Generation of Recombinant Nucleosomes Containing Site-Specific DNA Damage.

Eukaryotic DNA exists in chromatin, where the genomic DNA is packaged into a fundamental repeating unit known as the nucleosome. In this chromatin environment, our genomic DNA is constantly under attack by exogenous and endogenous stressors that can lead to DNA damage. Importantly, this DNA damage must be repaired to prevent the accumulation of mutations and ensure normal cellular function. To date, most in-depth biochemical studies of DNA repair proteins have been performed in the context of free duplex DNA. However, chromatin can serve as a barrier that DNA repair enzymes must navigate in order find, access, and process DNA damage in the cell. To facilitate future studies of DNA repair in chromatin, we describe a protocol for generating nucleosome containing site-specific DNA damage that can be utilized for a variety of in vitro applications. This protocol describes several key steps including how to generate damaged DNA oligonucleotides, the expression and purification of recombinant histones, the refolding of histone complexes, and the reconstitution of nucleosomes containing site-specific DNA damage. These methods will enable researchers to generate nucleosomes containing site-specific DNA damage for extensive biochemical and structural studies of DNA repair in the nucleosome.

Structural basis for APE1 processing DNA damage in the nucleosome.

Genomic DNA is continually exposed to endogenous and exogenous factors that promote DNA damage. Eukaryotic genomic DNA is packaged into nucleosomes, which present a barrier to accessing and effectively repairing DNA damage. The mechanisms by which DNA repair proteins overcome this barrier to repair DNA damage in the nucleosome and protect genomic stability is unknown. Here, we determine how the base excision repair (BER) endonuclease AP-endonuclease 1 (APE1) recognizes and cleaves DNA damage in the nucleosome. Kinetic assays determine that APE1 cleaves solvent-exposed AP sites in the nucleosome with 3 - 6 orders of magnitude higher efficiency than occluded AP sites. A cryo-electron microscopy structure of APE1 bound to a nucleosome containing a solvent-exposed AP site reveal that APE1 uses a DNA sculpting mechanism for AP site recognition, where APE1 bends the nucleosomal DNA to access the AP site. Notably, additional biochemical and structural characterization of occluded AP sites identify contacts between the nucleosomal DNA and histone octamer that prevent efficient processing of the AP site by APE1. These findings provide a rationale for the position-dependent activity of BER proteins in the nucleosome and suggests the ability of BER proteins to sculpt nucleosomal DNA drives efficient BER in chromatin.