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 with fluorescently tagged proteins isolated from nuclear extracts of human cells. We demonstrated the wide applicability of this novel technique on undamaged DNA and 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), and 8-oxoguanine glycosylase 1 (OGG1). We found that PARP1 binding to DNA nicks is altered by tension, and that UV-DDB did not act as an obligate heterodimer of DDB1 and DDB2 on UV-irradiated DNA. UV-DDB bound to UV photoproducts with an average lifetime of 39 seconds (corrected for photobleaching, τ _c), whereas binding lifetimes to 8-oxoG adducts were < 1 second. Catalytically inactive OGG1 variant K249Q bound oxidative damage 23-fold longer than WT OGG1, at 47 and 2.0 s, respectively. By measuring three fluorescent colors simultaneously, we also characterized the assembly and disassembly kinetics of UV-DDB and OGG1 complexes on DNA. Hence, the SMADNE technique represents a novel, scalable, and universal method to obtain single-molecule mechanistic insights into key protein–DNA interactions in an environment containing physiologically-relevant nuclear proteins.