Systematic Study of the Impact of DNA Interstrand Cross-Links on Nucleosome Structure, Sliding, and Transcription

DNA interstrand cross-links (ICLs) covalently link complementary DNA strands and represent one of the most cytotoxic forms of DNA damage. While their impact on free DNA has been extensively characterized, how ICLs influence nucleosomes─the fundamental units of chromatin─remains largely unexplored. Here, we generated nucleosomes containing site-specific ICLs using click chemistry and systematically examined their effects on nucleosome structure, dynamics, and transcription. Biophysical assays revealed that ICLs did not impair nucleosome assembly, DNA accessibility, or ATP-dependent sliding, and only slightly reduced nucleosome stability. However, in vitro transcription assays demonstrated that ICLs function as absolute barriers to RNA polymerase elongation, producing truncated transcripts that terminate precisely at the cross-linking site in both free DNA and nucleosomal contexts. Restriction enzyme protection assays further showed that transcription-induced nucleosome translocation was unaffected, indicating that arrest results from the inability of the elongation complex to separate cross-linked strands rather than impaired nucleosome mobility. These findings provide mechanistic insight into ICL-induced cytotoxicity at the chromatin level and establish site-specific cross-linked nucleosomes as valuable tools for probing DNA damage responses.