PMID:24101514

Citation	Hura, GL, Tsai, CL, Claridge, SA, Mendillo, ML, Smith, JM, Williams, GJ, Mastroianni, AJ, Alivisatos, AP, Putnam, CD, Kolodner, RD and Tainer, JA (2013) DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals. Proc. Natl. Acad. Sci. U.S.A. 110:17308-13
Abstract	DNA metabolism and processing frequently require transient or metastable DNA conformations that are biologically important but challenging to characterize. We use gold nanocrystal labels combined with small angle X-ray scattering to develop, test, and apply a method to follow DNA conformations acting in the Escherichia coli mismatch repair (MMR) system in solution. We developed a neutral PEG linker that allowed gold-labeled DNAs to be flash-cooled and stored without degradation in sample quality. The 1,000-fold increased gold nanocrystal scattering vs. DNA enabled investigations at much lower concentrations than otherwise possible to avoid concentration-dependent tetramerization of the MMR initiation enzyme MutS. We analyzed the correlation scattering functions for the nanocrystals to provide higher resolution interparticle distributions not convoluted by the intraparticle distribution. We determined that mispair-containing DNAs were bent more by MutS than complementary sequence DNA (csDNA), did not promote tetramer formation, and allowed MutS conversion to a sliding clamp conformation that eliminated the DNA bends. Addition of second protein responder MutL did not stabilize the MutS-bent forms of DNA. Thus, DNA distortion is only involved at the earliest mispair recognition steps of MMR: MutL does not trap bent DNA conformations, suggesting migrating MutL or MutS/MutL complexes as a conserved feature of MMR. The results promote a mechanism of mismatch DNA bending followed by straightening in initial MutS and MutL responses in MMR. We demonstrate that small angle X-ray scattering with gold labels is an enabling method to examine protein-induced DNA distortions key to the DNA repair, replication, transcription, and packaging.
Links	PubMed PMC3808630 Online version:10.1073/pnas.1308595110
Keywords	Adenosine Triphosphatases/metabolism; Adenosine Triphosphate/metabolism; Algorithms; DNA Mismatch Repair; DNA, Bacterial/chemistry; DNA, Bacterial/metabolism; DNA, Bacterial/ultrastructure; Escherichia coli Proteins/metabolism; Gold/chemistry; Kinetics; Microscopy, Electron, Transmission; Models, Molecular; MutS DNA Mismatch-Binding Protein/metabolism; Nanoparticles/chemistry; Nanoparticles/ultrastructure; Nucleic Acid Conformation; Protein Binding; Scattering, Small Angle; Solutions; X-Ray Diffraction/methods
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ECOLI:MUTS		GO:0008301: DNA binding, bending	ECO:0000317:		F		Figure 2	Missing: with/from CACAO 10663

References

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