9ZQB

Nucleosome with an SSB at SHL -2.8 in complex with human PARP2 and HPF1, Class 1

Summary for 9ZQB

• Entry DOI: 10.2210/pdb9zqb/pdb
• EMDB information: 74563
• Descriptor: Histone H2A, Histone H2B, Histone H3, ... (9 entities in total)
• Functional Keywords: dna damage binding protein, dna binding protein, dna binding protein-dna complex, dna binding protein/dna
• Biological source: Drosophila melanogaster (fruit fly); More
• Total number of polymer chains: 14
• Total formula weight: 345795.66

Authors

Kim, T.H.,Jayathilake, C.,Virk, R.K.,Gregory-Lott, E.R. (deposition date: 2025-12-18, release date: 2026-03-25)

Primary citation

Jayathilake, C.,Mewhinney, C.E.,Gregory-Lott, E.R.,Virk, R.K.,Nair, R.,Yang, J.,Cho, E.,Day, A.G.,Taylor, D.J.,Kim, T.H.
High-Yield Production of Modified DNA Enables Structural Analysis of PARP2 Recognition of Nucleosomal Single-Strand Breaks.
J.Mol.Biol., :169753-169753, 2026

PubMed Abstract: Preparation 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.

PubMed: 41819421
DOI: 10.1016/j.jmb.2026.169753

Experimental method

ELECTRON MICROSCOPY (2.1 Å)