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AFM Studies of DNA Repair – Overview

 

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Research: Atomic Force Microscopy, Past Studies

Topic: DNA Mismatch Repair

AFM provides us unique capability in revealing structural and functional relationship of DNA repair, including but not limited to, the specificity of the protein binding complex, the binding affinity, the DNA bending through bend angle analysis, the conformation analysis, and how they relate to the biological context of its functions. The following figure gives you a walk-through over what image information we are seeking and the analysis that we could get from these images.

Figure. (B-J) AFM images of hMutSa-DNA complexes. Yellow and blue arrows identify, respectively, single and multiple MutSa proteins. Z-scale is 4nm. B. MutSa + ADP, C. MutSa + ATPgS + ADP, E&F. MutSa+ATP on linear and circular DNA, D&G. MutSa+ATP, zoomed view of a mismatch-bound complex containing multiple MutSa. DNA is 2030 bp with a GT mismatch located at 372 bp (120 nm) from one end when linearized. (H-J) MutSa induced DNA bending in the presence of ADP (H) or ATP (I&J). K. Distribution of MutSa on linearized GT DNA: position (left), number of MutSa proteins per complex (middle), and MutSa induced DNA bending at the mismatch (right). Stoichiometries (middle) were estimated by dividing the volume of the observed complex by the volume single MutSa.

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MutS

The DNA binding properties of MutS and its homologs, the initiation protein in the DNA mismatch repair pathway, is the center piece of our AFM studies. For example, we use atomic force microscopy to examine the DNA binding properties of yeast Msh2-Msh6, the initiation protein in the DNA mismatch repair pathway.

Figure. Top(a, b) and side(c, d) views of yMsh2-yMsh6 bound to mismatched DNA. Here yMsh2-yMsh6 is shown bound to bent(a,c) and unbent(b, d) DNA.

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These results support our model for prokaryotic mismatch repair initiation, which shows that the DNA exists in two conformations when MutS is bound to the mismatch: one in which the DNA is bent and one in which the DNA is unbent. We are now able to relate structure-function properties of mismatch repair initiation in prokaryotes with eukaryotic MutS homologs.

MutL

We also study the downstream mismatch repair protein MutL and its homologs. For example, we use atomic force microscopy to image Saccharomyces cerevisiae yMutLα under a variety of conditions (presence and absence of DNA and/or adenine nucleotide, varying salt concentrations) in order to understand the role yMutLα plays in mismatch repair.

Figure. 3-D Image of yMutLα in the absence of ATP and DNA. The inset shows a 2D 1×1 µm image. By varying the conditions, conformational changes in the protein are observed.

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Figure: AFM image of E. coli MutL.

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MutS-MutL

We also use AFM to study the interaction of MutS and MutL related to DNA mismatch repair initiation. MutS-MutL complexes recruit downstream proteins in the mismatch repair system to complete the repair process.

Figure: AFM images of hMutSa-hMutLa-DNA complexes.

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