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. 2021 Dec 22;601(7892):268–273. doi: 10.1038/s41586-021-04261-0

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 6 — a. Native gel (longer 6% polyacrylamide) showing RPA stripping assay with HELQ (10 nM) alone and with indicated concentrations of RPA. b, Native gel (longer 6% polyacrylamide) showing RPA stripping assay with indicated concentrations of HELQ in the absence and presence of RPA (40 nM). c, Schematic of immobilized dual labelled (Cy3 and Cy5) DNA in the absence of RPA. d–e, Representative intensity trajectory (top) and corresponding FRET trajectory (bottom) of dual labelled DNA in the absence of RPA. f, Time-binned FRET histogram of DNA only, fit with gaussian. g, Schematic of immobilized dual labelled DNA in the presence of RPA. h–i, Representative intensity trajectory (top) and corresponding FRET trajectory (bottom) of dual labelled DNA in the presence of RPA. Stable high FRET is observed. j, Time-binned FRET histogram of DNA in the presence of RPA. Fit with gaussian. k, Schematic of the experimental set up of single-molecule FRET-based RPA striping assay. DNA dual-labelled with the FRET pair Cy3 and Cy5 is immobilized on the microscope slide. In the absence of RPA, a short 6 nt sequence of homology causes the DNA to fold into a high FRET state. Upon RPA binding, the DNA unfolds resulting in a low FRET state. Addition of HELQ results in cycling between the low (bound) and high (free) FRET states as RPA is bound and removed respectively. l, Example of single-molecule fluorescence trajectory (top, Cy3 in blue, Cy5 in red) and corresponding FRET (bottom) showing the transition from low FRET (bound) to high FRET (free). m, Representative FRET trajectory of DNA in the presence of 1 nM RPA and 200 nM HELQ, spikes of high FRET correspond to RPA removal events. n, Plot of dwell time of RPA bound (t_off), low FRET, state with increasing HELQ concentration. o, Plot of dwell time of free state (t_on), high FRET, on RPA removal with increasing HELQ concentration. p, Representative FRET trajectory of DNA in the presence of 1 nM RPA and 200 nM HELQ KM. q, Representative gel of DNA annealing assay with HELQ (3 nM), RPA (40 nM) and indicated concentrations of RAD51. r, Quantification of experiments such as shown in q. n = 3 independent experiments; mean ± S.E.M.