Issue 6, 2021

Precise sequencing of single protected-DNA fragment molecules for profiling of protein distribution and assembly on DNA

Abstract

Multiple DNA-interacting protein molecules are often dynamically distributed and/or assembled along a DNA molecule to adapt to their intricate functions temporally. However, analytical technology for measuring such binding behaviours is still missing. Here, we demonstrate the unique capacity of a supernuclease for a highly efficient cutting of the unprotected-DNA segments and with complete preservation of the protein-occluded DNA segments at near single-nucleotide resolution. By exploring this high-resolution cutting, an unprecedented assay that allows a precise sequencing of single protected-DNA fragment molecules (SPDFMS) was developed. As relevant applications, relevant information was gained on the respective distribution/assembly patterns and coordinated displacement of single-stranded DNA-binding protein and recombinase RecA, two model proteins, on DNA. Benefiting from this assay, we also for the first time provide direct measurement of the length of single RecA nucleofilaments, showing the predominant stoichiometry of 5–7 RecA monomers per RecA nucleofilament under physiologically relevant conditions. This innovative assay appears as a promising analytical tool for studying diverse protein–DNA interactions implicated in DNA replication, transcription, recombination, repair, and gene editing.

Graphical abstract: Precise sequencing of single protected-DNA fragment molecules for profiling of protein distribution and assembly on DNA

Supplementary files

Article information

Article type
Edge Article
Submitted
26 mar. 2020
Accepted
31 des. 2020
First published
02 jan. 2021
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2021,12, 2039-2049

Precise sequencing of single protected-DNA fragment molecules for profiling of protein distribution and assembly on DNA

Z. Yuan, D. Zhang, F. Yu, Y. Ma, Y. Liu, X. Li and H. Wang, Chem. Sci., 2021, 12, 2039 DOI: 10.1039/D0SC01742F

This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. You can use material from this article in other publications, without requesting further permission from the RSC, provided that the correct acknowledgement is given and it is not used for commercial purposes.

To request permission to reproduce material from this article in a commercial publication, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party commercial publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements