Structural highlights
FunctionPublication Abstract from PubMedPreparation of high-quality nucleosomal DNA substrates in milligram quantities remains a major bottleneck for mechanistic studies of chromatin-associated processes. Here, we present an optimized large-scale PCR workflow that enables rapid, low-cost production of diverse nucleosomal DNAs suitable for biochemical assays and high-resolution cryo-EM. Systematic optimization of amplification conditions yields milligram quantities of homogeneous DNA that can be fluorescently or biotin-labeled and enzymatically modified to introduce site-specific single-strand breaks (SSBs) or epigenetic marks. We also engineered an improved Nt.BsmAI nickase variant (R386D) that minimizes undesired double-strand cleavage while maintaining robust nicking activity. Using nucleosomes reconstituted with these engineered DNAs, we demonstrate the versatility of this platform across EMSA, biolayer interferometry, and cryo-EM. Structural analysis reveals how the PARP2 WGR domain engages an SSB within the nucleosome and uncovers associated shifts in H2B tail conformation that facilitate access to lesions positioned near the tail. Overall, this workflow provides a robust and scalable method for generating precisely modified nucleosomal substrates, enabling quantitative and structural dissection of PARP2-mediated DNA damage recognition and the coupled histone H2B tail rearrangements that facilitate lesion accessibility in chromatin. High-Yield Production of Modified DNA Enables Structural Analysis of PARP2 Recognition of Nucleosomal Single-Strand Breaks.,Jayathilake C, Mewhinney CE, Gregory-Lott ER, Virk RK, Nair R, Yang J, Cho E, Day AG, Taylor DJ, Kim TH J Mol Biol. 2026 Mar 10;438(10):169753. doi: 10.1016/j.jmb.2026.169753. PMID:41819421[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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This page was last modified 08:09, 25 March 2026.