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

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 su...

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Bibliographic Details
Published in:Chemical science (Cambridge) 2021-01, Vol.12 (6), p.239-249
Main Authors: Yuan, Zheng, Zhang, Dapeng, Yu, Fangzhi, Ma, Yangde, Liu, Yan, Li, Xiangjun, Wang, Hailin
Format: Article
Language:English
Subjects:
Assaying
Assembly
Chemistry
Cutting
Deoxyribonucleic acid
DNA
Gene sequencing
Genetic modification
Mathematical analysis
Nucleotides
Proteins
Segments
Stoichiometry
Technology assessment
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Summary: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. A single molecule footprinting-Seq assay at single nucleotide resolution was developed for measuring the distribution and kinetic binding of proteins on DNA.
ISSN:2041-6520
2041-6539
DOI:10.1039/d0sc01742f