Dataset Information

Comparison of the Base Excision and Direct Reversal Repair Pathways for Correcting 1,N6-Ethenoadenine in Strongly Positioned Nucleosome Core Particles.

ABSTRACT: 1,N6-ethenoadenine (?A) is a mutagenic lesion and biomarker observed in numerous cancerous tissues. Two pathways are responsible for its repair: base excision repair (BER) and direct reversal repair (DRR). Alkyladenine DNA glycosylase (AAG) is the primary enzyme that excises ?A in BER, generating stable intermediates that are processed by downstream enzymes. For DRR, the Fe(II)/?-ketoglutarate-dependent ALKBH2 enzyme repairs ?A by direct conversion of ?A to A. While the molecular mechanism of each enzyme is well understood on unpackaged duplex DNA, less is known about their actions on packaged DNA. The nucleosome core particle (NCP) forms the minimal packaging unit of DNA in eukaryotic organisms and is composed of 145-147 base pairs wrapped around a core of eight histone proteins. In this work, we investigated the activity of AAG and ALKBH2 on ?A lesions globally distributed at positions throughout a strongly positioned NCP. Overall, we examined the repair of ?A at 23 unique locations in packaged DNA. We observed a strong correlation between rotational positioning of ?A and AAG activity but not ALKBH2 activity. ALKBH2 was more effective than AAG at repairing occluded ?A lesions, but only AAG was capable of full repair of any ?A in the NCP. However, notable exceptions to these trends were observed, highlighting the complexity of the NCP as a substrate for DNA repair. Modeling of binding of the repair enzymes to NCPs revealed that some of these observations can be explained by steric interference caused by DNA packaging. Specifically, interactions between ALKBH2 and the histone proteins obstruct binding to DNA, which leads to diminished activity. Taken together, these results support in vivo observations of alkylation damage profiles and contribute to our understanding of mutational hotspots.

SUBMITTER: Caffrey PJ

PROVIDER: S-EPMC7374743 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Publications

Comparison of the Base Excision and Direct Reversal Repair Pathways for Correcting 1,N6-Ethenoadenine in Strongly Positioned Nucleosome Core Particles.

Caffrey Paul J PJ Kher Raadhika R Bian Ke K Li Deyu D Delaney Sarah S

Chemical research in toxicology 20200501 7

1,N6-ethenoadenine (εA) is a mutagenic lesion and biomarker observed in numerous cancerous tissues. Two pathways are responsible for its repair: base excision repair (BER) and direct reversal repair (DRR). Alkyladenine DNA glycosylase (AAG) is the primary enzyme that excises εA in BER, generating stable intermediates that are processed by downstream enzymes. For DRR, the Fe(II)/α-ketoglutarate-dependent ALKBH2 enzyme repairs εA by direct conversion of εA to A. While the molecul ...[more]

PMID: 32293880

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Project description:Base excision repair is hindered by nucleosomes.Outwardly oriented uracils near the nucleosome center are efficiently cleaved; however, polymerase ? 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 ? 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.

Dataset Information

Comparison of the Base Excision and Direct Reversal Repair Pathways for Correcting 1,N6-Ethenoadenine in Strongly Positioned Nucleosome Core Particles.

Publications

Comparison of the Base Excision and Direct Reversal Repair Pathways for Correcting 1,<i>N</i><sup>6</sup>-Ethenoadenine in Strongly Positioned Nucleosome Core Particles.

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