The ionic environment of the eukaryotic cell is dynamic and can broadly affect cellular processes. Fluctuations in ionic strength can arise from extracellular factors and intracellular events that can influence interactions between cellular components [1], [2]. Ionic strength affects protein structure and function [3], [4], including protein aggregation [5], and nucleic acid binding [6]. Pockets of liquid-liquid phase separation within the nucleus [7], [8] also contribute to the dynamic nature of the cell’s ionic environment. Genetic material of eukaryotic cells is highly packaged into chromatin within these nuclear regions of changing ionic strength, which may impact how transcriptional, repair, and other cellular machinery interacts with DNA.

Chromatin fibers consist of DNA packaging units known as nucleosome core particles (NCPs) and are compacted in high ionic strength environments [9], [10], [11], [12]. An NCP is comprised of ~145 base pairs (bp) of DNA wrapped around two copies each of the histone proteins H2A, H2B, H3, and H4, with a central axis of pseudo-symmetry referred to as the dyad axis (Fig. 1) [13]. The overall stability of the NCP is in part driven by interactions between positively charged histone proteins as well as between histones and the negatively charged backbone of DNA. Such electrostatic interactions are sensitive to changes in ionic strength to maintain a balance between histone core integrity and NCP stability [14], [15]. The NCP is a dynamic structure that can undergo unwrapping of DNA in the entry/exit regions [16], compaction and gaping of the NCP gyres [10], [17], and association of histone N-terminal tails with the nucleosomal DNA [18], [19], [20], all of which are influenced by ionic strength. Histone octamers are also subjected to post-translational modifications (PTMs), exchange of histones for variant versions, as well as interactions with histone chaperones and chromatin remodelers that contribute the dynamic nature of NCPs [21], [22], [23].

DNA repair is impacted by the packaging of DNA into NCPs, which poses a significant barrier to the repair machinery. In this work we focus on initiation of the base excision repair (BER) pathway. The initiation is catalyzed by a glycosylase, which excises a modified nucleobase from the sugar-phosphate backbone. Using NCPs, biochemical experiments have shown that glycosylase activity is modulated by the rotational and translational positioning of DNA [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37]. Typically, glycosylases can more easily access lesions that face outward towards solution compared to those that face inward towards the histone protein core. Additionally, lesions in the entry/exit region of NCPs are more accessible to glycosylases due to transient and spontaneous unwrapping of the DNA from the histones [25], [28], [31], [32], [33], [34], [38], [39]. Notably, these results of biochemical experiments are consistent with in vivo genome-wide mutation mapping [40].

Single-strand selective monofunctional uracil DNA glycosylase (SMUG1) is one of eleven known human glycosylases [41], [42]. SMUG1 has a wide substrate range, including uracil (U), and can excise lesions from both single- and double-stranded DNA [42], [43]. SMUG1 uses an intercalating “helical wedge” that distorts DNA to access and repair a lesion [44]. SMUG1 has been shown to excise U from U:G [39], [45], [46] and U:A [47] bp in NCPs, although in many locations of the NCP its activity is significantly diminished compared to duplex DNA.

Here we use an in vitro NCP model system to determine the effects of ionic strength on the excision of U by SMUG1 (Fig. 1). The U:G bp represents deamination of cytosine in a C:G bp and was chosen as a prototypic damage type for BER. Ranges of ionic strength were chosen in the context of reported cellular concentrations of various ions, and in particular K+ which can reach 150 mM [48], [49], [50], [51], [52], [53]. Total physiological ionic strength concentrations of monovalent cations have been reported to reach ~100-200 mM, with fluctuations surrounding compartments of the nucleus [48], [49], [50], [51], [52], [53]. We employ complementary footprinting techniques using hydroxyl radicals and DNase to assess the NCP structure. SMUG1 reactions were performed on U-containing duplex and NCPs in different ionic strength environments. We find that SMUG1 activity on NCPs is modulated by ionic strength and that lesion excision is better at lower ionic strength, likely due to NCP structure and/or dynamics.