DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals

Greg L. Hura, Chi Lin Tsai, Shelley A. Claridge, Marc L. Mendillo, Jessica M. Smith, Gareth J. Williams, Alexander J. Mastroianni, A. Paul Alivisatos, Christopher D. Putnam, Richard D. Kolodner, John A. Tainer*

*Corresponding author for this work

Research output: Contribution to journalArticle

38 Scopus citations

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 flashcooled 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 ofMMR. 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.

Original languageEnglish (US)
Pages (from-to)17308-17313
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume110
Issue number43
DOIs
StatePublished - Oct 22 2013

ASJC Scopus subject areas

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    Hura, G. L., Tsai, C. L., Claridge, S. A., Mendillo, M. L., Smith, J. M., Williams, G. J., Mastroianni, A. J., Alivisatos, A. P., Putnam, C. D., Kolodner, R. D., & Tainer, J. A. (2013). DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals. Proceedings of the National Academy of Sciences of the United States of America, 110(43), 17308-17313. https://doi.org/10.1073/pnas.1308595110