Abstract
A modified SELEX (Systematic Evolution of Ligands by Exponential Enrichment) pr,otocol (referred to as PT SELEX) was used to select primer–template (P/T) sequences that bound to the vaccinia virus polymerase catalytic subunit (E9) with enhanced affinity. A single selected P/T sequence (referred to as E9-R5-12) bound in physiological salt conditions with an apparent equilibrium dissociation constant (KD,app) of 93 ± 7 nM. The dissociation rate constant (koff) and binding half-life (t1/2) for E9-R5-12 were 0.083 ± 0.019 min−1 and 8.6 ± 2.0 min, respectively. The values indicated a several-fold greater binding ability compared to controls, which bound too weakly to be accurately measured under the conditions employed. Loop-back DNA constructs with 3′-recessed termini derived from E9-R5-12 also showed enhanced binding when the hybrid region was 21 nucleotides or more. Although the sequence of E9-R5-12 matched perfectly over a 12-base-pair segment in the coding region of the virus B20 protein, there was no clear indication that this sequence plays any role in vaccinia virus biology, or a clear reason why it promotes stronger binding to E9. In addition to E9, five other polymerases (HIV-1, Moloney murine leukemia virus, and avian myeloblastosis virus reverse transcriptases (RTs), and Taq and Klenow DNA polymerases) have demonstrated strong sequence binding preferences for P/Ts and, in those cases, there was biological or potential evolutionary relevance. For the HIV-1 RT, sequence preferences were used to aid crystallization and study viral inhibitors. The results suggest that several other DNA polymerases may have P/T sequence preferences that could potentially be exploited in various protocols.
Highlights
Introduction published maps and institutional affilThe recognition of recessed 30 termini is a hallmark of all DNA polymerases, except for some viral enzymes that can initiate synthesis using specific viral proteins
The sequences fell into three distinct lineages, represented by E9-R5-12 (Protein-Systematic Evolution of Ligands by Exponential Enrichment (SELEX) round #-sequence clone), which was recovered twice, along with four other closely related sequences, E9-R5-4, which was recovered twice, and E9-R5-3, which was recovered five times, along with one other closely related sequence (E9-R5-2)
We show that E9, like other DNA polymerases [2,3,7], demonstrates preferential binding to specific P/T sequences
Summary
The recognition of recessed 30 termini is a hallmark of all DNA polymerases, except for some viral enzymes that can initiate synthesis using specific viral proteins Our group has shown that viral RTs, including those from human immunodeficiency virus-1 (HIV-1), Maloney murine leukemia virus (MuLV). Avian myeloblastosis virus (AMV), bind more tightly to DNA-DNA primer–templates with runs of G (G-tract) at the 30 primer end [2,3]. The G-tract on the DNA primer mimics the G-tract found on the polypurine tract (PPT) RNAs of these viruses. The findings suggest that RTs and PPTs may have co-evolved, leading to strong interactions and the proper orientation of the RT at the 30 end of the PPT.
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