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Loading dynamics of a sliding DNA clamp

Journal Article


Abstract


  • Sliding DNA clamps are loaded at a ss/dsDNA junction by a clamp loader that depends on ATP binding for clamp opening. Sequential ATP hydrolysis results in closure of the clamp so that it completely encircles and diffuses on dsDNA. We followed events during loading of an E.coli βclamp in real time by using single-molecule FRET (smFRET). Three successive FRET states were retained for 0.3s, 0.7s, and 9min: Hydrolysis of the first ATP molecule by the γ clamp loader resulted in closure of the clamp in 0.3s, and after 0.7s in the closed conformation, the clamp was released to diffuse on the dsDNA for at least 9min. An additional single-molecule polarization study revealed that the interfacial domain of the clamp rotated in plane by approximately 8° during clamp closure. The single-molecule polarization and FRET studies thus revealed the real-time dynamics of the ATP-hydrolysis-dependent 3D conformational change of the βclamp during loading at a ss/dsDNA junction. When one clamp closes. Single-molecule Förster resonance energy transfer and single-molecule fluorescence polarization were used to monitor the loading of a sliding DNA clamp onto double-stranded DNA by the clamp loader (see picture). Thus, the dynamic features of a DNA clamp in the DNA/DNA-clamp/clamp-loader ternary complex were revealed. © 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Publication Date


  • 2014

Citation


  • Cho, W. K., Jergic, S., Kim, D., Dixon, N. E., & Lee, J. B. (2014). Loading dynamics of a sliding DNA clamp. Angewandte Chemie - International Edition, 53(26), 6768-6771. doi:10.1002/anie.201403063

Scopus Eid


  • 2-s2.0-84903212525

Start Page


  • 6768

End Page


  • 6771

Volume


  • 53

Issue


  • 26

Abstract


  • Sliding DNA clamps are loaded at a ss/dsDNA junction by a clamp loader that depends on ATP binding for clamp opening. Sequential ATP hydrolysis results in closure of the clamp so that it completely encircles and diffuses on dsDNA. We followed events during loading of an E.coli βclamp in real time by using single-molecule FRET (smFRET). Three successive FRET states were retained for 0.3s, 0.7s, and 9min: Hydrolysis of the first ATP molecule by the γ clamp loader resulted in closure of the clamp in 0.3s, and after 0.7s in the closed conformation, the clamp was released to diffuse on the dsDNA for at least 9min. An additional single-molecule polarization study revealed that the interfacial domain of the clamp rotated in plane by approximately 8° during clamp closure. The single-molecule polarization and FRET studies thus revealed the real-time dynamics of the ATP-hydrolysis-dependent 3D conformational change of the βclamp during loading at a ss/dsDNA junction. When one clamp closes. Single-molecule Förster resonance energy transfer and single-molecule fluorescence polarization were used to monitor the loading of a sliding DNA clamp onto double-stranded DNA by the clamp loader (see picture). Thus, the dynamic features of a DNA clamp in the DNA/DNA-clamp/clamp-loader ternary complex were revealed. © 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Publication Date


  • 2014

Citation


  • Cho, W. K., Jergic, S., Kim, D., Dixon, N. E., & Lee, J. B. (2014). Loading dynamics of a sliding DNA clamp. Angewandte Chemie - International Edition, 53(26), 6768-6771. doi:10.1002/anie.201403063

Scopus Eid


  • 2-s2.0-84903212525

Start Page


  • 6768

End Page


  • 6771

Volume


  • 53

Issue


  • 26