, human immunodeficiency virus type 1; RT, reverse transcriptase; TLS, translesion synthesis; KF, Klenow fragment; dFTP, deoxyribose-fluorotoluene triphosphate
Figure 1. Interactions between HIV-1 RT and the newly formed base-pair. (a) Position of the β3–β4 loop within the fingers subdomain of HIV-RT. The primer and template strands are indicated in dark and light brown, respectively. Fingers, Palm and Thumb subdomains of reverse transcriptase are colored orange, blue and green, respectively. Atomic coordinates were from 1rtd.33 (b) Interaction between the K65 residue in fingers β3–β4 loop and the γ-phosphate of the incoming dNTP. The hydrogen bond between residue K65 and the γ-phosphate of the incoming nucleotide is shown as a broken green line. (c) Diagrammatic representation of the informational (indicated in red) and non-informational (indicated in green) interactions affecting the incoming nucleotide:template pair in the HIV RT active site. Coordinates used are from the published HIV-1 RT ternary structure 1rtd,33 and the Figures were created using VMD57 and Chimera.58,59
Figure 2. (a) The sequence of HIV-1 RT, and the residues deleted in the Δβ3-4L mutant are shown. The residues comprising the β3 and β4 strands are shown in inverse, and the position of lysine 65 is indicated. (b) Template-primers used in the running-start and standing-start primer extension assays. The position of the F or T residue in the template strand is indicated F/T. (c) Structure of the thymine shape analog, difluorotoluene.18
Figure 3. Binding of the wild-type and Δβ3-4L mutant enzyme to an RNA-DNA template-primer. Binding reactions were analyzed on native polyacrylamide gels, which were quantified, and the percentage of unbound template-primer was plotted against the protein concentration. Broken lines show the 95% confidence interval.
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Figure 4. Ability of wild-type and Δβ3-4L mutant of HIV-1 RT to utilize the thymidine analog difluorotoluene (F). (a) Incorporation of adenosine opposite either template thymidine or F base by Klenow, wild-type and Δβ3-4L mutant HIV-1 RT enzymes. Reactions contained the running-start template-primer (2 nM) and either dATP (100 μM), allowing extension up to and including the modified nucleotide, or dATP, dCTP and dGTP (100 μM each), allowing extension to proceed to the end of the template. Reactions contained 20 nM enzyme, and were incubated for 1.5, 5 or 15 min. The position of the F analog in the modified template is labeled F/T. (b) A basic residue is required at position 65 for HIV reverse transcriptase to utilize the F template nucleotide under either running-start or standing-start conditions. Reactions were incubated for 10 min, and contained 100 nM of the respective template-primer, and either 5 nM enzyme (wild-type and K65R) or 10 nM enzyme (Δβ3-4L and K65A mutants). Lanes contained running-start or standing-start primers, indicated R and S, respectively. The templates contained either a thymine (template T) or difluorotoluene analog (template F). (c) Utilization of F as either the template nucleotide or incoming nucleotide is impaired in the Δβ3-4L mutant. Reactions contained 100 nM template-primer, 5 nM wild-type HIV-1 RT or 10 nM Δβ3-4L mutant, and were incubated for 5 min. Reactions contained either dATP or dFTP at the concentrations indicated. The unextended primer is labeled p, and the extension product p+1. (d) The K65R mutant utilizes a template F, but K65A does not. Reactions contained standing-start primer and F containing template and dATP at the concentrations indicated, and 5 nM each enzyme.
Figure 5. Quantitative analysis of incorporation of dATP opposite template F or template T by wild-type and Δβ3-4L enzymes. Single-nucleotide extension assays were performed with a range of dATP substrate concentrations and an enzyme concentration of 5 nM (wild-type) or 10 nM (Δβ3-4L), and the proportion of extended primer determined by phophoimager analysis. The results were fitted to Michaelis-Menten curves using GraphPad Prism. The points are from at least two independent experiments. For clarity, separate graphs are displayed for the wild-type and Δβ3-4L results.
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Figure 6. Quantitative analysis of the incorporation of a non-complementary nucleotide opposite template thymidine by Δβ3-4L and wild-type enzymes. Single-nucleotide extension assays performed with wild-type (5 nM) and Δβ3-4L mutant (10 nM) RTs. (a) Reactions contained template-primer (100 nM) and either the next complementary nucleotide (dATP) 0.1, 1, 10 and 100 μM or incorrect nucleotides (dCTP, dGTP or dTTP) at 50, 250 or 1250 μM, respectively. The unextended primer and the expected extension product (primer+1) are labeled. (b) Single-nucleotide extension assays were performed as above, and the proportion of extended primer determined by phophoimager analysis. The results were fitted to a Michaelis-Menten curve using GraphPad Prism. The points are from at least two independent experiments. Wild-type fins were calculated to be 4.3 × 10− 4, 8.4 × 10− 4 and 2.9 × 10− 3 for misincorporation of dCTP, dGTP and dTTP opposite a template thymine; Δβ3-4L fins for the same misincorporations were calculated as 1.6 × 10− 4, 9.4 × 10− 5 and 1.4 × 10− 3.
Figure 7. (a) Single-nucleotide extension assays performed with K65R and K65A mutant enzymes (5 nM each). Reactions contained template-primer (100 nM) and either the next complementary nucleotide (dATP) at 10, 100 or 1000 μM or incorrect nucleotides (dCTP, dGTP or dTTP) at 5, 50 or 500 μM, respectively. The unextended primer and the expected extension product (primer+1) are labeled. (b) Single nucleotide extension assays performed with the K65R mutant and wild-type reverse transcriptase (both at 5 nM). Reactions contained template-primer in which the next complimentary nucleotide was dTTP, which was included at 0.1, 1, 10 or 100 μM. Incorrect nucleotides (dATP, dCTP or dGTP) were included at concentrations of 0.05, 0.25, 1 or 2 mM. The template-primer pairs used in each case are shown at the bottom of the panels.
Figure 8. Nucleotide binding by wild-type, Δβ3-4L, K65R and K65A HIV reverse transcriptase, analyzed by dead-end complex formation. Ternary complexes consisting of reverse transcriptase, template annealed with 5′ end-labeled, 3′-blocked primer and the next complementary nucleotide, dATP (in a concentration range between 0.5 μM and 1 mM) were formed, and challenged with an unlabeled nucleic acid competitor. The dead end complex was separated from unbound primer-template by native PAGE.
Table 1.
Steady-state kinetic analysis of dAMP incorporation by wild-type HIV RT and the Δβ3-4L mutant, templated by either thymidine or difluorotoluene
a The incoming dNTP is specified first, followed by the template nucleotide.
b Template nucleotide F, a non-polar thymidine analog.
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-amino group of K65 was positioned 2.7 ± 0.1 Å from the γ-phosphate of the dTTP ligand and stabilized the triphosphate. In the R65 mutant, this distance increased to 4.2 ± 0.4 Å and the interaction energy with the ligand was less favorable, but the K70 
