Many protein interactions with DNA require specific sequences; however, how these sequences are located remains uncertain. DNA normally appears bundled in solution but, to study DNA–protein interactions, the DNA needs to be elongated. Using fluidics single DNA strands can be efficiently and rapidly elongated between beads immobilized on a microscope slide surface. Such “DNA tightropes” offer a valuable method to study protein search mechanisms. Real-time fluorescence imaging of these interactions provides quantitative descriptions of search mechanism at the single molecule level. In our lab, we use this method to study the complex process of nucleotide excision DNA repair to determine mechanisms of damage detection, lesion removal, and DNA excision.
Single molecule imaging DNA tightropes Quantum dots Diffusion DNA repair Search mechanisms Nucleotide excision repair
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This work was supported by the BBSRC [BB/I003460/1] and [BB/M019144/1] to N.M.K., BHF [FS/13/69/30504] to A.V.I., and University of Kent for L.S.
Dunn AR, Kad NM, Nelson SR, Warshaw DM, Wallace SS (2011) Single Qdot-labeled glycosylase molecules use a wedge amino acid to probe for lesions while scanning along DNA. Nucleic Acids Res 39:7487–7498CrossRefPubMedPubMedCentralGoogle Scholar
Kad NM, Wang H, Kennedy GG, Warshaw DM, Van Houten B (2010) Collaborative dynamic DNA scanning by nucleotide excision repair proteins investigated by single- molecule imaging of quantum-dot-labeled proteins. Mol Cell 37:702–713CrossRefPubMedPubMedCentralGoogle Scholar
Wang H, Tessmer I, Croteau DL, Erie DA, Van Houten B (2008) Functional characterization and atomic force microscopy of a DNA repair protein conjugated to a quantum dot. Nano Lett 8:1631–1637CrossRefPubMedPubMedCentralGoogle Scholar
Hughes CD, Wang H, Ghodke H, Simons M, Towheed A, Peng Y, Van Houten B, Kad NM (2013) Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes. Nucleic Acids Res 41:4901–4912CrossRefPubMedPubMedCentralGoogle Scholar
Berg HC (1993) Random walks in biology. Princeton University Press, Princeton, NJGoogle Scholar
Hohng S, Ha T (2004) Near-complete suppression of quantum dot blinking in ambient conditions. J Am Chem Soc 126:1324–1325CrossRefPubMedGoogle Scholar
Tokunaga M, Imamoto N, Sakata-Sogawa K (2008) Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat Methods 5:159–161CrossRefPubMedGoogle Scholar