Single-Molecule Analysis of DNA-binding proteins from Nuclear Extracts (SMADNE)
Single-Molecule Analysis of DNA-binding proteins from Nuclear Extracts (SMADNE)
Abstract Single-molecule characterization of protein-DNA dynamics provides unprecedented mechanistic details about numerous nuclear processes. Here, we describe a new method that rapidly generates single-molecule information for fluorescently tagged proteins isolated from nuclear extracts of human cells. This approach determines binding lifetimes (koff), events per second (～kon), positional dependence (specificity), and characterizes 1D diffusion along DNA. We demonstrated the wide applicability of this approach on three forms of DNA damage using seven native DNA repair proteins and two structural variants, including: poly(ADP-ribose) polymerase (PARP1), heterodimeric ultraviolet-damaged DNA-binding protein (UV-DDB), xeroderma pigmentosum complementation group C protein (XPC), and 8-oxoguanine glycosylase 1 (OGG1). By measuring multiple fluorescent colors simultaneously, we additionally characterized the assembly and disassembly kinetics of multi-protein complexes on DNA. Thus, Single-Molecule Analysis of DNA-binding proteins from Nuclear Extracts (SMADNE) provides new insights about damage recognition and represents a universal technique that can be used to rapidly characterize numerous protein-DNA interactions.
Roginskaya Vera、Van Houten Bennett、Kumar Namrata、Jakielski Rachel C.、Schaich Matthew A.、Zhong Zhou、Urban Roman、Schnable Brittani L.、Kad Neil M.
UPMC-Hillman Cancer Center, University of Pittsburgh School of MedicineDepartment of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine||UPMC-Hillman Cancer Center, University of Pittsburgh School of Medicine||Molecular Genetics and Developmental Biology Graduate Program, University of Pittsburgh School of Medicine||Molecular Biophysics and Structural Biology Program, University of PittsburghUPMC-Hillman Cancer Center, University of Pittsburgh School of Medicine||Molecular Genetics and Developmental Biology Graduate Program, University of Pittsburgh School of MedicineDepartment of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine||UPMC-Hillman Cancer Center, University of Pittsburgh School of MedicineDepartment of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine||UPMC-Hillman Cancer Center, University of Pittsburgh School of MedicineDepartment of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine||UPMC-Hillman Cancer Center, University of Pittsburgh School of Medicine||LUMICKS, WalthamSchool of Biosciences, University of KentUPMC-Hillman Cancer Center, University of Pittsburgh School of Medicine||Molecular Biophysics and Structural Biology Program, University of PittsburghSchool of Biosciences, University of Kent
生物科学研究方法、生物科学研究技术分子生物学基础医学
Single molecule fluorescence microscopynuclear extractnucleotide excision repairbase excision repairLUMICKS C-trap
Roginskaya Vera,Van Houten Bennett,Kumar Namrata,Jakielski Rachel C.,Schaich Matthew A.,Zhong Zhou,Urban Roman,Schnable Brittani L.,Kad Neil M..Single-Molecule Analysis of DNA-binding proteins from Nuclear Extracts (SMADNE)[EB/OL].(2025-03-28)[2025-05-18].https://www.biorxiv.org/content/10.1101/2022.04.01.486290.点此复制
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