Abstract
Base excision repair is hindered by nucleosomes. Outwardly oriented uracils near the nucleosome center are efficiently cleaved; however, polymerase β is strongly inhibited at these sites. The histone octamer presents different levels of constraints on BER, dependent on the structural requirements for enzyme activity. Chromatin remodeling is necessary to prevent accumulation of aborted intermediates in nucleosomes. Packaging of DNA into chromatin affects accessibility of DNA regulatory factors involved in transcription, replication, and repair. Evidence suggests that even in the nucleosome core particle (NCP), accessibility to damaged DNA is hindered by the presence of the histone octamer. Base excision repair is the major pathway in mammalian cells responsible for correcting a large number of chemically modified bases. We have measured the repair of site-specific uracil and single nucleotide gaps along the surface of the NCP. Our results indicate that removal of DNA lesions is greatly dependent on their rotational and translational positioning in NCPs. Significantly, the rate of uracil removal with outwardly oriented DNA backbones is 2-10-fold higher than those with inwardly oriented backbones. In general, uracils with inwardly oriented backbones farther away from the dyad center of the NCP are more accessible than those near the dyad. The translational positioning of outwardly oriented gaps is the key factor driving gap filling activity. An outwardly oriented gap near the DNA ends exhibits a 3-fold increase in gap filling activity as compared with one near the dyad with the same rotational orientation. Near the dyad, uracil DNA glycosylase/APE1 removes an outwardly oriented uracil efficiently; however, polymerase β activity is significantly inhibited at this site. These data suggest that the hindrance presented by the location of a DNA lesion is dependent on the structural requirements for enzyme catalysis. Therefore, remodeling at DNA damage sites in NCPs is critical for preventing accumulation of aborted intermediates and ensuring completion of base excision repair.
Highlights
Base excision repair is hindered by nucleosomes
Of Nucleosome Core Particles and Verification of Uracil Rotational Orientation—The design of the substrates is critical in correctly addressing the effects of rotational and translational positioning of DNA lesions on Base excision repair (BER) efficiency
The rotational setting of each uracil was verified by treatment of each construct with uracil DNA glycosylases (UDG) and APE1 to generate a single strand cleavage at the uracil site of interest, followed by comparison with the 1⁄7OH footprint of nucleosome core particle (NCP) on DNA sequencing gels (Fig. 1B)
Summary
Base excision repair is hindered by nucleosomes. Results: Outwardly oriented uracils near the nucleosome center are efficiently cleaved; polymerase  is strongly inhibited at these sites. Uracil DNA glycosylase/ APE1 removes an outwardly oriented uracil efficiently; polymerase  activity is significantly inhibited at this site These data suggest that the hindrance presented by the location of a DNA lesion is dependent on the structural requirements for enzyme catalysis. Given the numerous parameters that can influence accessibility to DNA damage, completion of the BER pathway to address how these parameters influence repair efficiency is essential in identifying the rate-limiting steps of BER in vivo This may be important for the pol  step because the requirement to bend the DNA template opposite the gap by ϳ90° [37] might not be achieved in the presence of the histones. The presence of APE1 did not affect pol  extension independent of translational and rotational positioning of the DNA gap
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