The same results were obtained when C6 and mCherry were substituted with A9 and GFP, respectively (Figure S2B,C). of a bivalent nanobody allowed us to precisely detect -H2AX foci in drug-treated cells as efficiently as with commercially available conventional antibodies. Furthermore, we tracked -H2AX foci in live cells upon intracellular delivery of the bivalent nanobody fused to the red fluorescent protein dTomato, making, consequently, this new cost-effective reagent useful for studying drug-induced replication stress in both fixed and living cancer cells. Abstract Histone H2AX phosphorylated at serine 139 (-H2AX) is usually a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While -H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to -H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of -H2AX and show the atomic constituents behind its specificity. CBL-0137 We designed a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize -H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect -H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize CBL-0137 with precision -H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual malignancy cells under a variety of conditions. Keywords: H2AX, phosphorylation, replication stress, nanobody, imaging, one-step detection, malignancy cells, genotoxicity assay in live cells 1. Introduction Histones constitute the core proteins of chromatin and their post-translational modifications (PTMs) contribute to the molecular basis of epigenetic gene regulation and cellular memory [1]. In humans, several variant forms of histones have been described [2] and this is particularly relevant for the H2A histone. The H2A variants represent the largest and most diverse family of histones; there is overwhelming evidence that their unstructured N- and C-termini, which protrude out of the core structure of the nucleosome, harbor several sites for PTMs in response to varying stimuli [3]. The H2AX variant shares high amino acid similarity with H2A and is characterized by an extended C-terminus, which is usually phosphorylated when the cells become injured by brokers that provoke DNA replication stress (RS) and genome instability [4]. The Gimap6 phosphorylation of serine at position 139 (S139) of H2AX has been particularly well studied and represents a key event in the detection and response to DNA damage [5,6]. Phosphorylation of histone H2AX at S139, which gives rise to what is usually generally referred to as -H2AX, is in fact a very early step in the DNA damage response (DDR) and an essential signal for the recruitment and retention of DDR complexes at the site of damage [7]. Three different phosphatidylinositol 3 kinase (PI3K)-related kinases mediate S139 phosphorylation on H2AX: ATM (ataxia-telangiectasia mutated), ATR (ATM and Rad3-related), and DNA-PK (DNA-dependent protein kinase) [8]. ATM and DNA-PK share functional redundancy upon ionizing radiation, while ATR may preferentially phosphorylate H2AX during RS [9]. This PTM of H2AX is usually highly CBL-0137 dynamic and a number of phosphatases, including those of the PPP family and Wip1, are able to dephosphorylate -H2AX to fine-tune the duration and intensity of the DDR signaling [10]. It has also been found that H2AX can be phosphorylated at the threonine residue at position 136 (T136) and at the C-terminal tyrosine residue CBL-0137 at position 142 (Y142) to facilitate DNA repair,.
