Stable DNA-protein cross-links are products of DNA charge transport in a nucleosome core particle

DNA-protein cross-links (DPCs) in nucleosome core particles (NCPs), the fundamental building block of chromatin, arise during times of cellular oxidative stress. These lesions are expected to be detrimental to the cell due to interference with processes like chromatin remodeling, transcription, DNA replication, and epigenetic marking. However, much is still unknown about the mechanisms leading to the formation of DPCs in NCPs, and the exact sites of these lesions in chromatin have not been delineated. During DNA charge transport (CT), an oxidant leads to the formation of a guanine radical cation (G(center dot+)) which then becomes mobile and migrates away from the initial site of damage. Since previous studies have established that reactions between a G(center dot+) and some amino acids lead to DPC formation in both DNA-peptide and DNA-protein complexes, we hypothesized that DNA CT could lead to DPC fort-nation within NCPs. To test this hypothesis, we studied DNA CT reactions in NCPs reconstituted with DNA containing (i) the 601 NCP positioning sequence and (ii) 14 bp of a linker DNA with a covalently attached anthraquinone (AQ) photooxidant. Collectively, the results from Western blotting, EMSAs, and DNA footprinting reactions lead to the conclusion that AQ-initiated DNA CT is responsible for DNA-H3 cross-linking in one specific region of these NCPs. Furthermore, these DPCs are stable for days at 37 degrees C, indicating that DNA CT in chromatin can lead to long-lived DNA lesions which the cell must somehow find and excise.