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Production of long linear DNA substrates with site-specific chemical lesions for single-molecule replisome studies

Journal Article


Abstract


  • Single-molecule imaging studies using long linear DNA substrates have revealed unanticipated insights into the dynamics of multi-protein systems. The use of long DNA substrates allows for the study of protein���DNA interactions with observation of the movement and behavior of proteins over distances accessible by fluorescence microscopy. Generalized methods can be exploited to generate and optimize a variety of linear DNA substrates with plasmid DNA as a simple starting point using standard biochemical techniques. Here, we present protocols to produce high-quality plasmid-based 36-kb linear DNA substrates that support DNA replication by the Escherichia coli replisome and that contain chemical lesions at well-defined positions. These substrates can be used to visualize replisome���lesion encounters at the single-molecule level, providing mechanistic details of replisome stalling and dynamics occurring during replication rescue and restart.

Publication Date


  • 2022

Citation


  • Kaur, G., Spenkelink, L. M., Lewis, J. S., Jergic, S., Dixon, N. E., & van Oijen, A. M. (2022). Production of long linear DNA substrates with site-specific chemical lesions for single-molecule replisome studies. Unknown Journal, 672, 299-315. doi:10.1016/bs.mie.2022.02.022

Scopus Eid


  • 2-s2.0-85128308770

Web Of Science Accession Number


Start Page


  • 299

End Page


  • 315

Volume


  • 672

Issue


Place Of Publication


Abstract


  • Single-molecule imaging studies using long linear DNA substrates have revealed unanticipated insights into the dynamics of multi-protein systems. The use of long DNA substrates allows for the study of protein���DNA interactions with observation of the movement and behavior of proteins over distances accessible by fluorescence microscopy. Generalized methods can be exploited to generate and optimize a variety of linear DNA substrates with plasmid DNA as a simple starting point using standard biochemical techniques. Here, we present protocols to produce high-quality plasmid-based 36-kb linear DNA substrates that support DNA replication by the Escherichia coli replisome and that contain chemical lesions at well-defined positions. These substrates can be used to visualize replisome���lesion encounters at the single-molecule level, providing mechanistic details of replisome stalling and dynamics occurring during replication rescue and restart.

Publication Date


  • 2022

Citation


  • Kaur, G., Spenkelink, L. M., Lewis, J. S., Jergic, S., Dixon, N. E., & van Oijen, A. M. (2022). Production of long linear DNA substrates with site-specific chemical lesions for single-molecule replisome studies. Unknown Journal, 672, 299-315. doi:10.1016/bs.mie.2022.02.022

Scopus Eid


  • 2-s2.0-85128308770

Web Of Science Accession Number


Start Page


  • 299

End Page


  • 315

Volume


  • 672

Issue


Place Of Publication