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Studying DNA and DNA/Protein Interaction

Dorothy Erie in Chemistry and other collaborators are studying the shape of DNA and the behavior of DNA/Protein complexes. Within the Resource, we are developing image-analysis tools to help build quantitative models of DNA systems, AFM simulation tools to provide precise descriptions of what should be visible in experiment scans, and visualization tools to enable the direct visual comparison of experiment output with the expected result of scanning a model with an AFM tip.

Extracting DNA shape from images

The image above right shows a model of a tube that was extracted from an AFM image using MIDAG-produced CORE-tracking software. The model is drawn in green raised above the image from which it was extracted. This software (Yonatan Fridman's dissertation work) is able to extract the 3D shape of the tube along with its width and curvature information at each point along the tube. This enables us to extract and analyze tubular objects such as DNA, fibrin, and mucin from images.

The image on the left shows the centerline trace (in red) and boundaries (in blue) of one of several strands of DNA on a surface. The DNA was scanned by Dorothy Erie's group in a Digital Instruments AFM. The user selects points on the ends of the strand and a starting direction and the tube tracing code finds the centerline, reporting orientation (for curvature) and radius along the entire strand. The code used here is available on the CISMM software download page.

The image below shows a trace of DNA on an AFM image as it passes through a protein. This tracing was produced by code that was custom-designed for quantitatively producing the length of DNA outside the protein, the length of the interpolated line through the protein, and the angle between the incoming and outgoing DNA strands. The length information enables the scientists to estimate how much DNA is free to wrap around the protein (since they know the total strand length) while the angle information provides hints as to the geometry of wrapping (see below for why this is important). The boundary of the DNA as detected by the Resource code is shown as a thin blue line. The medial axis of the DNA is shown as a thin red line. The interpolated trace of the DNA and an estimated bend angle as the DNA leaves the protein is indicated by a thin green line passing through the protein. The use of this code produced an order-of-magnitude decrease in analysis time and more accuracy than the manual estimation being routinely used to extract this parameter.

By feeding these extracted models to the AFM simulator (described next), a scientist can investigate which portions of the AFM scans are well-explained by the model. Using direct visual comparison between the model and scan (described on the Mixing Model and Simulation core project page), a scientist can compare the model's fit and discover where it needs improving.

Which way does the DNA wrap?

One question that is being investigated by Dorothy Erie's group is what conformation DNA makes as it wraps around the lac repressor enzyme. The binding sites on the DNA, the binding sites on lac repressor, and the length of the DNA strand bound to the protein are all known. What is not known is the path taken by the DNA around or through the lac repressor when it is bound. Dorothy and her team are studying three candidate wrappings by binding lac repressor to DNA, depositing the bound complexes on a surface, and scanning the complex with an AFM.

As described in our SPIE paper, hardware acceleration enables interactive calculation of imaging artifacts that would be expected from scanning a model with an atomic force microscope (AFM). This enables direct comparison between experimental results and expected microscope scans for both hand-made models and atom-coordinate data. The images to the left show this technique applied to a DNA/lac-repressor complex. The four images in the upper right corner show the result of imaging the crystallographics atom coordinates with increasingly coarse AFM tips. The bottom image shows a simplified model of a DNA strand wrapped around a protein; the image above it shows the AFM scan expected when this model is scanned. The image in the upper left shows an actual AFM scan of DNA wrapped around a protein. Such comparisons have enabled Dorothy Erie's chemistry group to determine which of several possible wrappings occur in actual experiments by showing that conformations which were not seen experimentally would have been resolvable with AFM had they occured, ruling out their being hidden by imaging or reconstruction artifacts.