Assessment of DNA Susceptibility to Denaturation as a Marker of Chromatin Structure

Abstract

The susceptibility of DNA in situ to denaturation is modulated by its interactions with histone and nonhistone proteins, as well as with other chromatin components related to the maintenance of the 3D nuclear structure. Measurement of DNA proclivity to denature by cytometry provides insight into chromatin structure and thus can be used to recognize cells in different phases of the cell cycle, including mitosis, quiescence (G 0 ), and apoptosis, as well as to identify the effects of drugs that modify chromatin structure. Particularly useful is the method's ability to detect chromatin changes in sperm cells related to DNA fragmentation and infertility. This article presents a flow cytometric procedure for assessing DNA denaturation based on application of the metachromatic property of acridine orange (AO) to differentially stain singleversus double-stranded DNA. This approach circumvents limitations of biochemical methods of examining DNA denaturation, in particular the fact that the latter destroy higher orders of chromatin structure and that, being applied to bulk cell populations, they cannot detect heterogeneity of individual cells. Because the metachromatic properties of AO have also found application in other cytometric procedures, such as differential staining of RNA versus DNA and assessment of lysosomal proton pump including autophagy, to avert confusion between these approaches and the use of this dye in the DNA denaturation assay, these AO applications are briefly outlined in this unit as well. C 2019 by John Wiley & Sons, Inc. Basic Protocol: Differential staining of single-versus double-stranded DNA with acridine orange Keywords: acridine orange r apoptosis r autophagy r cell cycle r lysosomes r mitosis r sperm chromatin fertility assay How to cite this article: Darzynkiewicz, Z., Halicka, D. H., Zhao, H., & Li, J. (2019). Assessment of DNA susceptibility to denaturation as a marker of chromatin structure. BASIC PROTOCOL DNA DenaturationThe process of splitting double-stranded DNA (dsDNA) into single strands, which involves breaking hydrogen bonds between the bases in the DNA double-helical structure, is defined as DNA denaturation. DNA in solution undergoes denaturation when heated, and the temperature at which half remains in double-stranded conformation while the other half is single stranded is called the melting temperature (T m ). Because the G-C base pair, maintained by three hydrogen bonds, is stronger that the two-base pairing of AO has unique metachromatic properties: under the correct conditions (AO concentration, ionic strength, temperature), it can differentially stain ssDNA and dsDNA. Specifically, when AO intercalates between the base pairs of dsDNA, its fluorescence is maximally excited at 480 nm, its emission is at 530 nm, and its excitation lifetime is ß2 ns (Kapuscinski & Darzynkiewicz, 1984Kapuscinski, Darzynkiewicz, & Melamed, 1982. These values reflect singlet excitation of AO. The interaction of AO with ssDNA is more complex: it involves the insertion of the AO cation between adj...