Abstract
Long terminal repeat-containing retrotransposons encode reverse transcriptases (RTs) that replicate their RNA into integratable, double-stranded DNA. A mutant version of the RT from Saccharomyces cerevisiae retrotransposon Ty1, in which one of the three active site aspartates has been changed to asparagine (D211N), is still capable of in vitro polymerization, although it is blocked for in vivo transposition. We generated recombinant WT and D211N Ty1 RTs to study RT function and determine specific roles for the Asp(211) residue. Presteady-state kinetic analysis of the two enzymes shows that the D211N mutation has minimal effect on nucleotide binding but reduces the k(pol) by approximately 230-fold. The mutation reduces binding affinity for both Mn(2+) and Mg(2+), indicating that the Asp(211) side chain helps create a tight metal binding pocket. Although both enzymes are highly processive and tend to remain bound to their initial substrate, each shows distinctive patterns of pausing, attributable to interactions between metal ions and the active site residue. These results provide insights to specific roles for the Asp(211) residue during polymerization and indicate unusual enzymatic properties that bear on the Ty1 replication pathway.
Highlights
Reverse transcriptases (RTs)1 are DNA polymerases that can use either RNA or DNA as their template
Examining mutant enzymes within Ty1 virus-like particles (VLPs), we found that none were capable of in vitro polymerization except for D211N, using a standard homopolymer assay [12]
Pre-steady-state Kinetics of dATP Incorporation of WT and Mutant D211N Ty1 RT—Recombinant versions of both WT and D211N mutant Ty1 RT enzymes were purified by nickel affinity chromatography (Fig. 1)
Summary
Plasmids and Strains—The plasmid p6H Ty1 IN-RT-RH (construct 2 in Ref. 16) contained WT Ty1 RT-RH plus a 115-amino acid contiguous C-terminal portion of Ty1 integrase fused to the N terminus of the RT-RH domain, all preceded by 6 histidine residues. Determination of the Kd of the Divalent Ions—Reactions were carried out in the presence of various concentrations of divalent ions, 0 –228 mM Mg2ϩ (as MgCl2) and 0 – 49 mM Mn2ϩ (as MnCl2), in a 10-min extension assay using the 5Ј-32P-end-labeled 14-mer/28-mer substrate, with 8.0 M each dNTP and equivalent amounts of active WT or D211N enzymes. Measurement of the Dissociation Rate for the Ty1 RT1⁄7DNA Complex— Ty1 RT (18 nM active WT or mutant enzyme) and 40 nM 5Ј-labeled 14-mer/28-mer were combined in a reaction mixture containing Mg2ϩ (final concentration, 10 mM) and preincubated in the extension buffer for 10 min to allow the enzyme1⁄7DNA complex to reach equilibrium. The obtained curves were fitted to a single exponential equation using a three-parameter fit for the decay (Equation 5; see Supplemental Material)
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