Protein-DNA interactions

Recent advances in single-molecule enzymatic assays have profoundly changed how biochemical reactions are studied, revealing new insights into established problems and opening new lines of investigation. Through the removal of ensemble averaging, the distributions and fluctuations of molecular properties can be characterized, transient intermediates observed and identified, and catalytic mechanisms elucidated. The following shows ongoing efforts to study DNA-protein interactions at the single-molecule level.

We developed a simple experimental scheme based on flow-stretched DNA to monitor the activity of many individual nucleic acid enzymes on DNA simultaneously. Individual DNA molecules are attached at one end of the glass surface of a flow cell.

When a laminar flow is applied above the surface, the DNA molecules are stretched. If beads are attached to the free ends of the DNA, the molecules are stretched to a uniform tension by the drag force on the beads, allowing careful measurement of the DNA lengths by by tracking the positions of the beads with a CCD camera (accuracy ~ 10 nm for seconds, ~ 100 nm for minutes).

The force-extension curves of a single-stranded (ssDNA) and a double-stranded (dsDNA) l phage DNA (48,502 nt) are shown. The coiling of ssDNA causes it to be shorter than dsDNA at low stretching forces (< 6 pN). Consequently, a (enzymatic) conversion between dsDNA and ssDNA can be monitored by the variation of the DNA length at a constant stretching force.

Much of the pioneering work of studying the mechanical properties of individual DNA molecules is done in Carlos Bustamante's Lab at UC Berkeley. See their recent review in Nature.

DNA digestion by an individual λ exonuclease enzyme

We used this multiplexed single-molecule method to study the enzymatic activity of λ exonuclease, a homotrimeric, ringshaped enzyme required for recombination in bacteriophage λ, which degrades each strand of duplex DNA in the 5' to 3' direction.

Some of the individual λ exonuclease enzyme molecules digest processively all 48 knt of the λ phage DNA substrate. The digestion rate of the individual enzymes exhibits large fluctuations. These fluctuations are partially dependent on the sequence of the λ DNA. We calculate the variations in the enzymatic rate caused by the modulation on the free energy required to melt the cleaved bases (green trace*) and compare it to the average of multiple single-molecule rate traces (black trace). The high degree of correlation identifies the base melting as a rate-limiting step in the catalytic cycle.

*Kool, Ann.Rev.Bioph.Biom.Str. 30 (2001) 1; Bommarito et al., NAR 28 (2000) 1934