Biomedical subjects

Jason V Cristofaro

Publications and source records attributed to Jason V Cristofaro.

2 recordsLinked to original sources

PubMed2006

Selection of primer-template sequences that bind human immunodeficiency virus reverse transcriptase with high affinity.

A SELEX (systematic evolution of ligands by exponential enrichment)-based approach was developed to determine whether HIV-RT showed preference for particular primer-template sequences. A 70 nt duplex DNA was designed with 20 nt fixed flanking sequences at the 3' and 5' ends and a randomized 30 nt internal sequence. The fixed sequence at the 5' end contained a BbsI site six bases removed from the randomized region. BbsI cuts downstream of its recognition site generating four base 5' overhangs with recessed 3' termini. Cleavage produced a 50 nt template and 46 nt primer with the 3' terminus within the randomized region. HIV-RT was incubated with this substrate and material that bound RT was isolated by gel-shift. The recovered material was treated to regenerate the BbsI site, amplified by PCR, cleaved with BbsI and selected with HIV-RT again. This was repeated for 12 rounds. Material from round 12 bound approximately 10-fold more tightly than starting material. All selected round 12 primer-templates had similar sequence configuration with a 6-8 base G run at the 3' primer terminus, similar to the HIV polypurine tract. Further modifications indicate that the Gs were necessary and sufficient for strong binding.

Aptamers, Nucleotide↗

PubMed2002

Mutations in the ribonuclease H active site of HIV-RT reveal a role for this site in stabilizing enzyme-primer-template binding.

The RNase H activity of HIV-RT is coordinated by a catalytic triad (E478, D443, D498) of acidic residues that bind divalent cations. We examined the effect of RNase H deficient E(478)-->Q and D(549)-->N mutations that do not alter polymerase activity on binding of enzyme to various nucleic acid substrates. Binding of the mutant and wild-type enzymes to various nucleic acid substrates was examined by determining dissociation rate constants (k(off)) by titrating both Mg(2+) and salt concentrations. In agreement with the unaltered polymerase activity of the mutant, the k(off) values for the wild-type and mutant enzymes were essentially identical using DNA-DNA templates in the presence of 6 mM Mg(2+). However, with lower concentrations of Mg(2+) and in the absence of Mg(2+), although both enzymes dissociated more rapidly, the mutant enzymes dissociated several-fold more slowly than the wild type. This was also observed on RNA-DNA templates. These results indicate that alterations in residues essential for Mg(2+) binding have a pronounced positive effect on enzyme-template stability and that the negative residues in the RNase H region of the enzyme have a negative influence on binding in the absence of Mg(2+). In this regard RT is similar to other nucleic acid cleaving enzymes that show enhanced binding upon mutation of active site residues.

Binding Sites↗