Biomedical subjects

David J Keller

Publications and source records attributed to David J Keller.

4 recordsLinked to original sources

PubMed2005

Stepping statistics of single HIV-1 reverse transcriptase molecules during DNA polymerization.

DNA polymerases are protein machines that processively incorporate complimentary nucleotides into a growing double-stranded DNA (ds-DNA). Single-base nucleotide incorporation rates have been determined by stalling and restarting various polymerases, but intrinsic processive rates have been difficult to obtain, particularly for polymerases with low processivity, such as the human immunodeficiency virus type 1 reverse transcriptase (HIV RT) polymerase. Here we find, using a new fluorescence-based single-molecule polymerization assay, that the intrinsic processive DNA-dependent polymerization of HIV RT is approximately Poissionian (i.e. each nucleotide is added sequentially) with a rate of about 100 bases per second at 21 degrees C. From the same experiments, based on the stepping statistics of polymerization, we also estimate the rates for HIV RT early termination and final release of completely replicated primer-template DNA. In addition, by measuring the rate of polymerization as a function of temperature, we have estimated the activation energy for processive nucleotide incorporation.

DNA Replication↗

PubMed2005

Framework model for DNA polymerases.

DNA polymerases are complex machines with both chemical and mechanical functions. Recent crystal structures, ensemble kinetics, and single-molecule investigations have helped to elucidate the main properties of several DNA polymerases, all of which share common structural elements and a common basic mechanism, despite wide variations in amino acid sequence. The framework model is intended to aid in the understanding of these common features (and differences). It defines a class of models that automatically incorporates most of what is known about DNA polymerases within a single theoretical structure so that it is easier to make comparisons between them and to generate detailed models for specific polymerases. The framework model has three main elements: (1) a set of four key variables that describe the important motions within the protein-DNA-nucleotide complex, (2) a complete set of conformational states for the protein-DNA-nucleotide system, and (3) an approximate potential energy surface that controls the motions and transition rates between states. As an example application, we use the general framework ideas to build a detailed model for the HIV reverse transcriptase that is consistent with existing data, and predicts force-velocity curves and stepping-statistics histograms that can be directly compared to experiment.

Animals↗

PubMed2004

Closing of the fingers domain generates motor forces in the HIV reverse transcriptase.

Using the force sensor of an atomic force microscope, motor forces of the human immunodeficiency virus-1 reverse transcriptase were measured during active replication of a short DNA transcript. At low load forces the polymerase is mechanically slowed, whereas at high force (approximately 15 piconewton) it stalls. From recordings of estimated polymerase turnover velocity versus load force, an approximate force-velocity curve has been constructed. The shape of the curve suggests that load force strongly inhibits the rate-limiting step of the polymerase turnover cycle and that the combined effect of load on all steps involves an effective motion of about 1.6 nm. Earlier results from pre-steady-state kinetics experiments have identified the rate-limiting step as the closing of the fingers domain to form a tight catalytic complex. Together these findings indicate that the closing of the fingers domain is a major force-generating step for human immunodeficiency virus reverse transcriptase and, by extension, for all DNA polymerase machines.

Binding Sites↗

PubMed2004

Reversible control of free energy and topography of nanostructured surfaces.

We describe a facile method for the formation of dynamic nanostructured surfaces based on the modification of porous anodic aluminum oxide with poly(N-isopropyl acrylamide) (PNIPAAm) via surface-initiated atom transfer radical polymerization. The dynamic structure of these surfaces was investigated by atomic force microscopy (AFM), which showed dramatic changes in the surface nanostructure above and below the aqueous lower critical solution temperature of PNIPAAm. These changes in surface structure are correlated with changes in the macroscopic wettability of the surfaces, which was probed by water contact angle measurements. Principal component analysis was used to develop a quantitative correlation between AFM image intensity histograms and macroscopic wettability. Such correlations and dynamic nanostructured surfaces may have a variety of uses.

Acrylic Resins↗